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Surgical and molecular characterization of primary and metastatic disease in a neuroendocrine tumor arising in a tailgut cyst. Cold Spring Harbor molecular case studies Erdrich, J., Schaberg, K., Khodadoust, M. S., Zhou, L., Shelton, A. A., Visser, B. C., Ford, J. M., Alizadeh, A. A., Quake, S. R., Kunz, P. L., Beausang, J. F. 2018
Abstract

Neuroendocrine tumors arising from tailgut cysts are rare but increasingly reported entity with gene expression profiles that may be indicative of the gastrointestinal cell of origin. We present a case report describing the unique pathological and genomic characteristics of a tailgut cyst neuroendocrine tumor that metastasized to liver. The histologic and immunohistochemical findings were consistent with a well-differentiated neuroendocrine tumor. Genomic testing indicates a germline frame-shift in BRCA1 and a few somatic mutations of unknown significance. Transcriptomic analysis suggests an enteroendocrine L-cell in the tailgut as a putative cell-of-origin. Genomic profiling of a rare neuroendocrine tumor and metastasis provides insight into its origin, development and potential therapeutic options.

View details for PubMedID 30087100
Noninvasive blood tests for fetal development predict gestational age and preterm delivery SCIENCE Ngo, T. M., Moufarrej, M. N., Rasmussen, M. H., Camunas-Soler, J., Pan, W., Okamoto, J., Neff, N. F., Liu, K., Wong, R. J., Downes, K., Tibshirani, R., Shaw, G. M., Skotte, L., Stevenson, D. K., Biggio, J. R., Elovitz, M. A., Melbye, M., Quake, S. R. 2018; 360 (6393): 1133?36
Abstract

Noninvasive blood tests that provide information about fetal development and gestational age could potentially improve prenatal care. Ultrasound, the current gold standard, is not always affordable in low-resource settings and does not predict spontaneous preterm birth, a leading cause of infant death. In a pilot study of 31 healthy pregnant women, we found that measurement of nine cell-free RNA (cfRNA) transcripts in maternal blood predicted gestational age with comparable accuracy to ultrasound but at substantially lower cost. In a related study of 38 women (23 full-term and 15 preterm deliveries), all at elevated risk of delivering preterm, we identified seven cfRNA transcripts that accurately classified women who delivered preterm up to 2 months in advance of labor. These tests hold promise for prenatal care in both the developed and developing worlds, although they require validation in larger, blinded clinical trials.

View details for PubMedID 29880692
High fidelity hypothermic preservation of primary tissues in organ transplant preservative for single cell transcriptome analysis BMC GENOMICS Wang, W., Penland, L., Gokce, O., Croote, D., Quake, S. R. 2018; 19: 140
Abstract

High-fidelity preservation strategies for primary tissues are in great demand in the single cell RNAseq community. A reliable method would greatly expand the scope of feasible multi-site collaborations and maximize the utilization of technical expertise. When choosing a method, standardizability and fidelity are important factors to consider due to the susceptibility of single-cell RNAseq analysis to technical noise. Existing approaches such as cryopreservation and chemical fixation are less than ideal for failing to satisfy either or both of these standards.Here we propose a new strategy that leverages preservation schemes developed for organ transplantation. We evaluated the strategy by storing intact mouse kidneys in organ transplant preservative solution at hypothermic temperature for up to 4�days (6�h, 1, 2, 3, and 4�days), and comparing the quality of preserved and fresh samples using FACS and single cell RNAseq. We demonstrate that the strategy effectively maintained cell viability, transcriptome integrity, cell population heterogeneity, and transcriptome landscape stability for samples after up to 3�days of preservation. The strategy also facilitated the definition of the diverse spectrum of kidney resident immune cells, to our knowledge the first time at single cell resolution.Hypothermic storage of intact primary tissues in organ transplant preservative maintains the quality and stability of the transcriptome of cells for single cell RNAseq analysis. The strategy is readily generalizable to primary specimens from other tissue types for single cell RNAseq analysis.

View details for PubMedID 29439658
Noninvasive Prenatal Diagnosis of Single-Gene Disorders by Use of Droplet Digital PCR CLINICAL CHEMISTRY Camunas-Soler, J., Lee, H., Hudgins, L., Hintz, S. R., Blumenfeld, Y. J., El-Sayed, Y. Y., Quake, S. R. 2018; 64 (2): 336?45
View details for DOI 10.1373/clinchem.2017.278101

View details for Web of Science ID 000424396200017
Antigen Identification for Orphan T Cell Receptors Expressed on Tumor-Infiltrating Lymphocytes CELL Gee, M. H., Han, A., Lofgren, S. M., Beausang, J. F., Mendoza, J. L., Birnbaum, M. E., Bethune, M. T., Fischer, S., Yang, X., Gomez-Eerland, R., Bingham, D. B., Sibener, L. V., Fernandes, R. A., Velasco, A., Baltimore, D., Schumacher, T. N., Khatri, P., Quake, S. R., Davis, M. M., Garcia, K. 2018; 172 (3): 549-+
Abstract

The immune system can mount T�cell responses against tumors; however, the antigen specificities of tumor-infiltrating lymphocytes (TILs) are not well understood. We used yeast-display libraries of peptide-human leukocyte antigen (pHLA) to screen for antigens of "orphan" T�cell receptors (TCRs) expressed on TILs from human colorectal adenocarcinoma. Four TIL-derived TCRs exhibited strong selection for peptides presented in a highly diverse pHLA-A?02:01 library. Three of the TIL TCRs were specific for non-mutated self-antigens, two of which were present in separate patient tumors, and shared specificity for a non-mutated self-antigen derived from U2AF2. These results show that the exposed recognition surface of MHC-bound peptides accessible to the TCR contains sufficient structural information to enable the reconstruction of sequences of peptide targets for pathogenic TCRs of unknown specificity. This finding underscores the surprising specificity of TCRs for their cognate antigens and enables the facile indentification of tumor antigens through unbiased screening.

View details for PubMedID 29275860

View details for PubMedCentralID PMC5786495
Single-cell transcriptional dynamics of flavivirus infection. eLife Zanini, F., Pu, S. Y., Bekerman, E., Einav, S., Quake, S. R. 2018; 7
Abstract

Dengue and Zika viral infections affect millions of people annually and can be complicated by hemorrhage and shock or neurological manifestations, respectively. However, a thorough understanding of the host response to these viruses is lacking, partly because conventional approaches ignore heterogeneity in virus abundance across cells. We present viscRNA-Seq (virus-inclusive single cell RNA-Seq), an approach to probe the host transcriptome together with intracellular viral RNA at the single cell level. We applied viscRNA-Seq to monitor dengue and Zika virus infection in cultured cells and discovered extreme heterogeneity in virus abundance. We exploited this variation to identify host factors that show complex dynamics and a high degree of specificity for either virus, including proteins involved in the endoplasmic reticulum translocon, signal peptide processing, and membrane trafficking. We validated the viscRNA-Seq hits and discovered novel proviral and antiviral factors. viscRNA-Seq is a powerful approach to assess the genome-wide virus-host dynamics at single cell level.

View details for PubMedID 29451494
Single-cell analysis of early progenitor cells that build coronary arteries. Nature Su, T., Stanley, G., Sinha, R., D'Amato, G., Das, S., Rhee, S., Chang, A. H., Poduri, A., Raftrey, B., Dinh, T. T., Roper, W. A., Li, G., Quinn, K. E., Caron, K. M., Wu, S., Miquerol, L., Butcher, E. C., Weissman, I., Quake, S., Red-Horse, K. 2018
Abstract

Arteries and veins are specified by antagonistic transcriptional programs. However, during development and regeneration, new arteries can arise from pre-existing veins through a poorly understood process of cell fate conversion. Here, using single-cell RNA sequencing and mouse genetics, we show that vein cells of the developing heart undergo an early cell fate switch to create a pre-artery population that subsequently builds coronary arteries. Vein cells underwent a gradual and simultaneous switch from venous to arterial fate before a subset of cells crossed a transcriptional threshold into the pre-artery state. Before the onset of coronary blood flow, pre-artery cells appeared in the immature vessel plexus, expressed mature artery markers, and decreased cell cycling. The vein-specifying transcription factor COUP-TF2 (also known as NR2F2) prevented plexus cells from overcoming the pre-artery threshold by inducing cell cycle genes. Thus, vein-derived coronary arteries are built by pre-artery cells that can differentiate independently of blood flow upon the release of inhibition mediated by COUP-TF2 and cell cycle factors.

View details for PubMedID 29973725
The Human Cell Atlas ELIFE Regev, A., Teichmann, S. A., Lander, E. S., Amt, I., Benoist, C., Birney, E., Bodenmiller, B., Campbell, P., Carninci, P., Clatworthy, M., Clevers, H., Deplancke, B., Dunham, I., Eberwine, J., Elis, R., Enard, W., Farmer, A., Fugger, L., Gottgens, B., Hacohen, N., Haniffa, M., Hemberg, M., Kim, S., Klenerman, P., Kriegstein, A., Lein, E. D., Linnarsson, S., Lundberg, E., Lundeberg, J., Majumder, P., Marioni, J. C., Merad, M., Mhlanga, M., Nawijin, M., Netea, M., Nolan, G., Pe'er, D., Phillipakis, A., Ponting, C. P., Quake, S., Reik, W., Rozenblatt-Rosen, O., Sanes, J., Satija, R., Schumacher, T. N., Shalek, A., Shapiro, E., Sharma, P., Shin, J. W., Stegle, O., Stratton, M., Stubbington, M. T., Theis, F. J., Uhlen, M., Van Oudenaarden, A., Wagner, A., Watt, F., Weissman, J., Wold, B., Xavier, R., Yosef, N., HUMAN CELL ATLAS MEETING 2017; 6
Abstract

The recent advent of methods for high-throughput single-cell molecular profiling has catalyzed a growing sense in the scientific community that the time is ripe to complete the 150-year-old effort to identify all cell types in the human body. The Human Cell Atlas Project is an international collaborative effort that aims to define all human cell types in terms of distinctive molecular profiles (such as gene expression profiles) and to connect this information with classical cellular descriptions (such as location and morphology). An open comprehensive reference map of the molecular state of cells in healthy human tissues would propel the systematic study of physiological states, developmental trajectories, regulatory circuitry and interactions of cells, and also provide a framework for understanding cellular dysregulation in human disease. Here we describe the idea, its potential utility, early proofs-of-concept, and some design considerations for the Human Cell Atlas, including a commitment to open data, code, and community.

View details for PubMedID 29206104
Single-cell RNA sequencing reveals intrinsic and extrinsic regulatory heterogeneity in yeast responding to stress PLOS BIOLOGY Gasch, A. P., Yu, F., Hose, J., Escalante, L. E., Place, M., Bacher, R., Kanbar, J., Ciobanu, D., Sandor, L., Grigoriev, I. V., Kendziorski, C., Quake, S. R., McClean, M. N. 2017; 15 (12): e2004050
Abstract

From bacteria to humans, individual cells within isogenic populations can show significant variation in stress tolerance, but the nature of this heterogeneity is not clear. To investigate this, we used single-cell RNA sequencing to quantify transcript heterogeneity in single Saccharomyces cerevisiae cells treated with and without salt stress to explore population variation and identify cellular covariates that influence the stress-responsive transcriptome. Leveraging the extensive knowledge of yeast transcriptional regulation, we uncovered significant regulatory variation in individual yeast cells, both before and after stress. We also discovered that a subset of cells appears to decouple expression of ribosomal protein genes from the environmental stress response in a manner partly correlated with the cell cycle but unrelated to the yeast ultradian metabolic cycle. Live-cell imaging of cells expressing pairs of fluorescent regulators, including the transcription factor Msn2 with Dot6, Sfp1, or MAP kinase Hog1, revealed both coordinated and decoupled nucleocytoplasmic shuttling. Together with transcriptomic analysis, our results suggest that cells maintain a cellular filter against decoupled bursts of transcription factor activation but mount a stress response upon coordinated regulation, even in a subset of unstressed cells.

View details for PubMedID 29240790
T cell receptor sequencing of early-stage breast cancer tumors identifies altered clonal structure of the T cell repertoire. Proceedings of the National Academy of Sciences of the United States of America Beausang, J. F., Wheeler, A. J., Chan, N. H., Hanft, V. R., Dirbas, F. M., Jeffrey, S. S., Quake, S. R. 2017
Abstract

Tumor-infiltrating T cells play an important role in many cancers, and can improve prognosis and yield therapeutic targets. We characterized T cells infiltrating both breast cancer tumors and the surrounding normal breast tissue to identify T cells specific to each, as well as their abundance in peripheral blood. Using immune profiling of the T cell beta-chain repertoire in 16 patients with early-stage breast cancer, we show that the clonal structure of the tumor is significantly different from adjacent breast tissue, with the tumor containing 2.5-fold greater density of T cells and higher clonality compared with normal breast. The clonal structure of T cells in blood and normal breast is more similar than between blood and tumor, and could be used to distinguish tumor from normal breast tissue in 14 of 16 patients. Many T cell sequences overlap between tissue and blood from the same patient, including 50% of T cells between tumor and normal breast. Both tumor and normal breast contain high-abundance "enriched" sequences that are absent or of low abundance in the other tissue. Many of these T cells are either not detected or detected with very low frequency in the blood, suggesting the existence of separate compartments of T cells in both tumor and normal breast. Enriched T cell sequences are typically unique to each patient, but a subset is shared between many different patients. We show that many of these are commonly generated sequences, and thus unlikely to play an important role in the tumor microenvironment.

View details for PubMedID 29138313
Simultaneously Monitoring Immune Response and Microbial Infections during Pregnancy through Plasma cfRNA Sequencing CLINICAL CHEMISTRY Pan, W., Ngo, T. M., Camunas-Soler, J., Song, C., Kowarsky, M., Blumenfeld, Y. J., Wong, R. J., Shaw, G. M., Stevenson, D. K., Quake, S. R. 2017; 63 (11): 1695?1704
Abstract

Plasma cell-free RNA (cfRNA) encompasses a broad spectrum of RNA species that can be derived from both human cells and microbes. Because cfRNA is fragmented and of low concentration, it has been challenging to profile its transcriptome using standard RNA-seq methods.We assessed several recently developed RNA-seq methods on cfRNA samples. We then analyzed the dynamic changes of both the human transcriptome and the microbiome of plasma during pregnancy from 60 women.cfRNA reflects a well-orchestrated immune modulation during pregnancy: an up-regulation of antiinflammatory genes and an increased abundance of antimicrobial genes. We observed that the plasma microbiome remained relatively stable during pregnancy. The bacteria Ureaplasma shows an increased prevalence and increased abundance at postpartum, which is likely to be associated with postpartum infection. We demonstrated that cfRNA-seq can be used to monitor viral infections. We detected a number of human pathogens in our patients, including an undiagnosed patient with a high load of human parvovirus B19 virus (B19V), which is known to be a potential cause of complications in pregnancy.Plasma cfRNA-seq demonstrates the potential to simultaneously monitor immune response and microbial infections during pregnancy.

View details for PubMedID 28904056
Dynamics of the human antibody repertoire after B cell depletion in systemic sclerosis SCIENCE IMMUNOLOGY de Bourcy, C. A., Dekker, C. L., Davis, M. M., Nicolls, M. R., Quake, S. R. 2017; 2 (15)
View details for DOI 10.1126/sciimiunol.aan8289

View details for Web of Science ID 000434327200005
Microfluidic-based mini-metagenomics enables discovery of novel microbial lineages from complex environmental samples ELIFE Yu, F., Blainey, P. C., Schulz, F., Woyke, T., Horowitz, M. A., Quake, S. R. 2017; 6
Abstract

Metagenomics and single-cell genomics have enabled genome discovery from unknown branches of life. However, extracting novel genomes from complex mixtures of metagenomic data can still be challenging and represents an ill-posed problem which is generally approached with ad hoc methods. Here we present a microfluidic-based mini-metagenomic method which offers a statistically rigorous approach to extract novel microbial genomes while preserving single-cell resolution. We used this approach to analyze two hot spring samples from Yellowstone National Park and extracted 29 new genomes, including three deeply branching lineages. The single-cell resolution enabled accurate quantification of genome function and abundance, down to 1% in relative abundance. Our analyses of genome level SNP distributions also revealed low to moderate environmental selection. The scale, resolution, and statistical power of microfluidic-based mini-metagenomics make it a powerful tool to dissect the genomic structure of microbial communities while effectively preserving the fundamental unit of biology, the single cell.

View details for PubMedID 28678007
Quantitative Analysis of Synthetic Cell Lineage Tracing Using Nuclease Barcoding. ACS synthetic biology Schmidt, S. T., Zimmerman, S. M., Wang, J., Kim, S. K., Quake, S. R. 2017
Abstract

Lineage tracing by the determination and mapping of progeny arising from single cells is an important approach enabling the elucidation of mechanisms underlying diverse biological processes ranging from development to disease. We developed a dynamic sequence-based barcode system for synthetic lineage tracing and have demonstrated its performance in C.�elegans, a model organism whose lineage tree is well established. The strategy we use creates lineage trees based upon the introduction of synthetically controlled mutations into cells and the propagation of these mutations to daughter cells at each cell division. We analyzed this experimental proof of concept along with a corresponding simulation and analytical model to gain a deeper understanding of the coding capacity of the system. Our results provide specific bounds on the fidelity of lineage tracing using such approaches.

View details for DOI 10.1021/acssynbio.6b00309

View details for PubMedID 28264564
Long-term microfluidic tracking of coccoid cyanobacterial cells reveals robust control of division timing BMC BIOLOGY Yu, F. B., Willis, L., Chau, R. M., Zambon, A., Horowitz, M., Bhaya, D., Huang, K. C., Quake, S. R. 2017; 15
Abstract

Cyanobacteria are important agents in global carbon and nitrogen cycling and hold great promise for biotechnological applications. Model organisms such as Synechocystis sp. and Synechococcus sp. have advanced our understanding of photosynthetic capacity and circadian behavior, mostly using population-level measurements in which the behavior of individuals cannot be monitored. Synechocystis sp. cells are small and divide slowly, requiring long-term experiments to track single cells. Thus, the cumulative effects of drift over long periods can cause difficulties in monitoring and quantifying cell growth and division dynamics.To overcome this challenge, we enhanced a microfluidic cell-culture device and developed an image analysis pipeline for robust lineage reconstruction. This allowed simultaneous tracking of many cells over multiple generations, and revealed that cells expand exponentially throughout their cell cycle. Generation times were highly correlated for sister cells, but not between mother and daughter cells. Relationships between birth size, division size, and generation time indicated that cell-size control was inconsistent with the "sizer" rule, where division timing is based on cell size, or the "timer" rule, where division occurs after a fixed time interval. Instead, single cell growth statistics were most consistent with the "adder" rule, in which division occurs after a constant increment in cell volume. Cells exposed to light-dark cycles exhibited growth and division only during the light period; dark phases pause but do not disrupt cell-cycle control.Our analyses revealed that the "adder" model can explain both the growth-related statistics of single Synechocystis cells and the correlation between sister cell generation times. We also observed rapid phenotypic response to light-dark transitions at the single cell level, highlighting the critical role of light in cyanobacterial cell-cycle control. Our findings suggest that by monitoring the growth kinetics of individual cells we can build testable models of circadian control of the cell cycle in cyanobacteria.

View details for DOI 10.1186/s12915-016-0344-4

View details for Web of Science ID 000394057800001

View details for PubMedID 28196492
Converting Adult Pancreatic Islet a Cells into � Cells by Targeting Both Dnmt1 and Arx. Cell metabolism Chakravarthy, H., Gu, X., Enge, M., Dai, X., Wang, Y., Damond, N., Downie, C., Liu, K., Wang, J., Xing, Y., Chera, S., Thorel, F., Quake, S., Oberholzer, J., MacDonald, P. E., Herrera, P. L., Kim, S. K. 2017
Abstract

Insulin-producing pancreatic ? cells in mice can slowly regenerate from glucagon-producing ? cells in settings like ? cell loss, but the basis of this conversion is unknown. Moreover, it remains unclear if this intra-islet cell conversion is relevant to diseases like type 1 diabetes (T1D). We show that the ? cell regulators Aristaless-related homeobox (Arx) and DNA methyltransferase 1 (Dnmt1) maintain ? cell identity in mice. Within 3�months of Dnmt1 and Arx loss, lineage tracing and single-cell RNA sequencing revealed extensive ? cell conversion into progeny resembling native ? cells. Physiological studies demonstrated that converted ? cells acquire hallmark ? cell electrophysiology and show glucose-stimulated insulin secretion. In T1D patients, subsets of glucagon-expressing cells show loss of DNMT1 and ARX and produce insulin and other ? cell factors, suggesting that DNMT1 and ARX maintain ? cell identity in humans. Our work reveals pathways regulated by Arx and Dnmt1 that are sufficient for achieving targeted generation of ? cells from adult pancreatic ? cells.

View details for DOI 10.1016/j.cmet.2017.01.009

View details for PubMedID 28215845

View details for PubMedCentralID PMC5358097
Sanativo Wound Healing Product Does Not Accelerate Reepithelialization in a Mouse Cutaneous Wound Healing Model. Plastic and reconstructive surgery Marshall, C. D., Hu, M. S., Leavitt, T., Barnes, L. A., Cheung, A. T., Malhotra, S., Lorenz, H. P., Delp, S. L., Quake, S. R., Longaker, M. T. 2017; 139 (2): 343-352
Abstract

Sanativo is an over-the-counter Brazilian product derived from Amazon rainforest plant extract that is purported to improve the healing of skin wounds. Two experimental studies have shown accelerated closure of nonsplinted excisional wounds in rat models. However, these models allow for significant contraction of the wound and do not approximate healing in the tight skin of humans.Full-thickness excisional wounds were created on the dorsal skin of mice and were splinted with silicone rings, a model that forces the wound to heal by granulation and reepithelialization. Sanativo or a control solution was applied either daily or every other day to the wounds. Photographs were taken every other day, and the degree of reepithelialization of the wounds was determined.With both daily and every-other-day applications, Sanativo delayed reepithelialization of the wounds. Average time to complete healing was faster with control solution versus Sanativo in the daily application group (9.4 versus 15.2 days; p < 0.0001) and the every-other-day application group (11 versus 13 days; p = 0.017). The size of visible scar at the last time point of the study was not significantly different between the groups, and no differences were found on histologic examination.Sanativo wound healing compound delayed wound reepithelialization in a mouse splinted excisional wound model that approximates human wound healing. The size of visible scar after complete healing was not improved with the application of Sanativo. These results should cast doubt on claims that this product can improve wound healing in humans.

View details for DOI 10.1097/PRS.0000000000003013

View details for PubMedID 28121865
Phylogenetic analysis of the human antibody repertoire reveals quantitative signatures of immune senescence and aging PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA de Bourcy, C. F., Angel, C. J., Vollmers, C., Dekker, C. L., Davis, M. M., Quake, S. R. 2017; 114 (5): 1105-1110
Abstract

The elderly have reduced humoral immunity, as manifested by increased susceptibility to infections and impaired vaccine responses. To investigate the effects of aging on B-cell receptor (BCR) repertoire evolution during an immunological challenge, we used a phylogenetic distance metric to analyze Ig heavy-chain transcript sequences in both young and elderly individuals before and after influenza vaccination. We determined that BCR repertoires become increasingly specialized over a span of decades, but less plastic. In 50% of the elderly individuals, a large space in the repertoire was occupied by a small number of recall lineages that did not decline during vaccine response and contained hypermutated IgD(+) B cells. Relative to their younger counterparts, older subjects demonstrated a contracted naive repertoire and diminished intralineage diversification, signifying a reduced substrate for mounting novel responses and decreased fine-tuning of BCR specificities by somatic hypermutation. Furthermore, a larger proportion of the repertoire exhibited premature stop codons in some elderly subjects, indicating that aging may negatively affect the ability of B cells to discriminate between functional and nonfunctional receptors. Finally, we observed a decreased incidence of radical mutations compared with conservative mutations in elderly subjects' vaccine responses, which suggests that accumulating original antigenic sin may be limiting the accessible space for paratope evolution. Our findings shed light on the complex interplay of environmental and gerontological factors affecting immune senescence, and provide direct molecular characterization of the effects of senescence on the immune repertoire.

View details for DOI 10.1073/pnas.1617959114

View details for Web of Science ID 000393196300087

View details for PubMedID 28096374

View details for PubMedCentralID PMC5293037
B cell repertoires in HLA-sensitized kidney transplant candidates undergoing desensitization therapy JOURNAL OF TRANSLATIONAL MEDICINE Beausang, J. F., Fan, H. C., Sit, R., Hutchins, M. U., Jirage, K., Curtis, R., Hutchins, E., Quake, S. R., Yabu, J. M. 2017; 15
Abstract

Kidney transplantation is the most effective treatment for end-stage renal disease. Sensitization refers to pre-existing antibodies against human leukocyte antigen (HLA) protein and remains a major barrier to successful transplantation. Despite implementation of desensitization strategies, many candidates fail to respond. Our objective was to determine whether measuring B cell repertoires could differentiate candidates that respond to desensitization therapy.We developed an assay based on high-throughput DNA sequencing of the variable domain of the heavy chain of immunoglobulin genes to measure changes in B cell repertoires in 19 highly HLA-sensitized kidney transplant candidates undergoing desensitization and 7 controls with low to moderate HLA sensitization levels. Responders to desensitization had a decrease of 5% points or greater in cumulated calculated panel reactive antibody (cPRA) levels, and non-responders had no decrease in cPRA.Dominant B cell clones were not observed in highly sensitized candidates, suggesting that the B cells responsible for sensitization are either not present in peripheral blood or present at comparable levels to other circulating B cells. Candidates that responded to desensitization therapy had pre-treatment repertoires composed of a larger fraction of class-switched (IgG and IgA) isotypes compared to non-responding candidates. After B cell depleting therapy, the proportion of switched isotypes increased and the mutation frequencies of the remaining non-switched isotypes (IgM and IgD) increased in both responders and non-responders, perhaps representing a shift in the repertoire towards memory B cells or plasmablasts. Conversely, after transplantation, non-switched isotypes with fewer mutations increased, suggesting a shift in the repertoire towards na�ve B cells.Relative abundance of different B cell isotypes is strongly perturbed by desensitization therapy and transplantation, potentially reflecting changes in the relative abundance of memory and na�ve B cell compartments. Candidates that responded to therapy experienced similar changes to those that did not respond. Further studies are required to understand differences between these two groups of highly sensitized kidney transplant candidates.

View details for DOI 10.1186/s12967-017-1118-7

View details for Web of Science ID 000392448700001

View details for PubMedID 28086979

View details for PubMedCentralID PMC5237299
5-Hydroxymethylcytosine signatures in cell-free DNA provide information about tumor types and stages. Cell research Song, C. X., Yin, S., Ma, L., Wheeler, A., Chen, Y., Zhang, Y., Liu, B., Xiong, J., Zhang, W., Hu, J., Zhou, Z., Dong, B., Tian, Z., Jeffrey, S. S., Chua, M. S., So, S., Li, W., Wei, Y., Diao, J., Xie, D., Quake, S. R. 2017
Abstract

5-Hydroxymethylcytosine (5hmC) is an important mammalian DNA epigenetic modification that has been linked to gene regulation and cancer pathogenesis. Here we explored the diagnostic potential of 5hmC in circulating cell-free DNA (cfDNA) using a sensitive chemical labeling-based low-input shotgun sequencing approach. We sequenced cell-free 5hmC from 49 patients of seven different cancer types and found distinct features that could be used to predict cancer types and stages with high accuracy. Specifically, we discovered that lung cancer leads to a progressive global loss of 5hmC in cfDNA, whereas hepatocellular carcinoma and pancreatic cancer lead to disease-specific changes in the cell-free hydroxymethylome. Our proof-of-principle results suggest that cell-free 5hmC signatures may potentially be used not only to identify cancer types but also to track tumor stage in some cancers.Cell Research advance online publication 18 August 2017; doi:10.1038/cr.2017.106.

View details for PubMedID 28820176
Dynamics of the human antibody repertoire after B cell depletion in systemic sclerosis Dynamics of the human antibody repertoire after B cell depletion in systemic sclerosis de Bourcy, C. F., Dekker, C. L., Davis, M. M., Nicolls, M. R., Quake, S. R. 2017; 2 (15)
View details for DOI 10.1126/sciimmunol.aan8289
Risky Business: Meeting the Structural Needs of Transdisciplinary Science. The Journal of pediatrics Wise, P. H., Shaw, G. M., Druzin, M. L., Darmstadt, G. L., Quaintance, C., M�kinen, E., Relman, D. A., Quake, S. R., Butte, A. J., Angst, M. S., Muglia, L. J., Macones, G., Driscoll, D., Ober, C., Simpson, J. L., Katz, M., Howse, J., Stevenson, D. K. 2017; 191: 255?58
View details for PubMedID 29173314
Classifying Drosophila Olfactory Projection Neuron Subtypes by Single-Cell RNA Sequencing. Cell Li, H., Horns, F., Wu, B., Xie, Q., Li, J., Li, T., Luginbuhl, D. J., Quake, S. R., Luo, L. 2017; 171 (5): 1206?20.e22
Abstract

The definition of neuronal type and how it relates to the transcriptome are open questions. Drosophila olfactory projection neurons (PNs) are among the best-characterized neuronal types: different PN classes target dendrites to distinct olfactory glomeruli, while PNs of the same class exhibit indistinguishable anatomical and physiological properties. Using single-cell RNA sequencing, we comprehensively characterized the transcriptomes of most PN classes and unequivocally mapped transcriptomes to specific olfactory function for six classes. Transcriptomes of�closely related PN classes exhibit the largest differences during circuit assembly but become indistinguishable in adults, suggesting that neuronal subtype diversity peaks during development. Transcription factors and cell-surface molecules are the most differentially expressed genes between classes and are highly informative in encoding cell identity, enabling us to identify a new lineage-specific transcription factor that instructs PN dendrite targeting. These findings establish that neuronal transcriptomic identity corresponds with anatomical and physiological identity defined by connectivity and function.

View details for PubMedID 29149607
High-throughput full-length single-cell mRNA-seq of rare cells. PloS one Ooi, C. C., Mantalas, G. L., Koh, W., Neff, N. F., Fuchigami, T., Wong, D. J., Wilson, R. J., Park, S. M., Gambhir, S. S., Quake, S. R., Wang, S. X. 2017; 12 (11): e0188510
Abstract

Single-cell characterization techniques, such as mRNA-seq, have been applied to a diverse range of applications in cancer biology, yielding great insight into mechanisms leading to therapy resistance and tumor clonality. While single-cell techniques can yield a wealth of information, a common bottleneck is the lack of throughput, with many current processing methods being limited to the analysis of small volumes of single cell suspensions with cell densities on the order of 107 per mL. In this work, we present a high-throughput full-length mRNA-seq protocol incorporating a magnetic sifter and magnetic nanoparticle-antibody conjugates for rare cell enrichment, and Smart-seq2 chemistry for sequencing. We evaluate the efficiency and quality of this protocol with a simulated circulating tumor cell system, whereby non-small-cell lung cancer cell lines (NCI-H1650 and NCI-H1975) are spiked into whole blood, before being enriched for single-cell mRNA-seq by EpCAM-functionalized magnetic nanoparticles and the magnetic sifter. We obtain high efficiency (> 90%) capture and release of these simulated rare cells via the magnetic sifter, with reproducible transcriptome data. In addition, while mRNA-seq data is typically only used for gene expression analysis of transcriptomic data, we demonstrate the use of full-length mRNA-seq chemistries like Smart-seq2 to facilitate variant analysis of expressed genes. This enables the use of mRNA-seq data for differentiating cells in a heterogeneous population by both their phenotypic and variant profile. In a simulated heterogeneous mixture of circulating tumor cells in whole blood, we utilize this high-throughput protocol to differentiate these heterogeneous cells by both their phenotype (lung cancer versus white blood cells), and mutational profile (H1650 versus H1975 cells), in a single sequencing run. This high-throughput method can help facilitate single-cell analysis of rare cell populations, such as circulating tumor or endothelial cells, with demonstrably high-quality transcriptomic data.

View details for PubMedID 29186152

View details for PubMedCentralID PMC5706670
Role of epithelial to mesenchymal transition associated genes in mammary gland regeneration and breast tumorigenesis. Nature communications Sikandar, S. S., Kuo, A. H., Kalisky, T., Cai, S., Zabala, M., Hsieh, R. W., Lobo, N. A., Scheeren, F. A., Sim, S., Qian, D., Dirbas, F. M., Somlo, G., Quake, S. R., Clarke, M. F. 2017; 8 (1): 1669
Abstract

Previous studies have proposed that epithelial to mesenchymal transition (EMT) in breast cancer cells regulates metastasis, stem cell properties and chemo-resistance; most studies were based on in vitro culture of cell lines and mouse transgenic cancer models. However, the identity and function of cells expressing EMT-associated genes in normal murine mammary gland homeostasis and human breast cancer still remains under debate. Using in vivo lineage tracing and triple negative breast cancer (TNBC) patient derived xenografts we demonstrate that the repopulating capacity in normal mammary epithelial cells and tumorigenic capacity in TNBC is independent of expression of EMT-associated genes. In breast cancer, while a subset of cells with epithelial and mesenchymal phenotypes have stem cell activity, in many cells that have lost epithelial characteristics with increased expression of mesenchymal genes, have decreased tumor-initiating capacity and plasticity. These findings have implications for the development of effective therapeutic agents targeting tumor-initiating cells.

View details for PubMedID 29162812

View details for PubMedCentralID PMC5698470
Single-Cell RNA-Seq Analysis of Infiltrating Neoplastic Cells at the Migrating Front of Human Glioblastoma. Cell reports Darmanis, S., Sloan, S. A., Croote, D., Mignardi, M., Chernikova, S., Samghababi, P., Zhang, Y., Neff, N., Kowarsky, M., Caneda, C., Li, G., Chang, S. D., Connolly, I. D., Li, Y., Barres, B. A., Gephart, M. H., Quake, S. R. 2017; 21 (5): 1399?1410
Abstract

Glioblastoma (GBM) is the most common primary brain cancer in adults and is notoriously difficult to treat because of its diffuse nature. We performed single-cell RNA sequencing (RNA-seq) on 3,589 cells in a cohort of four patients. We obtained cells from the tumor core as well as surrounding peripheral tissue. Our analysis revealed cellular variation in the tumor's genome and transcriptome. We were also able to identify infiltrating neoplastic cells in regions peripheral to the core lesions. Despite the existence of significant heterogeneity among neoplastic cells, we found that infiltrating GBM cells share a consistent gene signature between patients, suggesting a common mechanism of infiltration. Additionally, in investigating the immunological response to the tumors, we found transcriptionally distinct myeloid cell populations residing in the tumor core and the surrounding peritumoral space. Our data provide a detailed dissection of GBM cell types, revealing an abundance of information about tumor formation and migration.

View details for PubMedID 29091775
Transcriptomic Profiling Maps Anatomically Patterned Subpopulations among Single Embryonic Cardiac Cells DEVELOPMENTAL CELL Li, G., Xu, A., Sim, S., Priest, J. R., Tian, X., Khan, T., Quertermous, T., Zhou, B., Tsao, P. S., Quake, S. R., Wu, S. M. 2016; 39 (4): 491-507
Abstract

Embryonic gene expression intricately reflects anatomical context, developmental stage, and cell type. To address whether the precise spatial origins of cardiac cells can be deduced solely from their transcriptional profiles, we established a genome-wide expression database from 118, 949, and 1,166 single murine heart cells at embryonic day 8.5 (e8.5), e9.5, and e10.5, respectively. We segregated these cells by type using unsupervised bioinformatics analysis and identified chamber-specific genes. Using a random forest algorithm, we reconstructed the spatial origin of single e9.5 and e10.5 cardiomyocytes with 92.0%�� 3.2% and 91.2%�� 2.8% accuracy, respectively (99.4%�� 1.0% and 99.1%�� 1.1% if a��1 zone margin is permitted) and predicted the second heart field distribution of Isl-1-lineage descendants. When applied to Nkx2-5(-/-) cardiomyocytes from murine e9.5 hearts, we showed their transcriptional alteration and lack of ventricular phenotype. Our database and zone classification algorithm will enable the discovery of novel mechanisms in early cardiac development and disease.

View details for DOI 10.1016/j.devcel.2016.10.014

View details for Web of Science ID 000389162800013

View details for PubMedID 27840109
Molecular-level analysis of the serum antibody repertoire in young adults before and after seasonal influenza vaccination. Nature medicine Lee, J., Boutz, D. R., Chromikova, V., Joyce, M. G., Vollmers, C., Leung, K., Horton, A. P., DeKosky, B. J., Lee, C., Lavinder, J. J., Murrin, E. M., Chrysostomou, C., Hoi, K. H., Tsybovsky, Y., Thomas, P. V., Druz, A., Zhang, B., Zhang, Y., Wang, L., Kong, W., Park, D., Popova, L. I., Dekker, C. L., Davis, M. M., Carter, C. E., Ross, T. M., Ellington, A. D., Wilson, P. C., Marcotte, E. M., Mascola, J. R., Ippolito, G. C., Krammer, F., Quake, S. R., Kwong, P. D., Georgiou, G. 2016
Abstract

Molecular understanding of serological immunity to influenza has been confounded by the complexity of the polyclonal antibody response in humans. Here we used high-resolution proteomics analysis of immunoglobulin (referred to as Ig-seq) coupled with high-throughput sequencing of transcripts encoding B cell receptors (BCR-seq) to quantitatively determine the antibody repertoire at the individual clonotype level in the sera of young adults before and after vaccination with trivalent seasonal influenza vaccine. The serum repertoire comprised between 40 and 147 clonotypes that were specific to each of the three monovalent components of the trivalent influenza vaccine, with boosted pre-existing clonotypes accounting for ?60% of the response. An unexpectedly high fraction of serum antibodies recognized both the H1 and H3 monovalent vaccines. Recombinant versions of these H1 + H3 cross-reactive antibodies showed broad binding to hemagglutinins (HAs) from previously circulating virus strains; several of these antibodies, which were prevalent in the serum of multiple donors, recognized the same conserved epitope in the HA head domain. Although the HA-head-specific H1 + H3 antibodies did not show neutralization activity in vitro, they protected mice against infection with the H1N1 and H3N2 virus strains when administered before or after challenge. Collectively, our data reveal unanticipated insights regarding the serological response to influenza vaccination and raise questions about the added benefits of using a quadrivalent vaccine instead of a trivalent vaccine.

View details for DOI 10.1038/nm.4224

View details for PubMedID 27820605
Early somatic mosaicism is a rare cause of long-QT syndrome PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Priest, J. R., Gawad, C., Kahlig, K. M., Yu, J. K., O'Hara, T., Boyle, P. M., Rajamani, S., Clark, M. J., Garcia, S. T., Ceresnak, S., Harris, J., Boyle, S., Dewey, F. E., Malloy-Walton, L., Dunn, K., Grove, M., Perez, M. V., Neff, N. F., Chen, R., Maeda, K., Dubin, A., Belardinelli, L., West, J., Antolik, C., Macaya, D., Quertermous, T., Trayanova, N. A., Quake, S. R., Ashley, E. A. 2016; 113 (41): 11555-11560
Abstract

Somatic mosaicism, the occurrence and propagation of genetic variation in cell lineages after fertilization, is increasingly recognized to play a causal role in a variety of human diseases. We investigated the case of life-threatening arrhythmia in a 10-day-old infant with long QT syndrome (LQTS). Rapid genome sequencing suggested a variant in the sodium channel NaV1.5 encoded by SCN5A, NM_000335:c.5284G > T predicting p.(V1762L), but read depth was insufficient to be diagnostic. Exome sequencing of the trio confirmed read ratios inconsistent with Mendelian inheritance only in the proband. Genotyping of single circulating leukocytes demonstrated the mutation in the genomes of 8% of patient cells, and RNA sequencing of cardiac tissue from the infant confirmed the expression of the mutant allele at mosaic ratios. Heterologous expression of the mutant channel revealed significantly delayed sodium current with a dominant negative effect. To investigate the mechanism by which mosaicism might cause arrhythmia, we built a finite element simulation model incorporating Purkinje fiber activation. This model confirmed the pathogenic consequences of cardiac cellular mosaicism and, under the presenting conditions of this case, recapitulated 2:1 AV block and arrhythmia. To investigate the extent to which mosaicism might explain undiagnosed arrhythmia, we studied 7,500 affected probands undergoing commercial gene-panel testing. Four individuals with pathogenic variants arising from early somatic mutation events were found. Here we establish cardiac mosaicism as a causal mechanism for LQTS and present methods by which the general phenomenon, likely to be relevant for all genetic diseases, can be detected through single-cell analysis and next-generation sequencing.

View details for DOI 10.1073/pnas.1607187113

View details for PubMedID 27681629
Single-cell multimodal profiling reveals cellular epigenetic heterogeneity NATURE METHODS Cheow, L. F., Courtois, E. T., Tan, Y., Viswanathan, R., Xing, Q., Tan, R. Z., Tan, D. S., Robson, P., Loh, Y., Quake, S. R., Burkholder, W. F. 2016; 13 (10): 833-?
Abstract

Sample heterogeneity often masks DNA methylation signatures in subpopulations of cells. Here, we present a method to genotype single cells while simultaneously interrogating gene expression and DNA methylation at multiple loci. We used this targeted multimodal approach, implemented on an automated, high-throughput microfluidic platform, to assess primary lung adenocarcinomas and human fibroblasts undergoing reprogramming by profiling epigenetic variation among cell types identified through genotyping and transcriptional analysis.

View details for DOI 10.1038/nmeth.3961

View details for Web of Science ID 000385194600017

View details for PubMedID 27525975
Single-cell RNAseq reveals cell adhesion molecule profiles in electrophysiologically defined neurons. Proceedings of the National Academy of Sciences of the United States of America F�ldy, C., Darmanis, S., Aoto, J., Malenka, R. C., Quake, S. R., S�dhof, T. C. 2016; 113 (35): E5222-31
Abstract

In brain, signaling mediated by cell adhesion molecules defines the identity and functional properties of synapses. The specificity of presynaptic and postsynaptic interactions that is presumably mediated by cell adhesion molecules suggests that there exists a logic that could explain neuronal connectivity at the molecular level. Despite its importance, however, the nature of such logic is poorly understood, and even basic parameters, such as the number, identity, and single-cell expression profiles of candidate synaptic cell adhesion molecules, are not known. Here, we devised a comprehensive list of genes involved in cell adhesion, and used single-cell RNA sequencing (RNAseq) to analyze their expression in electrophysiologically defined interneurons and projection neurons. We compared the cell type-specific expression of these genes with that of genes involved in transmembrane ion conductances (i.e., channels), exocytosis, and rho/rac signaling, which regulates the actin cytoskeleton. Using these data, we identified two independent, developmentally regulated networks of interacting genes encoding molecules involved in cell adhesion, exocytosis, and signal transduction. Our approach provides a framework for a presumed cell adhesion and signaling code in neurons, enables correlating electrophysiological with molecular properties of neurons, and suggests avenues toward understanding synaptic specificity.

View details for DOI 10.1073/pnas.1610155113

View details for PubMedID 27531958
Cellular Taxonomy of the Mouse Striatum as Revealed by Single-Cell RNA-Seq CELL REPORTS Gokce, O., Stanley, G. M., Treutlein, B., Neff, N. F., Camp, J. G., Malenka, R. C., Rothwell, P. E., Fuccillo, M. V., Sudhof, T. C., Quake, S. R. 2016; 16 (4): 1126-1137
Abstract

The striatum contributes to many cognitive processes and disorders, but its cell types are incompletely characterized. We show that microfluidic and FACS-based single-cell RNA sequencing of mouse striatum provides a well-resolved classification of striatal cell type diversity. Transcriptome analysis revealed ten differentiated, distinct cell types, including neurons, astrocytes, oligodendrocytes, ependymal, immune, and vascular cells, and enabled the discovery of numerous marker genes. Furthermore, we identified two discrete subtypes of medium spiny neurons (MSNs) that have specific markers and�that overexpress genes linked to cognitive disorders and addiction. We also describe continuous cellular identities, which increase heterogeneity within discrete cell types. Finally, we identified cell type-specific transcription and splicing factors that shape cellular identities by regulating splicing and expression patterns. Our findings suggest that functional diversity within a complex tissue arises from a small number of discrete cell types, which can exist in a continuous spectrum of functional states.

View details for DOI 10.1016/j.celrep.2016.06.059

View details for PubMedID 27425622
Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq NATURE Treutlein, B., Lee, Q. Y., Camp, J. G., Mall, M., Koh, W., Shariati, S. A., Sim, S., Neff, N. F., Skotheim, J. M., Wernig, M., Quake, S. R. 2016; 534 (7607): 391-?
Abstract

Direct lineage reprogramming represents a remarkable conversion of cellular and transcriptome states. However, the intermediate stages through which individual cells progress during reprogramming are largely undefined. Here we use single-cell RNA sequencing at multiple time points to dissect direct reprogramming from mouse embryonic fibroblasts to induced neuronal cells. By deconstructing heterogeneity at each time point and ordering cells by transcriptome similarity, we find that the molecular reprogramming path is remarkably continuous. Overexpression of the proneural pioneer factor Ascl1 results in a well-defined initialization, causing cells to exit the cell cycle and re-focus gene expression through distinct neural transcription factors. The initial transcriptional response is relatively homogeneous among fibroblasts, suggesting that the early steps are not limiting for productive reprogramming. Instead, the later emergence of a competing myogenic program and variable transgene dynamics over time appear to be the major efficiency limits of direct reprogramming. Moreover, a transcriptional state, distinct from donor and target cell programs, is transiently induced in cells undergoing productive reprogramming. Our data provide a high-resolution approach for understanding transcriptome states during lineage differentiation.

View details for DOI 10.1038/nature18323

View details for Web of Science ID 000377856800037

View details for PubMedID 27281220

View details for PubMedCentralID PMC4928860
Developmental cell death programs license cytotoxic cells to eliminate histocompatible partners PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Corey, D. M., rosental, B., Kowarsky, M., Sinha, R., Ishizuka, K. J., Palmeri, K. J., Quake, S. R., Voskoboynik, A., Weissman, I. L. 2016; 113 (23): 6520-6525
Abstract

In a primitive chordate model of natural chimerism, one chimeric partner is often eliminated in a process of allogeneic resorption. Here, we identify the cellular framework underlying loss of tolerance to one partner within a natural Botryllus schlosseri chimera. We show that the principal cell type mediating chimeric partner elimination is a cytotoxic morula cell (MC). Proinflammatory, developmental cell death programs render MCs cytotoxic and, in collaboration with activated phagocytes, eliminate chimeric partners during the "takeover" phase of blastogenic development. Among these genes, the proinflammatory cytokine IL-17 enhances cytotoxicity in allorecognition assays. Cellular transfer of FACS-purified MCs from allogeneic donors into recipients shows that the resorption response can be adoptively acquired. Transfer of 1 � 10(5) allogeneic MCs eliminated 33 of 78 (42%) recipient primary buds and 20 of 76 (20.5%) adult parental adult organisms (zooids) by 14 d whereas transfer of allogeneic cell populations lacking MCs had only minimal effects on recipient colonies. Furthermore, reactivity of transferred cells coincided with the onset of developmental-regulated cell death programs and disproportionately affected developing tissues within a chimera. Among chimeric partner "losers," severe developmental defects were observed in asexually propagating tissues, reflecting a pathologic switch in gene expression in developmental programs. These studies provide evidence that elimination of one partner in a chimera is an immune cell-based rejection that operates within histocompatible pairs and that maximal allogeneic responses involve the coordination of both phagocytic programs and the "arming" of cytotoxic cells.

View details for DOI 10.1073/pnas.1606276113

View details for Web of Science ID 000377155400052

View details for PubMedID 27217570
Tumor DNA in cerebral spinal fluid reflects clinical course in a patient with melanoma leptomeningeal brain metastases JOURNAL OF NEURO-ONCOLOGY Li, Y., Pan, W., Connolly, I. D., Reddy, S., Nagpal, S., Quake, S., Gephart, M. H. 2016; 128 (1): 93-100
Abstract

Cerebral spinal fluid (CSF) from brain tumor patients contains tumor cellular and cell-free DNA (cfDNA), which provides a less-invasive and routinely accessible method to obtain tumor genomic information. In this report, we used droplet digital PCR to test mutant tumor DNA in CSF of a patient to monitor the treatment response of metastatic melanoma leptomeningeal disease (LMD). The primary melanoma was known to have a BRAF (V600E) mutation, and the patient was treated with whole brain radiotherapy and BRAF inhibitors. We collected 9 CSF samples over 6�months. The mutant cfDNA fraction gradually decreased from 53�% (time of diagnosis) to 0 (time of symptom alleviation) over the first 6 time points. Three months after clinical improvement, the patient returned with severe symptoms and the mutant cfDNA was again detected in CSF at high levels. The mutant DNA fraction corresponded well with the patient's clinical response. We used whole exome sequencing to examine the mutation profiles of the LMD tumor DNA in CSF before therapeutic response and after disease relapse, and discovered a canonical cancer mutation PTEN (R130*) at both time points. The cellular and cfDNA revealed similar mutation profiles, suggesting cfDNA is representative of LMD cells. This study demonstrates the potential of using cellular or cfDNA in CSF to monitor treatment response for LMD.

View details for DOI 10.1007/s11060-016-2081-5

View details for PubMedID 26961773
Simultaneous single-molecule epigenetic imaging of DNA methylation and hydroxymethylation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Song, C., Diao, J., Brunger, A. T., Quake, S. R. 2016; 113 (16): 4338-4343
Abstract

The modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the two major DNA epigenetic modifications in mammalian genomes and play crucial roles in development and pathogenesis. Little is known about the colocalization or potential correlation of these two modifications. Here we present an ultrasensitive single-molecule imaging technology capable of detecting and quantifying 5hmC and 5mC from trace amounts of DNA. We used this approach to perform single-molecule fluorescence resonance energy transfer (smFRET) experiments which measure the proximity between 5mC and 5hmC in the same DNA molecule. Our results reveal high levels of adjacent and opposing methylated and hydroxymethylated CpG sites (5hmC/5mCpGs) in mouse genomic DNA across multiple tissues. This identifies the previously undetectable and unappreciated 5hmC/5mCpGs as one of the major states for 5hmC in the mammalian genome and suggest that they could function in promoting gene expression.

View details for DOI 10.1073/pnas.1600223113

View details for Web of Science ID 000374393800042

View details for PubMedID 27035984

View details for PubMedCentralID PMC4843451
Voices of biotech. Nature biotechnology Amit, I., Baker, D., Barker, R., Berger, B., Bertozzi, C., Bhatia, S., Biffi, A., Demichelis, F., Doudna, J., Dowdy, S. F., Endy, D., Helmstaedter, M., Junca, H., June, C., Kamb, S., Khvorova, A., Kim, D., Kim, J., Krishnan, Y., Lakadamyali, M., Lappalainen, T., Lewin, S., Liao, J., Loman, N., Lundberg, E., Lynd, L., Martin, C., Mellman, I., Miyawaki, A., Mummery, C., Nelson, K., Paz, J., Peralta-Yahya, P., Picotti, P., Polyak, K., Prather, K., Qin, J., Quake, S., Regev, A., Rogers, J. A., Shetty, R., Sommer, M., Stevens, M., Stolovitzky, G., Takahashi, M., Tang, F., Teichmann, S., Torres-Padilla, M., Tripathi, L., Vemula, P., Verdine, G., Vollmer, F., Wang, J., Ying, J. Y., Zhang, F., Zhang, T. 2016; 34 (3): 270-275
View details for DOI 10.1038/nbt.3502

View details for PubMedID 26963549
Single-cell genome sequencing: current state of the science NATURE REVIEWS GENETICS Gawad, C., Koh, W., Quake, S. R. 2016; 17 (3): 175-188
Abstract

The field of single-cell genomics is advancing rapidly and is generating many new insights into complex biological systems, ranging from the diversity of microbial ecosystems to the genomics of human cancer. In this Review, we provide an overview of the current state of the field of single-cell genome sequencing. First, we focus on the technical challenges of making measurements that start from a single molecule of DNA, and then explore how some of these recent methodological advancements have enabled the discovery of unexpected new biology. Areas highlighted include the application of single-cell genomics to interrogate microbial dark matter and to evaluate the pathogenic roles of genetic mosaicism in multicellular organisms, with a focus on cancer. We then attempt to predict advances we expect to see in the next few years.

View details for DOI 10.1038/nrg.2015.16

View details for Web of Science ID 000371739200012

View details for PubMedID 26806412
Tools for the Microbiome: Nano and Beyond ACS NANO Biteen, J. S., Blainey, P. C., Cardon, Z. G., Chun, M., Church, G. M., Dorrestein, P. C., Fraser, S. E., Gilbert, J. A., Jansson, J. K., Knight, R., Miller, J. F., Ozcan, A., Prather, K. A., Quake, S. R., Ruby, E. G., Silver, P. A., Taha, S., van den Engh, G., Weiss, P. S., Wong, G. C., Wright, A. T., Young, T. D. 2016; 10 (1): 6-37
Abstract

The microbiome presents great opportunities for understanding and improving the world around us and elucidating the interactions that compose it. The microbiome also poses tremendous challenges for mapping and manipulating the entangled networks of interactions among myriad diverse organisms. Here, we describe the opportunities, technical needs, and potential approaches to address these challenges, based on recent and upcoming advances in measurement and control at the nanoscale and beyond. These technical needs will provide the basis for advancing the largely descriptive studies of the microbiome to the theoretical and mechanistic understandings that will underpin the discipline of microbiome engineering. We anticipate that the new tools and methods developed will also be more broadly useful in environmental monitoring, medicine, forensics, and other areas.

View details for DOI 10.1021/acsnano.5b07826

View details for Web of Science ID 000369115800002

View details for PubMedID 26695070
Lineage tracing of human B cells reveals the in vivo landscape of human antibody class switching. eLife Horns, F., Vollmers, C., Croote, D., Mackey, S. F., Swan, G. E., Dekker, C. L., Davis, M. M., Quake, S. R. 2016; 5
Abstract

Antibody class switching is a feature of the adaptive immune system which enables diversification of the effector properties of antibodies. Even though class switching is essential for mounting a protective response to pathogens, the in vivo patterns and lineage characteristics of antibody class switching have remained uncharacterized in living humans. Here we comprehensively measured the landscape of antibody class switching in human adult twins using antibody repertoire sequencing. The map identifies how antibodies of every class are created and delineates a two-tiered hierarchy of class switch pathways. Using somatic hypermutations as a molecular clock, we discovered that closely related B cells often switch to the same class, but lose coherence as somatic mutations accumulate. Such correlations between closely related cells exist when purified B cells class switch in vitro, suggesting that class switch recombination is directed toward specific isotypes by a cell-autonomous imprinted state.

View details for DOI 10.7554/eLife.16578

View details for PubMedID 27481325

View details for PubMedCentralID PMC4970870
A reusable microfluidic device provides continuous measurement capability and improves the detection limit of digital biology LAB ON A CHIP Araci, I. E., Robles, M., Quake, S. R. 2016; 16 (9): 1573-1578
Abstract

Current digital biology platforms lack the ability to perform continuous measurements for transient analysis. A fundamental challenge is to perform complex fluidic manipulation processes such as washing and mixing in individual reaction volumes. Here, we present a reusable digital biology platform where the reaction compartmentalization and commencement are controlled by micromechanical valves fabricated in high density through microfluidic very large scale integration (mVLSI) technology. Background noise correction enabled by the platform improves signal-to-noise ratio and thus eliminates the need for sophisticated imaging technologies. We have used the detection platform for probing single molecules of the ?-galactosidase enzyme. The measurements were repeated hundreds of times at concentrations as low as 0.8 fM (resulting in a theoretical detection limit of 3 aM). We have also demonstrated multiple TNF-? measurements with a magnetic bead based digital ELISA assay. The assay showed that the average number of enzymes per magnetic bead is 0.55 for 10 pM TNF-? (compared to 0.08 for negative control). This automated and reusable digital platform allows on-chip assay preparation and continuous measurements; as a result, it will enable single cell/enzyme studies and clinical diagnostic tests (i.e. digital ELISA) to be performed in shorter time scales and with lower detection limits.

View details for DOI 10.1039/c6lc00194g

View details for Web of Science ID 000375569500006

View details for PubMedID 27072314
Microfluidics Technologies for Low Cell Number Chromatin Immunoprecipitation. Cold Spring Harbor protocols Wu, A. R., Quake, S. R. 2016; 2016 (4): pdb prot084996-?
Abstract

Protein-DNA interactions are responsible for numerous critical cellular events: For example, gene expression and silencing are mediated by transcription factor protein binding and histone protein modifications, and DNA replication and repair rely on site-specific protein binding. Chromatin immunoprecipitation (ChIP) is the only molecular assay that directly determines, in a living cell, the binding association between a protein of interest and specific genomic loci. It is an indispensible tool in the biologist's toolbox, but the many limitations of this technique prevent broad adoption of ChIP in biological studies. The typical ChIP assay can take up to 1 wk to complete, and the process is technically tricky, yet tedious. The ChIP assay yields are also low, thus requiring on the order of millions to billions of cells as starting material, which makes the assay unfeasible for studies using rare or precious samples. For example, fluorescence-activated cell sorting (FACS) of cancer stem cells (CSCs) obtained from primary tumors, rarely yields more than ~100,000 CSCs per tumor. This protocol describes a microfluidics-based strategy for performing ChIP, which uses automation and scalability to reduce both total and hands-on assay time, and improve throughput. It allows whole fixed cells as input, and enables automated ChIP from as few as 2000 cells.

View details for DOI 10.1101/pdb.prot084996

View details for PubMedID 26700100
High-dimensional immune profiling of total and rotavirus VP6-specific intestinal and circulating B cells by mass cytometry MUCOSAL IMMUNOLOGY Nair, N., Newell, E. W., Vollmers, C., Quake, S. R., Morton, J. M., DAVIS, M. M., He, X. S., Greenberg, H. B. 2016; 9 (1): 68-82
Abstract

In-depth phenotyping of human intestinal antibody secreting cells (ASCs) and their precursors is important for developing improved mucosal vaccines. We used single-cell mass cytometry to simultaneously analyze 34 differentiation and trafficking markers on intestinal and circulating B cells. In addition, we labeled rotavirus (RV) double-layered particles with a metal isotope and characterized B cells specific to the RV VP6 major structural protein. We describe the heterogeneity of the intestinal B-cell compartment, dominated by ASCs with some phenotypic and transcriptional characteristics of long-lived plasma cells. Using principal component analysis, we visualized the phenotypic relationships between major B-cell subsets in the intestine and blood, and revealed that IgM(+) memory B cells (MBCs) and naive B cells were phenotypically related as were CD27(-) MBCs and switched MBCs. ASCs in the intestine and blood were highly clonally related, but associated with distinct trajectories of phenotypic development. VP6-specific B cells were present among diverse B-cell subsets in immune donors, including naive B cells, with phenotypes representative of the overall B-cell pool. These data provide a high dimensional view of intestinal B cells and the determinants regulating humoral memory to a ubiquitous, mucosal pathogen at steady-state.Mucosal Immunology advance online publication, 22 April 2015; doi:10.1038/mi.2015.36.

View details for DOI 10.1038/mi.2015.36

View details for Web of Science ID 000367653800006
Noninvasive monitoring of infection and rejection after lung transplantation. Proceedings of the National Academy of Sciences of the United States of America De Vlaminck, I., Martin, L., Kertesz, M., Patel, K., Kowarsky, M., Strehl, C., Cohen, G., Luikart, H., Neff, N. F., Okamoto, J., Nicolls, M. R., Cornfield, D., Weill, D., Valantine, H., Khush, K. K., Quake, S. R. 2015; 112 (43): 13336-13341
Abstract

The survival rate following lung transplantation is among the lowest of all solid-organ transplants, and current diagnostic tests often fail to distinguish between infection and rejection, the two primary posttransplant clinical complications. We describe a diagnostic assay that simultaneously monitors for rejection and infection in lung transplant recipients by sequencing of cell-free DNA (cfDNA) in plasma. We determined that the levels of donor-derived cfDNA directly correlate with the results of invasive tests of rejection (area under the curve 0.9). We also analyzed the nonhuman cfDNA as a hypothesis-free approach to test for infections. Cytomegalovirus is most frequently assayed clinically, and the levels of CMV-derived sequences in cfDNA are consistent with clinical results. We furthermore show that hypothesis-free monitoring for pathogens using cfDNA reveals undiagnosed cases of infection, and that certain infectious pathogens such as human herpesvirus (HHV) 6, HHV-7, and adenovirus, which are not often tested clinically, occur with high frequency in this cohort.

View details for DOI 10.1073/pnas.1517494112

View details for PubMedID 26460048
Digital signaling decouples activation probability and population heterogeneity ELIFE Kellogg, R. A., Tian, C., Lipniacki, T., Quake, S. R., Tay, S. 2015; 4
Abstract

Digital signaling enhances robustness of cellular decisions in noisy environments, but it is unclear how digital systems transmit temporal information about a stimulus. To understand how temporal input information is encoded and decoded by the NF-?B system, we studied transcription factor dynamics and gene regulation under dose- and duration-modulated inflammatory inputs. Mathematical modeling predicted and microfluidic single-cell experiments confirmed that integral of the stimulus (or area, concentration � duration) controls the fraction of cells that activate NF-?B in the population. However, stimulus temporal profile determined NF-?B dynamics, cell-to-cell variability, and gene expression phenotype. A sustained, weak stimulation lead to heterogeneous activation and delayed timing that is transmitted to gene expression. In contrast, a transient, strong stimulus with the same area caused rapid and uniform dynamics. These results show that digital NF-?B signaling enables multidimensional control of cellular phenotype via input profile, allowing parallel and independent control of single-cell activation probability and population heterogeneity.

View details for DOI 10.7554/eLife.08931

View details for Web of Science ID 000363799000001

View details for PubMedID 26488364

View details for PubMedCentralID PMC4608393
Monitoring Pharmacologically Induced Immunosuppression by Immune Repertoire Sequencing to Detect Acute Allograft Rejection in Heart Transplant Patients: A Proof-of-Concept Diagnostic Accuracy Study PLOS MEDICINE Vollmers, C., De Vlaminck, I., Valantine, H. A., Penland, L., Luikart, H., Strehl, C., Cohen, G., Khush, K. K., Quake, S. R. 2015; 12 (10)
Abstract

It remains difficult to predict and to measure the efficacy of pharmacological immunosuppression. We hypothesized that measuring the B-cell repertoire would enable assessment of the overall level of immunosuppression after heart transplantation.In this proof-of-concept study, we implemented a molecular-barcode-based immune repertoire sequencing assay that sensitively and accurately measures the isotype and clonal composition of the circulating B cell repertoire. We used this assay to measure the temporal response of the B cell repertoire to immunosuppression after heart transplantation. We selected a subset of 12 participants from a larger prospective cohort study (ClinicalTrials.gov NCT01985412) that is ongoing at Stanford Medical Center and for which enrollment started in March 2010. This subset of 12 participants was selected to represent post-heart-transplant events, with and without acute rejection (six participants with moderate-to-severe rejection and six without). We analyzed 130 samples from these patients, with an average follow-up period of 15 mo. Immune repertoire sequencing enables the measurement of a patient's net state of immunosuppression (correlation with tacrolimus level, r = -0.867, 95% CI -0.968 to -0.523, p = 0.0014), as well as the diagnosis of acute allograft rejection, which is preceded by increased immune activity with a sensitivity of 71.4% (95% CI 30.3% to 94.9%) and a specificity of 82.0% (95% CI 72.1% to 89.1%) (cell-free donor-derived DNA as noninvasive gold standard). To illustrate the potential of immune repertoire sequencing to monitor atypical post-transplant trajectories, we analyzed two more patients, one with chronic infections and one with amyloidosis. A larger, prospective study will be needed to validate the power of immune repertoire sequencing to predict rejection events, as this proof-of-concept study is limited to a small number of patients who were selected based on several criteria including the availability of a large number of samples and the absence or presence of rejection events.If confirmed in larger, prospective studies, the method described here has potential applications in the tailored management of post-transplant immunosuppression and, more broadly, as a method for assessing the overall activity of the immune system.

View details for DOI 10.1371/journal.pmed.1001890

View details for Web of Science ID 000364466600008

View details for PubMedID 26466143

View details for PubMedCentralID PMC4605651
KIT Signaling Promotes Growth of Colon Xenograft Tumors in Mice and Is Up-Regulated in a Subset of Human Colon Cancers. Gastroenterology Chen, E. C., Karl, T. A., Kalisky, T., Gupta, S. K., O'Brien, C. A., Longacre, T. A., van de Rijn, M., Quake, S. R., Clarke, M. F., Rothenberg, M. E. 2015; 149 (3): 705-17 e2
Abstract

Receptor tyrosine kinase (RTK) inhibitors have advanced colon cancer treatment. We investigated the role of the RTK KIT in development of human colon cancer.An array of 137 patient-derived colon tumors and their associated xenografts were analyzed by immunohistochemistry to measure levels of KIT and its ligand KITLG. KIT and/or KITLG was stably knocked down by expression of small hairpin RNAs from lentiviral vectors in DLD1, HT29, LS174T, and COLO320 DM colon cancer cell lines, and in UM-COLON#8 and POP77 xenografts; cells transduced with only vector were used as controls. Cells were analyzed by real-time quantitative reverse transcription polymerase chain reaction, single-cell gene expression analysis, flow cytometry, and immunohistochemical, immunoblot, and functional assays. Xenograft tumors were grown from control and KIT-knockdown DLD1 and UM-COLON#8 cells in immunocompromised mice and compared. Some mice were given the RTK inhibitor imatinib after injection of cancer cells; tumor growth was measured based on bioluminescence. We assessed tumorigenicity using limiting dilution analysis.KIT and KITLG were expressed heterogeneously by a subset of human colon tumors. Knockdown of KIT decreased proliferation of colon cancer cell lines and growth of xenograft tumors in mice compared with control cells. KIT knockdown cells had increased expression of enterocyte markers, decreased expression of cycling genes, and, unexpectedly, increased expression of LGR5 associated genes. No activating mutations in KIT were detected in DLD1, POP77, or UM-COLON#8 cells. However, KITLG-knockdown DLD1 cells formed smaller xenograft tumors than control cells. Gene expression analysis of single CD44(+) cells indicated that KIT can promote growth via KITLG autocrine and/or paracrine signaling. Imatinib inhibited growth of KIT(+) colon cancer organoids in culture and growth of xenograft tumors in mice. Cancer cells with endogenous KIT expression were more tumorigenic in mice.KIT and KITLG are expressed by a subset of human colon tumors. KIT signaling promotes growth of colon cancer cells and organoids in culture and xenograft tumors in mice via its ligand, KITLG, in an autocrine or paracrine manner. Patients with KIT-expressing colon tumors can benefit from KIT RTK inhibitors.

View details for DOI 10.1053/j.gastro.2015.05.042

View details for PubMedID 26026391

View details for PubMedCentralID PMC4550533
KIT Signaling Promotes Growth of Colon Xenograft Tumors in Mice and Is Up-Regulated in a Subset of Human Colon Cancers GASTROENTEROLOGY Chen, E. C., Karl, T. A., Kalisky, T., Gupta, S. K., O'Brien, C. A., Longacre, T. A., De Rijn, M. V., Quake, S. R., Clarke, M. F., Rothenberg, M. E. 2015; 149 (3): 705-?
Abstract

Receptor tyrosine kinase (RTK) inhibitors have advanced colon cancer treatment. We investigated the role of the RTK KIT in development of human colon cancer.An array of 137 patient-derived colon tumors and their associated xenografts were analyzed by immunohistochemistry to measure levels of KIT and its ligand KITLG. KIT and/or KITLG was stably knocked down by expression of small hairpin RNAs from lentiviral vectors in DLD1, HT29, LS174T, and COLO320 DM colon cancer cell lines, and in UM-COLON#8 and POP77 xenografts; cells transduced with only vector were used as controls. Cells were analyzed by real-time quantitative reverse transcription polymerase chain reaction, single-cell gene expression analysis, flow cytometry, and immunohistochemical, immunoblot, and functional assays. Xenograft tumors were grown from control and KIT-knockdown DLD1 and UM-COLON#8 cells in immunocompromised mice and compared. Some mice were given the RTK inhibitor imatinib after injection of cancer cells; tumor growth was measured based on bioluminescence. We assessed tumorigenicity using limiting dilution analysis.KIT and KITLG were expressed heterogeneously by a subset of human colon tumors. Knockdown of KIT decreased proliferation of colon cancer cell lines and growth of xenograft tumors in mice compared with control cells. KIT knockdown cells had increased expression of enterocyte markers, decreased expression of cycling genes, and, unexpectedly, increased expression of LGR5 associated genes. No activating mutations in KIT were detected in DLD1, POP77, or UM-COLON#8 cells. However, KITLG-knockdown DLD1 cells formed smaller xenograft tumors than control cells. Gene expression analysis of single CD44(+) cells indicated that KIT can promote growth via KITLG autocrine and/or paracrine signaling. Imatinib inhibited growth of KIT(+) colon cancer organoids in culture and growth of xenograft tumors in mice. Cancer cells with endogenous KIT expression were more tumorigenic in mice.KIT and KITLG are expressed by a subset of human colon tumors. KIT signaling promotes growth of colon cancer cells and organoids in culture and xenograft tumors in mice via its ligand, KITLG, in an autocrine or paracrine manner. Patients with KIT-expressing colon tumors can benefit from KIT RTK inhibitors.

View details for DOI 10.1053/j.gastro.2015.05.042

View details for Web of Science ID 000360269800039

View details for PubMedCentralID PMC4550533
Read count-based method for high-throughput allelic genotyping of transposable elements and structural variants BMC GENOMICS Kuhn, A., Ong, Y. M., Quake, S. R., Burkholder, W. F. 2015; 16
Abstract

Like other structural variants, transposable element insertions can be highly polymorphic across individuals. Their functional impact, however, remains poorly understood. Current genome-wide approaches for genotyping insertion-site polymorphisms based on targeted or whole-genome sequencing remain very expensive and can lack accuracy, hence new large-scale genotyping methods are needed.We describe a high-throughput method for genotyping transposable element insertions and other types of structural variants that can be assayed by breakpoint PCR. The method relies on next-generation sequencing of multiplex, site-specific PCR amplification products and read count-based genotype calls. We show that this method is flexible, efficient (it does not require rounds of optimization), cost-effective and highly accurate.This method can benefit a wide range of applications from the routine genotyping of animal and plant populations to the functional study of structural variants in humans.

View details for DOI 10.1186/s12864-015-1700-4

View details for Web of Science ID 000357449400001

View details for PubMedID 26153459

View details for PubMedCentralID PMC4494700
A survey of human brain transcriptome diversity at the single cell level PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Darmanis, S., Sloan, S. A., Zhang, Y., Enge, M., Caneda, C., Shuer, L. M., Gephart, M. G., Barres, B. A., Quake, S. R. 2015; 112 (23): 7285-7290
Abstract

The human brain is a tissue of vast complexity in terms of the cell types it comprises. Conventional approaches to classifying cell types in the human brain at single cell resolution have been limited to exploring relatively few markers and therefore have provided a limited molecular characterization of any given cell type. We used single cell RNA sequencing on 466 cells to capture the cellular complexity of the adult and fetal human brain at a whole transcriptome level. Healthy adult temporal lobe tissue was obtained during surgical procedures where otherwise normal tissue was removed to gain access to deeper hippocampal pathology in patients with medical refractory seizures. We were able to classify individual cells into all of the major neuronal, glial, and vascular cell types in the brain. We were able to divide neurons into individual communities and show that these communities preserve the categorization of interneuron subtypes that is typically observed with the use of classic interneuron markers. We then used single cell RNA sequencing on fetal human cortical neurons to identify genes that are differentially expressed between fetal and adult neurons and those genes that display an expression gradient that reflects the transition between replicating and quiescent fetal neuronal populations. Finally, we observed the expression of major histocompatibility complex type I genes in a subset of adult neurons, but not fetal neurons. The work presented here demonstrates the applicability of single cell RNA sequencing on the study of the adult human brain and constitutes a first step toward a comprehensive cellular atlas of the human brain.

View details for DOI 10.1073/pnas.1507125112

View details for Web of Science ID 000355823200055

View details for PubMedID 26060301

View details for PubMedCentralID PMC4466750
Clonal Deletion Prunes but Does Not Eliminate Self-Specific alpha beta CD8(+) T Lymphocytes IMMUNITY Yu, W., Jiang, N., Ebert, P. J., Kidd, B. A., Mueller, S., Lund, P. J., Juang, J., Adachi, K., Tse, T., Birnbaum, M. E., Newell, E. W., Wilson, D. M., Grotenbreg, G. M., Valitutti, S., Quake, S. R., Davis, M. M. 2015; 42 (5): 929-941
Abstract

It has long been thought that clonal deletion efficiently removes almost all self-specific T cells from the peripheral repertoire. We found that self-peptide MHC-specific CD8(+) T cells in the blood of healthy humans were present in frequencies similar to those specific for non-self antigens. For the Y chromosome-encoded SMCY antigen, self-specific T cells exhibited only a 3-fold lower average frequency in males versus females and were anergic with respect to peptide activation, although this inhibition could be overcome by a stronger stimulus. We conclude that clonal deletion prunes but does not eliminate self-specific T cells and suggest that to do so would create holes in the repertoire that pathogens could readily exploit. In support of this hypothesis, we detected T cells specific for all 20 amino acid variants at the p5 position of a hepatitis C virus epitope in a random group of blood donors.

View details for DOI 10.1016/j.immuni.2015.05.001

View details for PubMedID 25992863
Multiplexed locus-specific analysis of DNA methylation in single cells NATURE PROTOCOLS Cheow, L. F., Quake, S. R., Burkholder, W. F., Messerschmidt, D. M. 2015; 10 (4): 619-631
Abstract

This protocol details a method for measuring the DNA methylation state of multiple target sites in single cells, otherwise known as single-cell restriction analysis of methylation (SCRAM). The basic steps include isolating and lysing single cells, digesting genomic DNA with a methylation-sensitive restriction endonuclease (MSRE) and amplification of multiple targets by two rounds of PCR to determine the methylation status of target sites. The method can reliably and accurately detect the methylation status of multiple target sites in each single cell, and it can be completed in a relatively short time (<2 d) at low cost. Consequently, the method may be preferable over whole-genome methods in applications requiring highly reliable and cost-effective coverage of specific target sites in all cells from a sample and in cases when the DNA methylation states of single CpG sites are representative of the methylation status of corresponding regions of interest.

View details for DOI 10.1038/nprot.2015.041

View details for Web of Science ID 000351932400008

View details for PubMedID 25811896
A Biotic Game Design Project for Integrated Life Science and Engineering Education PLOS BIOLOGY Cira, N. J., Chung, A. M., Denisin, A. K., Rensi, S., Sanchez, G. N., Quake, S. R., Riedel-Kruse, I. H. 2015; 13 (3)
Abstract

Engaging, hands-on design experiences are key for formal and informal Science, Technology, Engineering, and Mathematics (STEM) education. Robotic and video game design challenges have been particularly effective in stimulating student interest, but equivalent experiences for the life sciences are not as developed. Here we present the concept of a "biotic game design project" to motivate student learning at the interface of life sciences and device engineering (as part of a cornerstone bioengineering devices course). We provide all course material and also present efforts in adapting the project's complexity to serve other time frames, age groups, learning focuses, and budgets. Students self-reported that they found the biotic game project fun and motivating, resulting in increased effort. Hence this type of design project could generate excitement and educational impact similar to robotics and video games.

View details for DOI 10.1371/journal.pbio.1002110

View details for Web of Science ID 000352095700019

View details for PubMedID 25807212

View details for PubMedCentralID PMC4373802
Brain Tumor Mutations Detected in Cerebral Spinal Fluid CLINICAL CHEMISTRY Pan, W., Gu, W., Nagpal, S., Gephart, M. H., Quake, S. R. 2015; 61 (3): 514-522
Abstract

Detecting tumor-derived cell-free DNA (cfDNA) in the blood of brain tumor patients is challenging, presumably owing to the blood-brain barrier. Cerebral spinal fluid (CSF) may serve as an alternative "liquid biopsy" of brain tumors by enabling measurement of circulating DNA within CSF to characterize tumor-specific mutations. Many aspects about the characteristics and detectability of tumor mutations in CSF remain undetermined.We used digital PCR and targeted amplicon sequencing to quantify tumor mutations in the cfDNA of CSF and plasma collected from 7 patients with solid brain tumors. Also, we applied cancer panel sequencing to globally characterize the somatic mutation profile from the CSF of 1 patient with suspected leptomeningeal disease.We detected tumor mutations in CSF samples from 6 of 7 patients with solid brain tumors. The concentration of the tumor mutant alleles varied widely between patients, from <5 to nearly 3000 copies/mL CSF. We identified 7 somatic mutations from the CSF of a patient with leptomeningeal disease by use of cancer panel sequencing, and the result was concordant with genetic testing on the primary tumor biopsy.Tumor mutations were detectable in cfDNA from the CSF of patients with different primary and metastatic brain tumors. We designed 2 strategies to characterize tumor mutations in CSF for potential clinical diagnosis: the targeted detection of known driver mutations to monitor brain metastasis and the global characterization of genomic aberrations to direct personalized cancer care.

View details for DOI 10.1373/clinchem.2014.235457

View details for Web of Science ID 000352161300013

View details for PubMedID 25605683
Novel Exons and Splice Variants in the Human Antibody Heavy Chain Identified by Single Cell and Single Molecule Sequencing PLOS ONE Vollmers, C., Penland, L., Kanbar, J. N., Quake, S. R. 2015; 10 (1)
Abstract

Antibody heavy chains contain a variable and a constant region. The constant region of the antibody heavy chain is encoded by multiple groups of exons which define the isotype and therefore many functional characteristics of the antibody. We performed both single B cell RNAseq and long read single molecule sequencing of antibody heavy chain transcripts and were able to identify novel exons for IGHA1 and IGHA2 as well as novel isoforms for IGHM antibody heavy chain.

View details for DOI 10.1371/journal.pone.0117050

View details for Web of Science ID 000348562900030

View details for PubMedID 25611855

View details for PubMedCentralID PMC4303433
Dissecting noncoding and pathogen RNA-protein interactomes RNA-A PUBLICATION OF THE RNA SOCIETY Flynn, R. A., Martin, L., Spitale, R. C., Do, B. T., Sagan, S. M., Zarnegar, B., Qu, K., Khavari, P. A., Quake, S. R., Sarnow, P., Chang, H. Y. 2015; 21 (1): 135-143
Abstract

RNA-protein interactions are central to biological regulation. Cross-linking immunoprecipitation (CLIP)-seq is a powerful tool for genome-wide interrogation of RNA-protein interactomes, but current CLIP methods are limited by challenging biochemical steps and fail to detect many classes of noncoding and nonhuman RNAs. Here we present FAST-iCLIP, an integrated pipeline with improved CLIP biochemistry and an automated informatic pipeline for comprehensive analysis across protein coding, noncoding, repetitive, retroviral, and nonhuman transcriptomes. FAST-iCLIP of Poly-C binding protein 2 (PCBP2) showed that PCBP2-bound CU-rich motifs in different topologies to recognize mRNAs and noncoding RNAs with distinct biological functions. FAST-iCLIP of PCBP2 in hepatitis C virus-infected cells enabled a joint analysis of the PCBP2 interactome with host and viral RNAs and their interplay. These results show that FAST-iCLIP can be used to rapidly discover and decipher mechanisms of RNA-protein recognition across the diversity of human and pathogen RNAs.

View details for DOI 10.1261/rna.047803.114

View details for PubMedID 25411354
The phylogenetic and geographic structure of Y-chromosome haplogroup R1a EUROPEAN JOURNAL OF HUMAN GENETICS Underhill, P. A., Poznik, G. D., Rootsi, S., Jaerve, M., Lin, A. A., Wang, J., Passarelli, B., Kanbar, J., Myres, N. M., King, R. J., Di Cristofaro, J., Sahakyan, H., Behar, D. M., Kushniarevich, A., Sarac, J., Saric, T., Rudan, P., Pathak, A. K., Chaubey, G., Grugni, V., Semino, O., Yepiskoposyan, L., Bahmanimehr, A., Farjadian, S., Balanovsky, O., Khusnutdinova, E. K., Herrera, R. J., Chiaroni, J., Bustamante, C. D., Quake, S. R., Kivisild, T., Villems, R. 2015; 23 (1): 124-131
Abstract

R1a-M420 is one of the most widely spread Y-chromosome haplogroups; however, its substructure within Europe and Asia has remained poorly characterized. Using a panel of 16?244 male subjects from 126 populations sampled across Eurasia, we identified 2923 R1a-M420 Y-chromosomes and analyzed them to a highly granular phylogeographic resolution. Whole Y-chromosome sequence analysis of eight R1a and five R1b individuals suggests a divergence time of ?25,000 (95% CI: 21,300-29,000) years ago and a coalescence time within R1a-M417 of ?5800 (95% CI: 4800-6800) years. The spatial frequency distributions of R1a sub-haplogroups conclusively indicate two major groups, one found primarily in Europe and the other confined to Central and South Asia. Beyond the major European versus Asian dichotomy, we describe several younger sub-haplogroups. Based on spatial distributions and diversity patterns within the R1a-M420 clade, particularly rare basal branches detected primarily within Iran and eastern Turkey, we conclude that the initial episodes of haplogroup R1a diversification likely occurred in the vicinity of present-day Iran.

View details for DOI 10.1038/ejhg.2014.50

View details for Web of Science ID 000346271500020

View details for PubMedID 24667786

View details for PubMedCentralID PMC4266736
Dissecting the clonal origins of childhood acute lymphoblastic leukemia by single-cell genomics. Proceedings of the National Academy of Sciences of the United States of America Gawad, C., Koh, W., Quake, S. R. 2014; 111 (50): 17947-17952
Abstract

Many cancers have substantial genomic heterogeneity within a given tumor, and to fully understand that diversity requires the ability to perform single cell analysis. We performed targeted sequencing of a panel of single nucleotide variants (SNVs), deletions, and IgH sequences in 1,479 single tumor cells from six acute lymphoblastic leukemia (ALL) patients. By accurately segregating groups of cooccurring mutations into distinct clonal populations, we identified codominant clones in the majority of patients. Evaluation of intraclonal mutation patterns identified clone-specific punctuated cytosine mutagenesis events, showed that most structural variants are acquired before SNVs, determined that KRAS mutations occur late in disease development but are not sufficient for clonal dominance, and identified clones within the same patient that are arrested at varied stages in B-cell development. Taken together, these data order the sequence of genetic events that underlie childhood ALL and provide a framework for understanding the development of the disease at single-cell resolution.

View details for DOI 10.1073/pnas.1420822111

View details for PubMedID 25425670
A cell-intrinsic role for TLR2-MYD88 in intestinal and breast epithelia and oncogenesis. Nature cell biology Scheeren, F. A., Kuo, A. H., van Weele, L. J., Cai, S., Glykofridis, I., Sikandar, S. S., Zabala, M., Qian, D., Lam, J. S., Johnston, D., Volkmer, J. P., Sahoo, D., van de Rijn, M., Dirbas, F. M., Somlo, G., Kalisky, T., Rothenberg, M. E., Quake, S. R., Clarke, M. F. 2014; 16 (12): 1238-1248
Abstract

It has been postulated that there is a link between inflammation and cancer. Here we describe a role for cell-intrinsic toll-like receptor-2 (TLR2; which is involved in inflammatory response) signalling in normal intestinal and mammary epithelial cells and oncogenesis. The downstream effectors of TLR2 are expressed by normal intestinal and mammary epithelia, including the stem/progenitor cells. Deletion of MYD88 or TLR2 in the intestinal epithelium markedly reduces DSS-induced colitis regeneration and spontaneous tumour development in mice. Limiting dilution transplantations of breast epithelial cells devoid of TLR2 or MYD88 revealed a significant decrease in mammary repopulating unit frequency compared with the control. Inhibition of TLR2, its co-receptor CD14, or its downstream targets MYD88 and IRAK1 inhibits growth of human breast cancers in vitro and in vivo. These results suggest that inhibitors of the TLR2 pathway merit investigation as possible therapeutic and chemoprevention agents.

View details for DOI 10.1038/ncb3058

View details for PubMedID 25362351
A cell-intrinsic role for TLR2 MYD88 in intestinal and breast epithelia and oncogenesis NATURE CELL BIOLOGY Scheeren, F. A., Kuo, A. H., van Weele, L. J., Cai, S., Glykofridis, I., Sikandar, S. S., Zabala, M., Qian, D., Lam, J. S., Johnston, D., Volkmer, J. P., Sahoo, D., van de Rijn, M., Dirbas, F. M., Somlo, G., Kalisky, T., Rothenberg, M. E., Quake, S. R., Clarke, M. F. 2014; 16 (12): 1238-U245
Abstract

It has been postulated that there is a link between inflammation and cancer. Here we describe a role for cell-intrinsic toll-like receptor-2 (TLR2; which is involved in inflammatory response) signalling in normal intestinal and mammary epithelial cells and oncogenesis. The downstream effectors of TLR2 are expressed by normal intestinal and mammary epithelia, including the stem/progenitor cells. Deletion of MYD88 or TLR2 in the intestinal epithelium markedly reduces DSS-induced colitis regeneration and spontaneous tumour development in mice. Limiting dilution transplantations of breast epithelial cells devoid of TLR2 or MYD88 revealed a significant decrease in mammary repopulating unit frequency compared with the control. Inhibition of TLR2, its co-receptor CD14, or its downstream targets MYD88 and IRAK1 inhibits growth of human breast cancers in vitro and in vivo. These results suggest that inhibitors of the TLR2 pathway merit investigation as possible therapeutic and chemoprevention agents.

View details for DOI 10.1038/ncb3058

View details for Web of Science ID 000345777300014

View details for PubMedID 25362351
Multiplexed Analysis of Protein-Ligand Interactions by Fluorescence Anisotropy in a Microfluidic Platform ANALYTICAL CHEMISTRY Cheow, L. F., Viswanathan, R., Chin, C., Jennifer, N., Jones, R. C., Guccione, E., Quake, S. R., Burkholder, W. F. 2014; 86 (19): 9901-9908
Abstract

Homogeneous assay platforms for measuring protein-ligand interactions are highly valued due to their potential for high-throughput screening. However, the implementation of these multiplexed assays in conventional microplate formats is considerably expensive due to the large amounts of reagents required and the need for automation. We implemented a homogeneous fluorescence anisotropy-based binding assay in an automated microfluidic chip to simultaneously interrogate >2300 pairwise interactions. We demonstrated the utility of this platform in determining the binding affinities between chromatin-regulatory proteins and different post-translationally modified histone peptides. The microfluidic chip assay produces comparable results to conventional microtiter plate assays, yet requires 2 orders of magnitude less sample and an order of magnitude fewer pipetting steps. This approach enables one to use small samples for medium-scale screening and could ease the bottleneck of large-scale protein purification.

View details for DOI 10.1021/ac502605f

View details for Web of Science ID 000343017100080

View details for PubMedID 25207435
RNA-guided endonuclease provides a therapeutic strategy to cure latent herpesviridae infection PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wang, J., Quake, S. R. 2014; 111 (36): 13157-13162
Abstract

Latent viral infection is a persistent cause of human disease. Although standard antiviral therapies can suppress active viral replication, no existing treatment can effectively eradicate latent infection and therefore a cure is lacking for many prevalent viral diseases. The prokaryotic immune system clustered regularly interspaced short palindromic repeat (CRISPR)/Cas evolved as a natural response to phage infections, and we demonstrate here that the CRISPR/Cas9 system can be adapted for antiviral treatment in human cells by specifically targeting the genomes of latent viral infections. Patient-derived cells from a Burkitt's lymphoma with latent Epstein-Barr virus infection showed dramatic proliferation arrest and a concomitant decrease in viral load after exposure to a CRISPR/Cas9 vector targeted to the viral genome.

View details for DOI 10.1073/pnas.1410785111

View details for Web of Science ID 000341625600048

View details for PubMedCentralID PMC4246930
An implantable microfluidic device for self-monitoring of intraocular pressure NATURE MEDICINE Araci, I. E., Su, B., Quake, S. R., Mandel, Y. 2014; 20 (9): 1074-1078
Abstract

Glaucoma is the second most common cause of blindness in the world. It is a multifactorial disease with several risk factors, of which intraocular pressure (IOP) is a primary contributing factor. IOP measurements are used for glaucoma diagnosis and patient monitoring. IOP has wide diurnal fluctuation and is dependent on body posture, so the occasional measurements done by the eye care expert in the clinic can be misleading. Here we show that microfluidic principles can be used to develop an implantable sensor that has a limit of detection of 1 mm Hg, high sensitivity and excellent reproducibility. This device has a simple optical interface that enables IOP to be read with a smartphone camera. This sensor, with its ease of fabrication and simple design, as well as its allowance for IOP home monitoring, offers a promising approach for better care of patients with glaucoma.

View details for DOI 10.1038/nm.3621

View details for Web of Science ID 000341404000022

View details for PubMedID 25150497
Noninvasive prenatal diagnosis in a fetus at risk for methylmalonic acidemia GENETICS IN MEDICINE Gu, W., Koh, W., Blumenfeld, Y. J., El-Sayed, Y. Y., Hudgins, L., Hintz, S. R., Quake, S. R. 2014; 16 (7): 564-567
View details for DOI 10.1038/gim.2013.194

View details for Web of Science ID 000338601400012
Circulating Cell-Free DNA Enables Noninvasive Diagnosis of Heart Transplant Rejection SCIENCE TRANSLATIONAL MEDICINE De Vlaminck, I., Valantine, H. A., Snyder, T. M., Strehl, C., Cohen, G., Luikart, H., Neff, N. F., Okamoto, J., Bernstein, D., Weisshaar, D., Quake, S. R., Khush, K. K. 2014; 6 (241)
Abstract

Monitoring allograft health is an important component of posttransplant therapy. Endomyocardial biopsy is the current gold standard for cardiac allograft monitoring but is an expensive and invasive procedure. Proof of principle of a universal, noninvasive diagnostic method based on high-throughput screening of circulating cell-free donor-derived DNA (cfdDNA) was recently demonstrated in a small retrospective cohort. We present the results of a prospective cohort study (65 patients, 565 samples) that tested the utility of cfdDNA in measuring acute rejection after heart transplantation. Circulating cell-free DNA was purified from plasma and sequenced (mean depth, 1.2 giga-base pairs) to quantify the fraction of cfdDNA. Through a comparison with endomyocardial biopsy results, we demonstrate that cfdDNA enables diagnosis of acute rejection after heart transplantation, with an area under the receiver operating characteristic curve of 0.83 and sensitivity and specificity that are comparable to the intrinsic performance of the biopsy itself. This noninvasive genome transplant dynamics approach is a powerful and informative method for routine monitoring of allograft health without incurring the risk, discomfort, and expense of an invasive biopsy.

View details for DOI 10.1126/scitranslmed.3007803

View details for Web of Science ID 000338711700003
Linkage disequilibrium and signatures of positive selection around LINE-1 retrotransposons in the human genome PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kuhn, A., Ong, Y. M., Cheng, C., Wong, T. Y., Quake, S. R., Burkholder, W. F. 2014; 111 (22): 8131-8136
Abstract

Insertions of the human-specific subfamily of LINE-1 (L1) retrotransposon are highly polymorphic across individuals and can critically influence the human transcriptome. We hypothesized that L1 insertions could represent genetic variants determining important human phenotypic traits, and performed an integrated analysis of L1 elements and single nucleotide polymorphisms (SNPs) in several human populations. We found that a large fraction of L1s were in high linkage disequilibrium with their surrounding genomic regions and that they were well tagged by SNPs. However, L1 variants were only partially captured by SNPs on standard SNP arrays, so that their potential phenotypic impact would be frequently missed by SNP array-based genome-wide association studies. We next identified potential phenotypic effects of L1s by looking for signatures of natural selection linked to L1 insertions; significant extended haplotype homozygosity was detected around several L1 insertions. This finding suggests that some of these L1 insertions may have been the target of recent positive selection.

View details for DOI 10.1073/pnas.1401532111

View details for Web of Science ID 000336687900064

View details for PubMedID 24847061

View details for PubMedCentralID PMC4050588
Noninvasive in vivo monitoring of tissue-specific global gene expression in humans. Proceedings of the National Academy of Sciences of the United States of America Koh, W., Pan, W., Gawad, C., Fan, H. C., Kerchner, G. A., Wyss-Coray, T., Blumenfeld, Y. J., El-Sayed, Y. Y., Quake, S. R. 2014; 111 (20): 7361-7366
Abstract

Circulating cell-free RNA in the blood provides a potential window into the health, phenotype, and developmental programs of a variety of human organs. We used high-throughput methods of RNA analysis such as microarrays and next-generation sequencing to characterize the global landscape circulating RNA in a cohort of human subjects. By focusing on genes whose expression is highly specific to certain tissues, we were able to identify the relative contributions of these tissues to circulating RNA and to monitor changes in tissue development and health. As one application of this approach, we performed a longitudinal study on pregnant women and analyzed their combined cell-free RNA transcriptomes across all three trimesters of pregnancy and after delivery. In addition to the analysis of mRNA, we observed and characterized noncoding species such as long noncoding RNA and circular RNA transcripts whose presence had not been previously observed in human plasma. We demonstrate that it is possible to track specific longitudinal phenotypic changes in both the mother and the fetus and that it is possible to directly measure transcripts from a variety of fetal tissues in the maternal blood sample. We also studied the role of neuron-specific transcripts in the blood of healthy adults and those suffering from the neurodegenerative disorder Alzheimer's disease and showed that disease specific neural transcripts are present at increased levels in the blood of affected individuals. Characterization of the cell-free transcriptome in its entirety may thus provide broad insights into human health and development without the need for invasive tissue sampling.

View details for DOI 10.1073/pnas.1405528111

View details for PubMedID 24799715
Reconstructing lineage hierarchies of the distal lung epithelium using single-cell RNA-seq. Nature Treutlein, B., Brownfield, D. G., Wu, A. R., Neff, N. F., Mantalas, G. L., Espinoza, F. H., Desai, T. J., Krasnow, M. A., Quake, S. R. 2014; 509 (7500): 371-375
Abstract

The mammalian lung is a highly branched network in which the distal regions of the bronchial tree transform during development into a densely packed honeycomb of alveolar air sacs that mediate gas exchange. Although this transformation has been studied by marker expression analysis and fate-mapping, the mechanisms that control the progression of lung progenitors along distinct lineages into mature alveolar cell types are still incompletely known, in part because of the limited number of lineage markers and the effects of ensemble averaging in conventional transcriptome analysis experiments on cell populations. Here we show that single-cell transcriptome analysis circumvents these problems and enables direct measurement of the various cell types and hierarchies in the developing lung. We used microfluidic single-cell RNA sequencing (RNA-seq) on 198 individual cells at four different stages encompassing alveolar differentiation to measure the transcriptional states which define the developmental and cellular hierarchy of the distal mouse lung epithelium. We empirically classified cells into distinct groups by using an unbiased genome-wide approach that did not require a priori knowledge of the underlying cell types or the previous purification of cell populations. The results confirmed the basic outlines of the classical model of epithelial cell-type diversity in the distal lung and led to the discovery of many previously unknown cell-type markers, including transcriptional regulators that discriminate between the different populations. We reconstructed the molecular steps during maturation of bipotential progenitors along both alveolar lineages and elucidated the full life cycle of the alveolar type?2 cell lineage. This single-cell genomics approach is applicable to any developing or mature tissue to robustly delineate molecularly distinct cell types, define progenitors and lineage hierarchies, and identify lineage-specific regulatory factors.

View details for DOI 10.1038/nature13173

View details for PubMedID 24739965
Reconstructing lineage hierarchies of the distal lung epithelium using single-cell RNA-seq. Nature Treutlein, B., Brownfield, D. G., Wu, A. R., Neff, N. F., Mantalas, G. L., Espinoza, F. H., Desai, T. J., Krasnow, M. A., Quake, S. R. 2014; 509 (7500): 371-375
View details for DOI 10.1038/nature13173

View details for PubMedID 24739965
Cartography of neurexin alternative splicing mapped by single-molecule long-read mRNA sequencing. Proceedings of the National Academy of Sciences of the United States of America Treutlein, B., Gokce, O., Quake, S. R., S�dhof, T. C. 2014; 111 (13): E1291-9
Abstract

Neurexins are evolutionarily conserved presynaptic cell-adhesion molecules that are essential for normal synapse formation and synaptic transmission. Indirect evidence has indicated that extensive alternative splicing of neurexin mRNAs may produce hundreds if not thousands of neurexin isoforms, but no direct evidence for such diversity has been available. Here we use unbiased long-read sequencing of full-length neurexin (Nrxn)1?, Nrxn1?, Nrxn2?, Nrxn3?, and Nrxn3? mRNAs to systematically assess how many sites of alternative splicing are used in neurexins with a significant frequency, and whether alternative splicing events at these sites are independent of each other. In sequencing more than 25,000 full-length mRNAs, we identified a novel, abundantly used alternatively spliced exon of Nrxn1? and Nrxn3? (referred to as alternatively spliced sequence 6) that encodes a 9-residue insertion in the flexible hinge region between the fifth LNS (laminin-?, neurexin, sex hormone-binding globulin) domain and the third EGF-like sequence. In addition, we observed several larger-scale events of alternative splicing that deleted multiple domains and were much less frequent than the canonical six sites of alternative splicing in neurexins. All of the six canonical events of alternative splicing appear to be independent of each other, suggesting that neurexins may exhibit an even larger isoform diversity than previously envisioned and comprise thousands of variants. Our data are consistent with the notion that ?-neurexins represent extracellular protein-interaction scaffolds in which different LNS and EGF domains mediate distinct interactions that affect diverse functions and are independently regulated by independent events of alternative splicing.

View details for DOI 10.1073/pnas.1403244111

View details for PubMedID 24639501
The promise and challenge of high-throughput sequencing of the antibody repertoire. Nature biotechnology Georgiou, G., Ippolito, G. C., Beausang, J., Busse, C. E., Wardemann, H., Quake, S. R. 2014; 32 (2): 158-168
Abstract

Efforts to determine the antibody repertoire encoded by B cells in the blood or lymphoid organs using high-throughput DNA sequencing technologies have been advancing at an extremely rapid pace and are transforming our understanding of humoral immune responses. Information gained from high-throughput DNA sequencing of immunoglobulin genes (Ig-seq) can be applied to detect B-cell malignancies with high sensitivity, to discover antibodies specific for antigens of interest, to guide vaccine development and to understand autoimmunity. Rapid progress in the development of experimental protocols and informatics analysis tools is helping to reduce sequencing artifacts, to achieve more precise quantification of clonal diversity and to extract the most pertinent biological information. That said, broader application of Ig-seq, especially in clinical settings, will require the development of a standardized experimental design framework that will enable the sharing and meta-analysis of sequencing data generated by different laboratories.

View details for DOI 10.1038/nbt.2782

View details for PubMedID 24441474
Mapping of Protein-Protein Interactions of E. coli RNA Polymerase with Microfluidic Mechanical Trapping. PloS one Bates, S. R., Quake, S. R. 2014; 9 (3)
Abstract

The biophysical details of how transcription factors and other proteins interact with RNA polymerase are of great interest as they represent the nexus of how structure and function interact to regulate gene expression in the cell. We used an in vitro microfluidic approach to map interactions between a set of ninety proteins, over a third of which are transcription factors, and each of the four subunits of E. coli RNA polymerase, and we compared our results to those of previous large-scale studies. We detected interactions between RNA polymerase and transcription factors that earlier high-throughput screens missed; our results suggest that such interactions can occur without DNA mediation more commonly than previously appreciated.

View details for DOI 10.1371/journal.pone.0091542

View details for PubMedID 24643045

View details for PubMedCentralID PMC3958368
BAsE-Seq: a method for obtaining long viral haplotypes from short sequence reads GENOME BIOLOGY Hong, L. Z., Hong, S., Wong, H. T., Aw, P. P., Cheng, Y., Wilm, A., de Sessions, P. F., Lim, S. G., Nagarajan, N., Hibberd, M. L., Quake, S. R., Burkholder, W. F. 2014; 15 (11)
Abstract

We present a method for obtaining long haplotypes, of over 3 kb in length, using a short-read sequencer, Barcode-directed Assembly for Extra-long Sequences (BAsE-Seq). BAsE-Seq relies on transposing a template-specific barcode onto random segments of the template molecule and assembling the barcoded short reads into complete haplotypes. We applied BAsE-Seq on mixed clones of hepatitis B virus and accurately identified haplotypes occurring at frequencies greater than or equal to 0.4%, with >99.9% specificity. Applying BAsE-Seq to a clinical sample, we obtained over 9,000 viral haplotypes, which provided an unprecedented view of hepatitis B virus population structure during chronic infection. BAsE-Seq is readily applicable for monitoring quasispecies evolution in viral diseases.

View details for DOI 10.1186/s13059-014-0517

View details for Web of Science ID 000346607300016

View details for PubMedID 25406369

View details for PubMedCentralID PMC4269956
Dissecting genomic diversity, one cell at a time NATURE METHODS Blainey, P. C., Quake, S. R. 2014; 11 (1): 19-21
View details for DOI 10.1038/nmeth.2783

View details for Web of Science ID 000329178200010

View details for PubMedID 24524132

View details for PubMedCentralID PMC3947563
A quantitative comparison of single-cell whole genome amplification methods. PloS one de Bourcy, C. F., De Vlaminck, I., Kanbar, J. N., Wang, J., Gawad, C., Quake, S. R. 2014; 9 (8)
Abstract

Single-cell sequencing is emerging as an important tool for studies of genomic heterogeneity. Whole genome amplification (WGA) is a key step in single-cell sequencing workflows and a multitude of methods have been introduced. Here, we compare three state-of-the-art methods on both bulk and single-cell samples of E. coli DNA: Multiple Displacement Amplification (MDA), Multiple Annealing and Looping Based Amplification Cycles (MALBAC), and the PicoPLEX single-cell WGA kit (NEB-WGA). We considered the effects of reaction gain on coverage uniformity, error rates and the level of background contamination. We compared the suitability of the different WGA methods for the detection of copy-number variations, for the detection of single-nucleotide polymorphisms and for de-novo genome assembly. No single method performed best across all criteria and significant differences in characteristics were observed; the choice of which amplifier to use will depend strongly on the details of the type of question being asked in any given experiment.

View details for DOI 10.1371/journal.pone.0105585

View details for PubMedID 25136831

View details for PubMedCentralID PMC4138190
Quantitative assessment of single-cell RNA-sequencing methods. Nature methods Wu, A. R., Neff, N. F., Kalisky, T., Dalerba, P., Treutlein, B., Rothenberg, M. E., Mburu, F. M., Mantalas, G. L., Sim, S., Clarke, M. F., Quake, S. R. 2014; 11 (1): 41-46
View details for DOI 10.1038/nmeth.2694

View details for PubMedID 24141493
Temporal response of the human virome to immunosuppression and antiviral therapy. Cell De Vlaminck, I., Khush, K. K., Strehl, C., Kohli, B., Luikart, H., Neff, N. F., Okamoto, J., Snyder, T. M., Cornfield, D. N., Nicolls, M. R., Weill, D., Bernstein, D., Valantine, H. A., Quake, S. R. 2013; 155 (5): 1178-1187
Abstract

There are few substantive methods to measure the health of the immune system, and the connection between immune strength and the viral component of the microbiome is poorly understood. Organ transplant recipients are treated with posttransplant therapies that combine immunosuppressive and antiviral drugs, offering a window into the effects of�immune modulation on the virome. We used sequencing of cell-free DNA in plasma to investigate drug-virome interactions in a cohort of organ transplant recipients (656 samples, 96 patients) and find that antivirals and immunosuppressants strongly affect the structure of the virome in plasma. We observe marked virome compositional dynamics at the onset of the therapy and find that the total viral load increases with immunosuppression, whereas the bacterial component of the microbiome remains largely unaffected. The data provide insight into the relationship between the human virome, the state of the immune system, and the effects of pharmacological treatment and offer a potential application of the virome state to predict immunocompetence.

View details for DOI 10.1016/j.cell.2013.10.034

View details for PubMedID 24267896
Single-Cell DNA-Methylation Analysis Reveals Epigenetic Chimerism in Preimplantation Embryos SCIENCE Lorthongpanich, C., Cheow, L. F., Balu, S., Quake, S. R., Knowles, B. B., Burkholder, W. F., Solter, D., Messerschmidt, D. M. 2013; 341 (6150): 1110-1112
Abstract

Epigenetic alterations are increasingly recognized as causes of human cancers and disease. These aberrations are likely to arise during genomic reprogramming in mammalian preimplantation embryos, when their epigenomes are most vulnerable. However, this process is only partially understood because of the experimental inaccessibility of early-stage embryos. Here, we introduce a methodologic advance, probing single cells for various DNA-methylation errors at multiple loci, to reveal failed maintenance of epigenetic mark results in chimeric mice, which display unpredictable phenotypes leading to developmental arrest. Yet we show that mouse pronuclear transfer can be used to ameliorate such reprogramming defects. This study not only details the epigenetic reprogramming dynamics in early mammalian embryos but also suggests diagnostic and potential future therapeutic applications.

View details for DOI 10.1126/science.1240617

View details for Web of Science ID 000323933100044

View details for PubMedID 24009393
Genetic measurement of memory B-cell recall using antibody repertoire sequencing PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Vollmers, C., Sit, R. V., Weinstein, J. A., Dekker, C. L., Quake, S. R. 2013; 110 (33): 13463-13468
Abstract

Annual influenza vaccinations aim to protect against seasonal infections, and vaccine strain compositions are updated every year. This protection is based on antibodies that are produced by either newly activated or memory B cells recalled from previous encounters with influenza vaccination or infection. The extent to which the B-cell repertoire responds to vaccination and recalls antibodies has so far not been analyzed at a genetic level-which is to say, at the level of antibody sequences. Here, we developed a consensus read sequencing approach that incorporates unique barcode labels on each starting RNA molecule. These labels allow one to combine multiple sequencing reads covering the same RNA molecule to reduce the error rate to a desired level, and they also enable accurate quantification of RNA and isotype levels. We validated this approach and analyzed the differential response of the antibody repertoire to live-attenuated or trivalent-inactivated influenza vaccination. Additionally, we analyzed the antibody repertoire in response to repeated yearly vaccinations with trivalent-inactivated influenza vaccination. We found antibody sequences that were present in both years, providing a direct genetic measurement of B-cell recall.

View details for DOI 10.1073/pnas.1312146110

View details for Web of Science ID 000323069200060

View details for PubMedID 23898164

View details for PubMedCentralID PMC3746854
Identification of a colonial chordate histocompatibility gene. Science Voskoboynik, A., Newman, A. M., Corey, D. M., Sahoo, D., Pushkarev, D., Neff, N. F., Passarelli, B., Koh, W., Ishizuka, K. J., Palmeri, K. J., Dimov, I. K., Keasar, C., Fan, H. C., Mantalas, G. L., Sinha, R., Penland, L., Quake, S. R., Weissman, I. L. 2013; 341 (6144): 384-387
Abstract

Histocompatibility is the basis by which multicellular organisms of the same species distinguish self from nonself. Relatively little is known about the mechanisms underlying histocompatibility reactions in lower organisms. Botryllus schlosseri is a colonial urochordate, a sister group of vertebrates, that exhibits a genetically determined natural transplantation reaction, whereby self-recognition between colonies leads to formation of parabionts with a common vasculature, whereas rejection occurs between incompatible colonies. Using genetically defined lines, whole-transcriptome sequencing, and genomics, we identified a single gene that encodes self-nonself and determines "graft" outcomes in this organism. This gene is significantly up-regulated in colonies poised to undergo fusion and/or rejection, is highly expressed in the vasculature, and is functionally linked to histocompatibility outcomes. These findings establish a platform for advancing the science of allorecognition.

View details for DOI 10.1126/science.1238036

View details for PubMedID 23888037
Identification of a colonial chordate histocompatibility gene. Science Voskoboynik, A., Newman, A. M., Corey, D. M., Sahoo, D., Pushkarev, D., Neff, N. F., Passarelli, B., Koh, W., Ishizuka, K. J., Palmeri, K. J., Dimov, I. K., Keasar, C., Fan, H. C., Mantalas, G. L., Sinha, R., Penland, L., Quake, S. R., Weissman, I. L. 2013; 341 (6144): 384-387
Abstract

Histocompatibility is the basis by which multicellular organisms of the same species distinguish self from nonself. Relatively little is known about the mechanisms underlying histocompatibility reactions in lower organisms. Botryllus schlosseri is a colonial urochordate, a sister group of vertebrates, that exhibits a genetically determined natural transplantation reaction, whereby self-recognition between colonies leads to formation of parabionts with a common vasculature, whereas rejection occurs between incompatible colonies. Using genetically defined lines, whole-transcriptome sequencing, and genomics, we identified a single gene that encodes self-nonself and determines "graft" outcomes in this organism. This gene is significantly up-regulated in colonies poised to undergo fusion and/or rejection, is highly expressed in the vasculature, and is functionally linked to histocompatibility outcomes. These findings establish a platform for advancing the science of allorecognition.

View details for DOI 10.1126/science.1238036

View details for PubMedID 23888037
A Microfluidic Device for Preparing Next Generation DNA Sequencing Libraries and for Automating Other Laboratory Protocols That Require One or More Column Chromatography Steps PLOS ONE Tan, S. J., Phan, H., Gerry, B. M., Kuhn, A., Hong, L. Z., Ong, Y. M., Poon, P. S., Unger, M. A., Jones, R. C., Quake, S. R., Burkholder, W. F. 2013; 8 (7)
Abstract

Library preparation for next-generation DNA sequencing (NGS) remains a key bottleneck in the sequencing process which can be relieved through improved automation and miniaturization. We describe a microfluidic device for automating laboratory protocols that require one or more column chromatography steps and demonstrate its utility for preparing Next Generation sequencing libraries for the Illumina and Ion Torrent platforms. Sixteen different libraries can be generated simultaneously with significantly reduced reagent cost and hands-on time compared to manual library preparation. Using an appropriate column matrix and buffers, size selection can be performed on-chip following end-repair, dA tailing, and linker ligation, so that the libraries eluted from the chip are ready for sequencing. The core architecture of the device ensures uniform, reproducible column packing without user supervision and accommodates multiple routine protocol steps in any sequence, such as reagent mixing and incubation; column packing, loading, washing, elution, and regeneration; capture of eluted material for use as a substrate in a later step of the protocol; and removal of one column matrix so that two or more column matrices with different functional properties can be used in the same protocol. The microfluidic device is mounted on a plastic carrier so that reagents and products can be aliquoted and recovered using standard pipettors and liquid handling robots. The carrier-mounted device is operated using a benchtop controller that seals and operates the device with programmable temperature control, eliminating any requirement for the user to manually attach tubing or connectors. In addition to NGS library preparation, the device and controller are suitable for automating other time-consuming and error-prone laboratory protocols requiring column chromatography steps, such as chromatin immunoprecipitation.

View details for DOI 10.1371/journal.pone.0064084

View details for Web of Science ID 000322167900001

View details for PubMedCentralID PMC3722208
Correlation of Gene Expression and Genome Mutation in Single B-Cells PLOS ONE Weinstein, J. A., Zeng, X., Chien, Y., Quake, S. R. 2013; 8 (6)
Abstract

High-throughput measurement of gene-expression and immune receptor repertoires have recently become powerful tools in the study of adaptive immune response. However, despite their now-widespread use, both tend to discard cell identity by treating cell populations in bulk, and therefore lose the correlation between genetic variability and gene-expression at the single cell level. In order to recover this information, we developed a method to simultaneously measure gene expression profiles and genome mutations in single cells. We applied this method by quantifying the relationships between gene expression and antibody mutation in ensembles of individual B-cells from immunized mice. The results reveal correlations reflecting the manner in which information propagates between a B-cell's antigen receptors, its gene expression, and its mutagenic machinery, and demonstrate the power of this approach to illuminate both heterogeneity and physiology in cell populations.

View details for DOI 10.1371/journal.pone.0067624

View details for Web of Science ID 000321148400117

View details for PubMedID 23840752

View details for PubMedCentralID PMC3695916
Single-cell and metagenomic analyses indicate a fermentative and saccharolytic lifestyle for members of the OP9 lineage NATURE COMMUNICATIONS Dodsworth, J. A., Blainey, P. C., Murugapiran, S. K., Swingley, W. D., Ross, C. A., Tringe, S. G., Chain, P. S., Scholz, M. B., Lo, C., Raymond, J., Quake, S. R., Hedlund, B. P. 2013; 4
Abstract

OP9 is a yet-uncultivated bacterial lineage found in geothermal systems, petroleum reservoirs, anaerobic digesters and wastewater treatment facilities. Here we use single-cell and metagenome sequencing to obtain two distinct, nearly complete OP9 genomes, one constructed from single cells sorted from hot spring sediments and the other derived from binned metagenomic contigs from an in situ-enriched cellulolytic, thermophilic community. Phylogenomic analyses support the designation of OP9 as a candidate phylum for which we propose the name 'Atribacteria'. Although a plurality of predicted proteins is most similar to those from Firmicutes, the presence of key genes suggests a diderm cell envelope. Metabolic reconstruction from the core genome suggests an anaerobic lifestyle based on sugar fermentation by Embden-Meyerhof glycolysis with production of hydrogen, acetate and ethanol. Putative glycohydrolases and an endoglucanase may enable catabolism of (hemi)cellulose in thermal environments. This study lays a foundation for understanding the physiology and ecological role of the 'Atribacteria'.

View details for DOI 10.1038/ncomms2884

View details for Web of Science ID 000320589900050

View details for PubMedID 23673639

View details for PubMedCentralID PMC3878185
Lineage structure of the human antibody repertoire in response to influenza vaccination. Science translational medicine Jiang, N., He, J., Weinstein, J. A., Penland, L., Sasaki, S., He, X., Dekker, C. L., Zheng, N., Huang, M., Sullivan, M., Wilson, P. C., Greenberg, H. B., Davis, M. M., Fisher, D. S., Quake, S. R. 2013; 5 (171): 171ra19-?
Abstract

The human antibody repertoire is one of the most important defenses against infectious disease, and the development of vaccines has enabled the conferral of targeted protection to specific pathogens. However, there are many challenges to measuring and analyzing the immunoglobulin sequence repertoire, including that each B cell's genome encodes a distinct antibody sequence, that the antibody repertoire changes over time, and the high similarity between antibody sequences. We have addressed these challenges by using high-throughput long read sequencing to perform immunogenomic characterization of expressed human antibody repertoires in the context of influenza vaccination. Informatic analysis of 5 million antibody heavy chain sequences from healthy individuals allowed us to perform global characterizations of isotype distributions, determine the lineage structure of the repertoire, and measure age- and antigen-related mutational activity. Our analysis of the clonal structure and mutational distribution of individuals' repertoires shows that elderly subjects have a decreased number of lineages but an increased prevaccination mutation load in their repertoire and that some of these subjects have an oligoclonal character to their repertoire in which the diversity of the lineages is greatly reduced relative to younger subjects. We have thus shown that global analysis of the immune system's clonal structure provides direct insight into the effects of vaccination and provides a detailed molecular portrait of age-related effects.

View details for DOI 10.1126/scitranslmed.3004794

View details for PubMedID 23390249
Lineage Structure of the Human Antibody Repertoire in Response to Influenza Vaccination SCIENCE TRANSLATIONAL MEDICINE Jiang, N., He, J., Weinstein, J. A., Penland, L., Sasaki, S., He, X., Dekker, C. L., Zheng, N., Huang, M., Sullivan, M., Wilson, P. C., Greenberg, H. B., Davis, M. M., Fisher, D. S., Quake, S. R. 2013; 5 (171)
Abstract

The human antibody repertoire is one of the most important defenses against infectious disease, and the development of vaccines has enabled the conferral of targeted protection to specific pathogens. However, there are many challenges to measuring and analyzing the immunoglobulin sequence repertoire, including that each B cell's genome encodes a distinct antibody sequence, that the antibody repertoire changes over time, and the high similarity between antibody sequences. We have addressed these challenges by using high-throughput long read sequencing to perform immunogenomic characterization of expressed human antibody repertoires in the context of influenza vaccination. Informatic analysis of 5 million antibody heavy chain sequences from healthy individuals allowed us to perform global characterizations of isotype distributions, determine the lineage structure of the repertoire, and measure age- and antigen-related mutational activity. Our analysis of the clonal structure and mutational distribution of individuals' repertoires shows that elderly subjects have a decreased number of lineages but an increased prevaccination mutation load in their repertoire and that some of these subjects have an oligoclonal character to their repertoire in which the diversity of the lineages is greatly reduced relative to younger subjects. We have thus shown that global analysis of the immune system's clonal structure provides direct insight into the effects of vaccination and provides a detailed molecular portrait of age-related effects.

View details for Web of Science ID 000314810000008

View details for PubMedID 23390249
Migration of Cells in a Social Context 57th Annual Meeting of the Biophysical-Society Vedel, S., Tay, S., Johnston, D. M., Bruus, H., Quake, S. R. CELL PRESS. 2013: 147A?147A
View details for Web of Science ID 000316074301249
Simultaneous Measurement of Amyloid Fibril Formation by Dynamic Light Scattering and Fluorescence Reveals Complex Aggregation Kinetics PLOS ONE Streets, A. M., Sourigues, Y., Kopito, R. R., Melki, R., Quake, S. R. 2013; 8 (1)
Abstract

An apparatus that combines dynamic light scattering and Thioflavin T fluorescence detection is used to simultaneously probe fibril formation in polyglutamine peptides, the aggregating subunit associated with Huntington's disease, in vitro. Huntington's disease is a neurodegenerative disorder in a class of human pathologies that includes Alzheimer's and Parkinson's disease. These pathologies are all related by the propensity of their associated protein or polypeptide to form insoluble, ?-sheet rich, amyloid fibrils. Despite the wide range of amino acid sequence in the aggregation prone polypeptides associated with these diseases, the resulting amyloids display strikingly similar physical structure, an observation which suggests a physical basis for amyloid fibril formation. Thioflavin T fluorescence reports ?-sheet fibril content while dynamic light scattering measures particle size distributions. The combined techniques allow elucidation of complex aggregation kinetics and are used to reveal multiple stages of amyloid fibril formation.

View details for DOI 10.1371/journal.pone.0054541

View details for Web of Science ID 000313738900076

View details for PubMedID 23349924

View details for PubMedCentralID PMC3547910
Proteome-wide protein interaction measurements of bacterial proteins of unknown function PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Meier, M., Sit, R. V., Quake, S. R. 2013; 110 (2): 477-482
Abstract

Despite the enormous proliferation of bacterial genome data, surprisingly persistent collections of bacterial proteins have resisted functional annotation. In a typical genome, roughly 30% of genes have no assigned function. Many of these proteins are conserved across a large number of bacterial genomes. To assign a putative function to these conserved proteins of unknown function, we created a physical interaction map by measuring biophysical interaction of these proteins. Binary protein--protein interactions in the model organism Streptococcus pneumoniae (TIGR4) are measured with a microfluidic high-throughput assay technology. In some cases, informatic analysis was used to restrict the space of potential binding partners. In other cases, we performed in vitro proteome-wide interaction screens. We were able to assign putative functions to 50 conserved proteins of unknown function that we studied with this approach.

View details for DOI 10.1073/pnas.1210634110

View details for Web of Science ID 000313906600027

View details for PubMedID 23267104

View details for PubMedCentralID PMC3545810
Migration of cells in a social context PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Vedel, S., Tay, S., Johnston, D. M., Bruus, H., Quake, S. R. 2013; 110 (1): 129-134
Abstract

In multicellular organisms and complex ecosystems, cells migrate in a social context. Whereas this is essential for the basic processes of life, the influence of neighboring cells on the individual remains poorly understood. Previous work on isolated cells has observed a stereotypical migratory behavior characterized by short-time directional persistence with long-time random movement. We discovered a much richer dynamic in the social context, with significant variations in directionality, displacement, and speed, which are all modulated by local cell density. We developed a mathematical model based on the experimentally identified "cellular traffic rules" and basic physics that revealed that these emergent behaviors are caused by the interplay of single-cell properties and intercellular interactions, the latter being dominated by a pseudopod formation bias mediated by secreted chemicals and pseudopod collapse following collisions. The model demonstrates how aspects of complex biology can be explained by simple rules of physics and constitutes a rapid test bed for future studies of collective migration of individual cells.

View details for DOI 10.1073/pnas.1204291110

View details for Web of Science ID 000313630300038

View details for PubMedID 23251032

View details for PubMedCentralID PMC3538227
Correlation of gene expression and genome mutation in single B-cells. PloS one Weinstein, J. A., Zeng, X., Chien, Y., Quake, S. R. 2013; 8 (6)
Abstract

High-throughput measurement of gene-expression and immune receptor repertoires have recently become powerful tools in the study of adaptive immune response. However, despite their now-widespread use, both tend to discard cell identity by treating cell populations in bulk, and therefore lose the correlation between genetic variability and gene-expression at the single cell level. In order to recover this information, we developed a method to simultaneously measure gene expression profiles and genome mutations in single cells. We applied this method by quantifying the relationships between gene expression and antibody mutation in ensembles of individual B-cells from immunized mice. The results reveal correlations reflecting the manner in which information propagates between a B-cell's antigen receptors, its gene expression, and its mutagenic machinery, and demonstrate the power of this approach to illuminate both heterogeneity and physiology in cell populations.

View details for DOI 10.1371/journal.pone.0067624

View details for PubMedID 23840752

View details for PubMedCentralID PMC3695916
Optofluidic Cell Selection from Complex Microbial Communities for Single-Genome Analysis MICROBIAL METAGENOMICS, METATRANSCRIPTOMICS, AND METAPROTEOMICS Landry, Z. C., Giovanonni, S. J., Quake, S. R., Blainey, P. C. 2013; 531: 61-90
Abstract

Genetic analysis of single cells is emerging as a powerful approach for studies of heterogeneous cell populations. Indeed, the notion of homogeneous cell populations is receding as approaches to resolve genetic and phenotypic variation between single cells are applied throughout the life sciences. A key step in single-cell genomic analysis today is the physical isolation of individual cells from heterogeneous populations, particularly microbial populations, which often exhibit high diversity. Here, we detail the construction and use of instrumentation for optical trapping inside microfluidic devices to select individual cells for analysis by methods including nucleic acid sequencing. This approach has unique advantages for analyses of rare community members, cells with irregular morphologies, small quantity samples, and studies that employ advanced optical microscopy.

View details for DOI 10.1016/B978-0-12-407863-5.00004-6

View details for Web of Science ID 000327361000005

View details for PubMedID 24060116

View details for PubMedCentralID PMC3886641
Single-cell and metagenomic analyses indicate a fermentative and saccharolytic lifestyle for members of the OP9 lineage. Nat Commun. Dodsworth, J. A., Blainey, P. C., Murugapiran, S. K., Swingley, W. D., Ross, C. A., Tringe, S. G., Quake, S. 2013; 4: 1854
View details for DOI 10.1038/ncomms2884.
The genome sequence of the colonial chordate, Botryllus schlosseri. eLife Voskoboynik, A., Neff, N. F., Sahoo, D., Newman, A. M., Pushkarev, D., Koh, W., Passarelli, B., Fan, H. C., Mantalas, G. L., Palmeri, K. J., Ishizuka, K. J., Gissi, C., Griggio, F., Ben-Shlomo, R., Corey, D. M., Penland, L., White, R. A., Weissman, I. L., Quake, S. R. 2013; 2
Abstract

Botryllus schlosseri is a colonial urochordate that follows the chordate plan of development following sexual reproduction, but invokes a stem cell-mediated budding program during subsequent rounds of asexual reproduction. As urochordates are considered to be the closest living invertebrate relatives of vertebrates, they are ideal subjects for whole genome sequence analyses. Using a novel method for high-throughput sequencing of eukaryotic genomes, we sequenced and assembled 580 Mbp of the B. schlosseri genome. The genome assembly is comprised of nearly 14,000 intron-containing predicted genes, and 13,500 intron-less predicted genes, 40% of which could be confidently parceled into 13 (of 16 haploid) chromosomes. A comparison of homologous genes between B. schlosseri and other diverse taxonomic groups revealed genomic events underlying the evolution of vertebrates and lymphoid-mediated immunity. The B. schlosseri genome is a community resource for studying alternative modes of reproduction, natural transplantation reactions, and stem cell-mediated regeneration. DOI:http://dx.doi.org/10.7554/eLife.00569.001.

View details for DOI 10.7554/eLife.00569

View details for PubMedID 23840927

View details for PubMedCentralID PMC3699833
The genome sequence of the colonial chordate Botryllus schlosseri. Elife. Voskoboynik, A., Neff, N. F., Sahoo, D., Newman, A. M., Pushkarev, D., Koh, W., Quake, S. 2013; 2: e00569
View details for DOI 10.7554/eLife. 00569.
Genetic measurement of memory B-cell recall using antibody repertoire sequencing. Vollmers, C., Sit, R. V., Weinstein, J. A., Dekker, C. L., Quake, S, R. 2013
View details for DOI 10.1073/pnas. 1312146110
Microfluidic serial digital to analog pressure converter for arbitrary pressure generation and contamination-free flow control LAB ON A CHIP Yu, F., Horowitz, M. A., Quake, S. R. 2013; 13 (10): 1911-1918
Abstract

Multilayer microfluidics based on PDMS (polydimethylsiloxane) soft lithography have offered parallelism and integration for biological and chemical sciences, where reduction in reaction volume and consistency of controlled variables across experiments translate into reduced cost, increased quantity and quality of data. One issue with push up or push down microfluidic control concept is the inability to provide multiple control pressures without adding more complex and expensive external pressure controls. We present here a microfluidic serial DAC (Digital to Analog Converter) that can be integrated with any PDMS device to expand the device's functionality by effectively adding an on-chip pressure regulator. The microfluidic serial DAC can be used with any incompressible fluids and operates in a similar fashion compared to an electronic serial DAC. It can be easily incorporated into any existing multilayer microfluidic devices, and the output pressure that the device generates could be held for extensive times. We explore in this paper various factors that affect resolution, speed, and linearity of the DAC output. As an application, we demonstrate microfluidic DAC's ability for on-chip manipulation of flow resistance when integrated with a simple flow network. In addition, we illustrate an added advantage of using the microfluidic serial DAC in preventing back flow and possible contamination.

View details for DOI 10.1039/c3lc41394b

View details for Web of Science ID 000317937300011

View details for PubMedID 23529280
Simultaneous measurement of amyloid fibril formation by dynamic light scattering and fluorescence reveals complex aggregation kinetics. PloS one Streets, A. M., Sourigues, Y., Kopito, R. R., Melki, R., Quake, S. R. 2013; 8 (1)
Abstract

An apparatus that combines dynamic light scattering and Thioflavin T fluorescence detection is used to simultaneously probe fibril formation in polyglutamine peptides, the aggregating subunit associated with Huntington's disease, in vitro. Huntington's disease is a neurodegenerative disorder in a class of human pathologies that includes Alzheimer's and Parkinson's disease. These pathologies are all related by the propensity of their associated protein or polypeptide to form insoluble, ?-sheet rich, amyloid fibrils. Despite the wide range of amino acid sequence in the aggregation prone polypeptides associated with these diseases, the resulting amyloids display strikingly similar physical structure, an observation which suggests a physical basis for amyloid fibril formation. Thioflavin T fluorescence reports ?-sheet fibril content while dynamic light scattering measures particle size distributions. The combined techniques allow elucidation of complex aggregation kinetics and are used to reveal multiple stages of amyloid fibril formation.

View details for DOI 10.1371/journal.pone.0054541

View details for PubMedID 23349924

View details for PubMedCentralID PMC3547910
The genome sequence of the colonial chordate, Botryllus schlosseri. eLife Voskoboynik, A., Neff, N. F., Sahoo, D., Newman, A. M., Pushkarev, D., Koh, W., Passarelli, B., Fan, H. C., Mantalas, G. L., Palmeri, K. J., Ishizuka, K. J., Gissi, C., Griggio, F., Ben-Shlomo, R., Corey, D. M., Penland, L., White, R. A., Weissman, I. L., Quake, S. R. 2013; 2
Abstract

Botryllus schlosseri is a colonial urochordate that follows the chordate plan of development following sexual reproduction, but invokes a stem cell-mediated budding program during subsequent rounds of asexual reproduction. As urochordates are considered to be the closest living invertebrate relatives of vertebrates, they are ideal subjects for whole genome sequence analyses. Using a novel method for high-throughput sequencing of eukaryotic genomes, we sequenced and assembled 580 Mbp of the B. schlosseri genome. The genome assembly is comprised of nearly 14,000 intron-containing predicted genes, and 13,500 intron-less predicted genes, 40% of which could be confidently parceled into 13 (of 16 haploid) chromosomes. A comparison of homologous genes between B. schlosseri and other diverse taxonomic groups revealed genomic events underlying the evolution of vertebrates and lymphoid-mediated immunity. The B. schlosseri genome is a community resource for studying alternative modes of reproduction, natural transplantation reactions, and stem cell-mediated regeneration. DOI:http://dx.doi.org/10.7554/eLife.00569.001.

View details for DOI 10.7554/eLife.00569

View details for PubMedID 23840927
Systematic reconstruction of RNA functional motifs with high-throughput microfluidics NATURE METHODS Martin, L., Meier, M., Lyons, S. M., Sit, R. V., Marzluff, W. F., Quake, S. R., Chang, H. Y. 2012; 9 (12): 1192-U85
Abstract

We present RNA-mechanically induced trapping of molecular interactions (RNA-MITOMI), a microfluidic platform that allows integrated synthesis and functional assays for programmable RNA libraries. The interaction of a comprehensive library of RNA mutants with stem-loop-binding protein precisely defined the RNA structural and sequence features that govern affinity. The functional motif reconstructed in a single experiment on our platform uncovers new binding specificities and enriches interpretation of phylogenetic data.

View details for DOI 10.1038/NMETH.2225

View details for PubMedID 23142872
A Single-Cell Genome for Thiovulum sp. APPLIED AND ENVIRONMENTAL MICROBIOLOGY Marshall, I. P., Blainey, P. C., Spormann, A. M., Quake, S. R. 2012; 78 (24): 8555-8563
Abstract

We determined a significant fraction of the genome sequence of a representative of Thiovulum, the uncultivated genus of colorless sulfur Epsilonproteobacteria, by analyzing the genome sequences of four individual cells collected from phototrophic mats from Elkhorn Slough, California. These cells were isolated utilizing a microfluidic laser-tweezing system, and their genomes were amplified by multiple-displacement amplification prior to sequencing. Thiovulum is a gradient bacterium found at oxic-anoxic marine interfaces and noted for its distinctive morphology and rapid swimming motility. The genomic sequences of the four individual cells were assembled into a composite genome consisting of 221 contigs covering 2.083 Mb including 2,162 genes. This single-cell genome represents a genomic view of the physiological capabilities of isolated Thiovulum cells. Thiovulum is the second-fastest bacterium ever observed, swimming at 615 ?m/s, and this genome shows that this rapid swimming motility is a result of a standard flagellar machinery that has been extensively characterized in other bacteria. This suggests that standard flagella are capable of propelling bacterial cells at speeds much faster than typically thought. Analysis of the genome suggests that naturally occurring Thiovulum populations are more diverse than previously recognized and that studies performed in the past probably address a wide range of unrecognized genotypic and phenotypic diversities of Thiovulum. The genome presented in this article provides a basis for future isolation-independent studies of Thiovulum, where single-cell and metagenomic tools can be used to differentiate between different Thiovulum genotypes.

View details for DOI 10.1128/AEM.02314-12

View details for Web of Science ID 000311213200007

View details for PubMedID 23023751

View details for PubMedCentralID PMC3502928
High-Performance Binary Protein Interaction Screening in a Microfluidic Format ANALYTICAL CHEMISTRY Meier, M., Sit, R., Pan, W., Quake, S. R. 2012; 84 (21): 9572-9578
Abstract

The standard procedure to increase microfluidic chip performance is to grow the number of parallel test systems on the chip. This process is accompanied by miniaturizing biochemical workflows and micromechanical elements, which is often a major challenge for both engineering fields. In this work, we show that it is possible to substantially increase the runtime performance of a microfluidic affinity assay for protein interactions by simultaneously engineering fluid logics and assay chemistry. For this, synergistic effects between the micro- and chemical architecture of the chip are exploited. The presented strategy of reducing the runtime rather than size and volume of the mechanical elements and biological reagent compartments will, in general, be of importance for future analytical test systems on microfluidic chips to overcome performance barriers.

View details for DOI 10.1021/ac302436y

View details for Web of Science ID 000310664600097

View details for PubMedID 23051662

View details for PubMedCentralID PMC3533494
Detecting Prenatal Microdeletions Non-Invasively: Case of a 22q11.2 Deletion Annual Meeting of the Association-for-Molecular-Pathology Gu, W., Fan, H., Blumenfeld, Y., El-Sayed, Y., Quake, S. ELSEVIER SCIENCE INC. 2012: 638?38
View details for Web of Science ID 000310178600021
Quantitative Analysis of the Human Airway Microbial Ecology Reveals a Pervasive Signature for Cystic Fibrosis SCIENCE TRANSLATIONAL MEDICINE Blainey, P. C., Milla, C. E., Cornfield, D. N., Quake, S. R. 2012; 4 (153)
Abstract

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the gene encoding the CF transmembrane conductance regulator. Disruption of electrolyte homeostasis at mucosal surfaces leads to severe lung, pancreatic, intestinal, hepatic, and reproductive abnormalities. Loss of lung function as a result of chronic lung disease is the primary cause of death from CF. Using high-throughput sequencing to survey microbes in the sputum of 16 CF patients and 9 control individuals, we identified diverse microbial communities in the healthy samples, contravening conventional wisdom that healthy airways are not significantly colonized. Comparing these communities with those from the CF patients revealed significant differences in microbial ecology, including differential representation of uncultivated phylotypes. Despite patient-specific differences, our analysis revealed a focal microbial profile characteristic of CF. The profile differentiated case and control groups even when classically recognized CF pathogens were excluded. As a control, lung explant tissues were also processed from a group of patients with pulmonary disease. The findings in lung tissue corroborated the presence of taxa identified in the sputum samples. Comparing the sequencing results with clinical data indicated that diminished microbial diversity is associated with severity of pulmonary inflammation within our adult CF cohort.

View details for DOI 10.1126/scitranslmed.3004458

View details for Web of Science ID 000309525600003

View details for PubMedID 23019655
Clonal Evolution of Preleukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia SCIENCE TRANSLATIONAL MEDICINE Jan, M., Snyder, T. M., Corces-Zimmerman, M. R., Vyas, P., Weissman, I. L., Quake, S. R., Majeti, R. 2012; 4 (149)
Abstract

Given that most bone marrow cells are short-lived, the accumulation of multiple leukemogenic mutations in a single clonal lineage has been difficult to explain. We propose that serial acquisition of mutations occurs in self-renewing hematopoietic stem cells (HSCs). We investigated this model through genomic analysis of HSCs from six patients with de novo acute myeloid leukemia (AML). Using exome sequencing, we identified mutations present in individual AML patients harboring the FLT3-ITD (internal tandem duplication) mutation. We then screened the residual HSCs and detected some of these mutations including mutations in the NPM1, TET2, and SMC1A genes. Finally, through single-cell analysis, we determined that a clonal progression of multiple mutations occurred in the HSCs of some AML patients. These preleukemic HSCs suggest the clonal evolution of AML genomes from founder mutations, revealing a potential mechanism contributing to relapse. Such preleukemic HSCs may constitute a cellular reservoir that should be targeted therapeutically for more durable remissions.

View details for DOI 10.1126/scitranslmed.3004315

View details for Web of Science ID 000308491600005

View details for PubMedID 22932223
Genome-wide Single-Cell Analysis of Recombination Activity and De Novo Mutation Rates in Human Sperm CELL Wang, J., Fan, H. C., Behr, B., Quake, S. R. 2012; 150 (2): 402-412
Abstract

Meiotic recombination and de novo mutation are the two main contributions toward gamete genome diversity, and many questions remain about how an individual human's genome is edited by these two processes. Here, we describe a high-throughput method for single-cell whole-genome analysis that was used to measure the genomic diversity in one individual's gamete genomes. A microfluidic system was used for highly parallel sample processing and to minimize nonspecific amplification. High-density genotyping results from 91 single cells were used to create a personal recombination map, which was consistent with population-wide data at low resolution but revealed significant differences from pedigree data at higher resolution. We used the data to test for meiotic drive and found evidence for gene conversion. High-throughput sequencing on 31 single cells was used to measure the frequency of large-scale genome instability, and deeper sequencing of eight single cells revealed de novo mutation rates with distinct characteristics.

View details for DOI 10.1016/j.cell.2012.06.030

View details for Web of Science ID 000306595700018

View details for PubMedID 22817899

View details for PubMedCentralID PMC3525523
Non-invasive prenatal measurement of the fetal genome NATURE Fan, H. C., Gu, W., Wang, J., Blumenfeld, Y. J., El-Sayed, Y. Y., Quake, S. R. 2012; 487 (7407): 320-?
Abstract

The vast majority of prenatal genetic testing requires invasive sampling. However, this poses a risk to the fetus, so one must make a decision that weighs the desire for genetic information against the risk of an adverse outcome due to hazards of the testing process. These issues are not required to be coupled, and it would be desirable to discover genetic information about the fetus without incurring a health risk. Here we demonstrate that it is possible to non-invasively sequence the entire prenatal genome. Our results show that molecular counting of parental haplotypes in maternal plasma by shotgun sequencing of maternal plasma DNA allows the inherited fetal genome to be deciphered non-invasively. We also applied the counting principle directly to each allele in the fetal exome by performing exome capture on maternal plasma DNA before shotgun sequencing. This approach enables non-invasive exome screening of clinically relevant and deleterious alleles that were paternally inherited or had arisen as de novo germline mutations, and complements the haplotype counting approach to provide a comprehensive view of the fetal genome. Non-invasive determination of the fetal genome may ultimately facilitate the diagnosis of all inherited and de novo genetic disease.

View details for DOI 10.1038/nature11251

View details for Web of Science ID 000306506500033

View details for PubMedID 22763444

View details for PubMedCentralID PMC3561905
Time-lapse Fluorescence Imaging of Arabidopsis Root Growth with Rapid Manipulation of The Root Environment Using The RootChip JOVE-JOURNAL OF VISUALIZED EXPERIMENTS Grossmann, G., Meier, M., Cartwright, H. N., Sosso, D., Quake, S. R., Ehrhardt, D. W., Frommer, W. B. 2012
Abstract

The root functions as the physical anchor of the plant and is the organ responsible for uptake of water and mineral nutrients such as nitrogen, phosphorus, sulfate and trace elements that plants acquire from the soil. If we want to develop sustainable approaches to producing high crop yield, we need to better understand how the root develops, takes up a wide spectrum of nutrients, and interacts with symbiotic and pathogenic organisms. To accomplish these goals, we need to be able to explore roots in microscopic detail over time periods ranging from minutes to days. We developed the RootChip, a polydimethylsiloxane (PDMS)- based microfluidic device, which allows us to grow and image roots from Arabidopsis seedlings while avoiding any physical stress to roots during preparation for imaging(1) (Figure 1). The device contains a bifurcated channel structure featuring micromechanical valves to guide the fluid flow from solution inlets to each of the eight observation chambers(2). This perfusion system allows the root microenvironment to be controlled and modified with precision and speed. The volume of the chambers is approximately 400 nl, thus requiring only minimal amounts of test solution. Here we provide a detailed protocol for studying root biology on the RootChip using imaging-based approaches with real time resolution. Roots can be analyzed over several days using time lapse microscopy. Roots can be perfused with nutrient solutions or inhibitors, and up to eight seedlings can be analyzed in parallel. This system has the potential for a wide range of applications, including analysis of root growth in the presence or absence of chemicals, fluorescence-based analysis of gene expression, and the analysis of biosensors, e.g. FRET nanosensors(3).

View details for DOI 10.3791/4290

View details for Web of Science ID 000209223200051

View details for PubMedCentralID PMC3471268
Single-cell sequencing provides clues about the host interactions of segmented filamentous bacteria (SFB) GENOME RESEARCH Pamp, S. J., Harrington, E. D., Quake, S. R., Relman, D. A., Blainey, P. C. 2012; 22 (6): 1107-1119
Abstract

Segmented filamentous bacteria (SFB) are host-specific intestinal symbionts that comprise a distinct clade within the Clostridiaceae, designated Candidatus Arthromitus. SFB display a unique life cycle within the host, involving differentiation into multiple cell types. The latter include filaments that attach intimately to intestinal epithelial cells, and from which "holdfasts" and spores develop. SFB induce a multifaceted immune response, leading to host protection from intestinal pathogens. Cultivation resistance has hindered characterization of these enigmatic bacteria. In the present study, we isolated five SFB filaments from a mouse using a microfluidic device equipped with laser tweezers, generated genome sequences from each, and compared these sequences with each other, as well as to recently published SFB genome sequences. Based on the resulting analyses, SFB appear to be dependent on the host for a variety of essential nutrients. SFB have a relatively high abundance of predicted proteins devoted to cell cycle control and to envelope biogenesis, and have a group of SFB-specific autolysins and a dynamin-like protein. Among the five filament genomes, an average of 8.6% of predicted proteins were novel, including a family of secreted SFB-specific proteins. Four ADP-ribosyltransferase (ADPRT) sequence types, and a myosin-cross-reactive antigen (MCRA) protein were discovered; we hypothesize that they are involved in modulation of host responses. The presence of polymorphisms among mouse SFB genomes suggests the evolution of distinct SFB lineages. Overall, our results reveal several aspects of SFB adaptation to the mammalian intestinal tract.

View details for DOI 10.1101/gr.131482.111

View details for Web of Science ID 000304728100012

View details for PubMedID 22434425

View details for PubMedCentralID PMC3371716
Single Cell Profiling of Circulating Tumor Cells: Transcriptional Heterogeneity and Diversity from Breast Cancer Cell Lines PLOS ONE Powell, A. A., Talasaz, A. H., Zhang, H., Coram, M. A., Reddy, A., Deng, G., Telli, M. L., Advani, R. H., Carlson, R. W., Mollick, J. A., Sheth, S., Kurian, A. W., Ford, J. M., Stockdale, F. E., Quake, S. R., Pease, R. F., Mindrinos, M. N., Bhanot, G., Dairkee, S. H., Davis, R. W., Jeffrey, S. S. 2012; 7 (5)
Abstract

To improve cancer therapy, it is critical to target metastasizing cells. Circulating tumor cells (CTCs) are rare cells found in the blood of patients with solid tumors and may play a key role in cancer dissemination. Uncovering CTC phenotypes offers a potential avenue to inform treatment. However, CTC transcriptional profiling is limited by leukocyte contamination; an approach to surmount this problem is single cell analysis. Here we demonstrate feasibility of performing high dimensional single CTC profiling, providing early insight into CTC heterogeneity and allowing comparisons to breast cancer cell lines widely used for drug discovery.We purified CTCs using the MagSweeper, an immunomagnetic enrichment device that isolates live tumor cells from unfractionated blood. CTCs that met stringent criteria for further analysis were obtained from 70% (14/20) of primary and 70% (21/30) of metastatic breast cancer patients; none were captured from patients with non-epithelial cancer (n = 20) or healthy subjects (n = 25). Microfluidic-based single cell transcriptional profiling of 87 cancer-associated and reference genes showed heterogeneity among individual CTCs, separating them into two major subgroups, based on 31 highly expressed genes. In contrast, single cells from seven breast cancer cell lines were tightly clustered together by sample ID and ER status. CTC profiles were distinct from those of cancer cell lines, questioning the suitability of such lines for drug discovery efforts for late stage cancer therapy.For the first time, we directly measured high dimensional gene expression in individual CTCs without the common practice of pooling such cells. Elevated transcript levels of genes associated with metastasis NPTN, S100A4, S100A9, and with epithelial mesenchymal transition: VIM, TGF�1, ZEB2, FOXC1, CXCR4, were striking compared to cell lines. Our findings demonstrate that profiling CTCs on a cell-by-cell basis is possible and may facilitate the application of 'liquid biopsies' to better model drug discovery.

View details for DOI 10.1371/journal.pone.0033788

View details for PubMedID 22586443
High Throughput Sequencing of the Human Antibody Repertoire in Response to Influenza Vaccination 99th Annual Meeting of the American-Association-of-Immunologists Jiang, N., He, J., Weinstein, J., Penland, L., Saaki, S., He, X., Dekker, C., Wilson, P., Greenberg, H., Davis, M., Fisher, D., Quake, S. AMER ASSOC IMMUNOLOGISTS. 2012
View details for Web of Science ID 000304659700423
Identification of a cKit(+) Colonic Crypt Base Secretory Cell That Supports Lgr5(+) Stem Cells in Mice GASTROENTEROLOGY Rothenberg, M. E., Nusse, Y., Kalisky, T., Lee, J. J., Dalerba, P., Scheeren, F., Lobo, N., Kulkarni, S., Sim, S., Qian, D., Beachy, P. A., Pasricha, P. J., Quake, S. R., Clarke, M. F. 2012; 142 (5): 1195-?
Abstract

Paneth cells contribute to the small intestinal niche of Lgr5(+) stem cells. Although the colon also contains Lgr5(+) stem cells, it does not contain Paneth cells. We investigated the existence of colonic Paneth-like cells that have a distinct transcriptional signature and support Lgr5(+) stem cells.We used multicolor fluorescence-activated cell sorting to isolate different subregions of colon crypts, based on known markers, from dissociated colonic epithelium of mice. We performed multiplexed single-cell gene expression analysis with quantitative reverse transcriptase polymerase chain reaction followed by hierarchical clustering analysis to characterize distinct cell types. We used immunostaining and fluorescence-activated cell sorting analyses with in vivo administration of a Notch inhibitor and in vitro organoid cultures to characterize different cell types.Multicolor fluorescence-activated cell sorting could isolate distinct regions of colonic crypts. Four major epithelial subtypes or transcriptional states were revealed by gene expression analysis of selected populations of single cells. One of these, the goblet cells, contained a distinct cKit/CD117(+) crypt base subpopulation that expressed Dll1, Dll4, and epidermal growth factor, similar to Paneth cells, which were also marked by cKit. In the colon, cKit(+) goblet cells were interdigitated with Lgr5(+) stem cells. In vivo, this colonic cKit(+) population was regulated by Notch signaling; administration of a ?-secretase inhibitor to mice increased the number of cKit(+) cells. When isolated from mouse colon, cKit(+) cells promoted formation of organoids from Lgr5(+) stem cells, which expressed Kitl/stem cell factor, the ligand for cKit. When organoids were depleted of cKit(+) cells using a toxin-conjugated antibody, organoid formation decreased.cKit marks small intestinal Paneth cells and a subset of colonic goblet cells that are regulated by Notch signaling and support Lgr5(+) stem cells.

View details for DOI 10.1053/j.gastro.2012.02.006

View details for Web of Science ID 000303113600038

View details for PubMedID 22333952

View details for PubMedCentralID PMC3911891
Microfluidic single-cell real-time PCR for comparative analysis of gene expression patterns NATURE PROTOCOLS Sanchez-Freire, V., Ebert, A. D., Kalisky, T., Quake, S. R., Wu, J. C. 2012; 7 (5): 829-838
Abstract

Single-cell quantitative real-time PCR (qRT-PCR) combined with high-throughput arrays allows the analysis of gene expression profiles at a molecular level in approximately 11 h after cell sample collection. We present here a high-content microfluidic real-time platform as a powerful tool for comparatively investigating the regulation of developmental processes in single cells. This approach overcomes the limitations involving heterogeneous cell populations and sample amounts, and may shed light on differential regulation of gene expression in normal versus disease-related contexts. Furthermore, high-throughput single-cell qRT-PCR provides a standardized, comparative assay for in-depth analysis of the mechanisms underlying human pluripotent stem cell self-renewal and differentiation.

View details for DOI 10.1038/nprot.2012.021

View details for Web of Science ID 000303359300002

View details for PubMedID 22481529

View details for PubMedCentralID PMC3657501
Sizing Up Cell-Free DNA CLINICAL CHEMISTRY Quake, S. 2012; 58 (3): 489-490
View details for DOI 10.1373/clinchem.2011.174250

View details for Web of Science ID 000300934900003

View details for PubMedID 22235102
DNA Sequencing Clinical Applications of New DNA Sequencing Technologies CIRCULATION Dewey, F. E., Pan, S., Wheeler, M. T., Quake, S. R., Ashley, E. A. 2012; 125 (7): 931-944
View details for DOI 10.1161/CIRCULATIONAHA.110.972828

View details for Web of Science ID 000300949800019

View details for PubMedID 22354974

View details for PubMedCentralID PMC3364518
High throughput automated chromatin immunoprecipitation as a platform for drug screening and antibody validation LAB ON A CHIP Wu, A. R., Kawahara, T. L., Rapicavoli, N. A., van Riggelen, J., Shroff, E. H., Xu, L., Felsher, D. W., Chang, H. Y., Quake, S. R. 2012; 12 (12): 2190-2198
Abstract

Chromatin immunoprecipitation (ChIP) is an assay for interrogating protein-DNA interactions that is increasingly being used for drug target discovery and screening applications. Currently the complexity of the protocol and the amount of hands-on time required for this assay limits its use to low throughput applications; furthermore, variability in antibody quality poses an additional obstacle in scaling up ChIP for large scale screening purposes. To address these challenges, we report HTChIP, an automated microfluidic-based platform for performing high-throughput ChIP screening measurements of 16 different targets simultaneously, with potential for further scale-up. From chromatin to analyzable PCR results only takes one day using HTChIP, as compared to several days up to one week for conventional protocols. HTChIP can also be used to test multiple antibodies and select the best performer for downstream ChIP applications, saving time and reagent costs of unsuccessful ChIP assays as a result of poor antibody quality. We performed a series of characterization assays to demonstrate that HTChIP can rapidly and accurately evaluate the epigenetic states of a cell, and that it is sensitive enough to detect the changes in the epigenetic state induced by a cytokine stimulant over a fine temporal resolution. With these results, we believe that HTChIP can introduce large improvements in routine ChIP, antibody screening, and drug screening efficiency, and further facilitate the use of ChIP as a valuable tool for research and discovery.

View details for DOI 10.1039/c2lc21290k

View details for PubMedID 22566096
Microfluidic very lage scale integration (mVLSI) with integrated micromechnical valves Lab on a Chip Araci, I. E., Quake, Stephen, R. 2012; 12 (16): 2803-2806
View details for DOI 10.1029/c21c40258k
Microfluidic very large scale integration (mVLSI) with integrated micromechanical valves LAB ON A CHIP Araci, I. E., Quake, S. R. 2012; 12 (16): 2803-2806
Abstract

Microfluidic chips with a high density of control elements are required to improve device performance parameters, such as throughput, sensitivity and dynamic range. In order to realize robust and accessible high-density microfluidic chips, we have fabricated a monolithic PDMS valve architecture with three layers, replacing the commonly used two-layer design. The design is realized through multi-layer soft lithography techniques, making it low cost and easy to fabricate. By carefully determining the process conditions of PDMS, we have demonstrated that 8 � 8 and 6 � 6 ?m(2) valve sizes can be operated at around 180 and 280 kPa differential pressure, respectively. We have shown that these valves can be fabricated at densities approaching 1 million valves per cm(2), substantially exceeding the current state of the art of microfluidic large-scale integration (mLSI) (thousands of valves per cm(2)). Because the density increase is greater than two orders of magnitude, we describe this technology as microfluidic very large scale integration (mVLSI), analogous to its electronic counterpart. We have captured and tracked fluorescent beads, and changed the electrical resistance of a fluidic channel by using these miniaturized valves in two different experiments, demonstrating that the valves are leakproof. We have also demonstrated that these valves can be addressed through multiplexing.

View details for DOI 10.1039/c2lc40258k

View details for Web of Science ID 000306523800007

View details for PubMedID 22714259
Profiling maternal plasma cell-free RNA by RNA-sequencing: a comprehensive approach 32nd Annual Pregnancy Meeting of the Society-for-Maternal-Fetal-Medicine (SMFM) Koh, W., Fan, C., Blumenfeld, Y., Wong, R., El-Sayed, Y., Kogut, E., Quake, S. MOSBY-ELSEVIER. 2012: S324?S324
View details for Web of Science ID 000298889900725
Digital PCR provides absolute quantitation of viral load for an occult RNA virus JOURNAL OF VIROLOGICAL METHODS White, R. A., Quake, S. R., Curr, K. 2012; 179 (1): 45-50
Abstract

Using a multiplexed LNA-based Taqman assay, RT-digital PCR (RT-dPCR) was performed in a prefabricated microfluidic device that monitored absolute viral load in native and immortalized cell lines, overall precision of detection, and the absolute detection limit of an occult RNA virus GB Virus Type C (GBV-C). RT-dPCR had on average a 10% lower overall coefficient of variation (CV, a measurement of precision) for viral load testing than RT-qPCR and had a higher overall detection limit, able to quantify as low as three 5'-UTR molecules of GBV-C genome. Two commercial high-yield in vitro transcription kits (T7 Ribomax Express by Promega and Ampliscribe T7 Flash by Epicentre) were compared to amplify GBV-C RNA genome with T7-mediated amplification. The Ampliscribe T7 Flash outperformed the T7 Ribomax Express in yield of full-length GBV-C RNA genome. THP-1 cells (a model of monocytic derived cells) were transfected with GBV-C, yielding infectious virions that replicated over a 120h time course and could be infected directly. This study provides the first evidence of GBV-C replication in monocytic derived clonal cells. Thus far, it is the only study using a microfluidic device that measures directly viral load of mammalian RNA virus in a digital format without need for a standard curve.

View details for DOI 10.1016/j.jviromet.2011.09.017

View details for Web of Science ID 000300207700008

View details for PubMedID 21983150
Single-cell dissection of transcriptional heterogeneity in human colon tumors NATURE BIOTECHNOLOGY Dalerba, P., Kalisky, T., Sahoo, D., Rajendran, P. S., Rothenberg, M. E., Leyrat, A. A., Sim, S., Okamoto, J., Johnston, D. M., Qian, D., Zabala, M., Bueno, J., Neff, N. F., Wang, J., Shelton, A. A., Visser, B., Hisamori, S., Shimono, Y., Van De Wetering, M., Clevers, H., Clarke, M. F., Quake, S. R. 2011; 29 (12): 1120-U11
Abstract

Cancer is often viewed as a caricature of normal developmental processes, but the extent to which its cellular heterogeneity truly recapitulates multilineage differentiation processes of normal tissues remains unknown. Here we implement single-cell PCR gene-expression analysis to dissect the cellular composition of primary human normal colon and colon cancer epithelia. We show that human colon cancer tissues contain distinct cell populations whose transcriptional identities mirror those of the different cellular lineages of normal colon. By creating monoclonal tumor xenografts from injection of a single (n = 1) cell, we demonstrate that the transcriptional diversity of cancer tissues is largely explained by in vivo multilineage differentiation and not only by clonal genetic heterogeneity. Finally, we show that the different gene-expression programs linked to multilineage differentiation are strongly associated with patient survival. We develop two-gene classifier systems (KRT20 versus CA1, MS4A12, CD177, SLC26A3) that predict clinical outcomes with hazard ratios superior to those of pathological grade and comparable to those of microarray-derived multigene expression signatures.

View details for DOI 10.1038/nbt.2038

View details for PubMedID 22081019
The RootChip: An Integrated Microfluidic Chip for Plant Science PLANT CELL Grossmann, G., Guo, W., Ehrhardt, D. W., Frommer, W. B., Sit, R. V., Quake, S. R., Meier, M. 2011; 23 (12): 4234-4240
Abstract

Studying development and physiology of growing roots is challenging due to limitations regarding cellular and subcellular analysis under controlled environmental conditions. We describe a microfluidic chip platform, called RootChip, that integrates live-cell imaging of growth and metabolism of Arabidopsis thaliana roots with rapid modulation of environmental conditions. The RootChip has separate chambers for individual regulation of the microenvironment of multiple roots from multiple seedlings in parallel. We demonstrate the utility of The RootChip by monitoring time-resolved growth and cytosolic sugar levels at subcellular resolution in plants by a genetically encoded fluorescence sensor for glucose and galactose. The RootChip can be modified for use with roots from other plant species by adapting the chamber geometry and facilitates the systematic analysis of root growth and metabolism from multiple seedlings, paving the way for large-scale phenotyping of root metabolism and signaling.

View details for DOI 10.1105/tpc.111.092577

View details for Web of Science ID 000299677700010

View details for PubMedID 22186371

View details for PubMedCentralID PMC3269862
Clonal Evolution of Pre-Leukemic Hematopoietic Stem Cells Precedes Human Acute Myeloid Leukemia 53rd Annual Meeting and Exposition of the American-Society-of-Hematology (ASH) Jan, M., Snyder, T. M., Corces-Zimmerman, M. R., Weissman, I. L., Quake, S. R., Majeti, R. AMER SOC HEMATOLOGY. 2011: 4?4
View details for Web of Science ID 000299597100005
Partial Genome Assembly for a Candidate Division OP11 Single Cell from an Anoxic Spring (Zodletone Spring, Oklahoma) APPLIED AND ENVIRONMENTAL MICROBIOLOGY Youssef, N. H., Blainey, P. C., Quake, S. R., Elshahed, M. S. 2011; 77 (21): 7804-7814
Abstract

Members of candidate division OP11 are widely distributed in terrestrial and marine ecosystems, yet little information regarding their metabolic capabilities and ecological role within such habitats is currently available. Here, we report on the microfluidic isolation, multiple-displacement-amplification, pyrosequencing, and genomic analysis of a single cell (ZG1) belonging to candidate division OP11. Genome analysis of the ?270-kb partial genome assembly obtained showed that it had no particular similarity to a specific phylum. Four hundred twenty-three open reading frames were identified, 46% of which had no function prediction. In-depth analysis revealed a heterotrophic lifestyle, with genes encoding endoglucanase, amylopullulanase, and laccase enzymes, suggesting a capacity for utilization of cellulose, starch, and, potentially, lignin, respectively. Genes encoding several glycolysis enzymes as well as formate utilization were identified, but no evidence for an electron transport chain was found. The presence of genes encoding various components of lipopolysaccharide biosynthesis indicates a Gram-negative bacterial cell wall. The partial genome also provides evidence for antibiotic resistance (?-lactamase, aminoglycoside phosphotransferase), as well as antibiotic production (bacteriocin) and extracellular bactericidal peptidases. Multiple mechanisms for stress response were identified, as were elements of type I and type IV secretion systems. Finally, housekeeping genes identified within the partial genome were used to demonstrate the OP11 affiliation of multiple hitherto unclassified genomic fragments from multiple database-deposited metagenomic data sets. These results provide the first glimpse into the lifestyle of a member of a ubiquitous, yet poorly understood bacterial candidate division.

View details for DOI 10.1128/AEM.06059-11

View details for Web of Science ID 000296568200043

View details for PubMedID 21908640

View details for PubMedCentralID PMC3209139
Tracking single hematopoietic stem cells in vivo using high-throughput sequencing in conjunction with viral genetic barcoding NATURE BIOTECHNOLOGY Lu, R., Neff, N. F., Quake, S. R., Weissman, I. L. 2011; 29 (10): 928-U229
Abstract

Disentangling cellular heterogeneity is a challenge in many fields, particularly in the stem cell and cancer biology fields. Here we demonstrate how to combine viral genetic barcoding with high-throughput sequencing to track single cells in a heterogeneous population. We use this technique to track the in vivo differentiation of unitary hematopoietic stem cells (HSCs). The results are consistent with single-cell transplantation studies but require two orders of magnitude fewer mice. In addition to its high throughput, the high sensitivity of the technique allows for a direct examination of the clonality of sparse cell populations such as HSCs. We show how these capabilities offer a clonal perspective of the HSC differentiation process. In particular, our data suggest that HSCs do not equally contribute to blood cells after irradiation-mediated transplantation, and that two distinct HSC differentiation patterns co-exist in the same recipient mouse after irradiation. This technique can be applied to any virus-accessible cell type for both in vitro and in vivo processes.

View details for DOI 10.1038/nbt.1977

View details for Web of Science ID 000296273000020

View details for PubMedID 21964413

View details for PubMedCentralID PMC3196379
The genomic sequence of the Chinese hamster ovary (CHO)-K1 cell line NATURE BIOTECHNOLOGY Xu, X., Nagarajan, H., Lewis, N. E., Pan, S., Cai, Z., Liu, X., Chen, W., Xie, M., Wang, W., Hammond, S., Andersen, M. R., Neff, N., Passarelli, B., Koh, W., Fan, H. C., Wang, J., Gui, Y., Lee, K. H., Betenbaugh, M. J., Quake, S. R., Famili, I., Palsson, B. O., Wang, J. 2011; 29 (8): 735-U131
Abstract

Chinese hamster ovary (CHO)-derived cell lines are the preferred host cells for the production of therapeutic proteins. Here we present a draft genomic sequence of the CHO-K1 ancestral cell line. The assembly comprises 2.45 Gb of genomic sequence, with 24,383 predicted genes. We associate most of the assembled scaffolds with 21 chromosomes isolated by microfluidics to identify chromosomal locations of genes. Furthermore, we investigate genes involved in glycosylation, which affect therapeutic protein quality, and viral susceptibility genes, which are relevant to cell engineering and regulatory concerns. Homologs of most human glycosylation-associated genes are present in the CHO-K1 genome, although 141 of these homologs are not expressed under exponential growth conditions. Many important viral entry genes are also present in the genome but not expressed, which may explain the unusual viral resistance property of CHO cell lines. We discuss how the availability of this genome sequence may facilitate genome-scale science for the optimization of biopharmaceutical protein production.

View details for DOI 10.1038/nbt.1932

View details for Web of Science ID 000293696500026

View details for PubMedID 21804562

View details for PubMedCentralID PMC3164356
An Information Theoretic, Microfluidic-Based Single Cell Analysis Permits Identification of Subpopulations among Putatively Homogeneous Stem Cells PLOS ONE Glotzbach, J. P., Januszyk, M., Vial, I. N., Wong, V. W., Gelbard, A., Kalisky, T., Thangarajah, H., Longaker, M. T., Quake, S. R., Chu, G., Gurtner, G. C. 2011; 6 (6)
Abstract

An incomplete understanding of the nature of heterogeneity within stem cell populations remains a major impediment to the development of clinically effective cell-based therapies. Transcriptional events within a single cell are inherently stochastic and can produce tremendous variability, even among genetically identical cells. It remains unclear how mammalian cellular systems overcome this intrinsic noisiness of gene expression to produce consequential variations in function, and what impact this has on the biologic and clinical relevance of highly 'purified' cell subgroups. To address these questions, we have developed a novel method combining microfluidic-based single cell analysis and information theory to characterize and predict transcriptional programs across hundreds of individual cells. Using this technique, we demonstrate that multiple subpopulations exist within a well-studied and putatively homogeneous stem cell population, murine long-term hematopoietic stem cells (LT-HSCs). These subgroups are defined by nonrandom patterns that are distinguishable from noise and are consistent with known functional properties of these cells. We anticipate that this analytic framework can also be applied to other cell types to elucidate the relationship between transcriptional and phenotypic variation.

View details for DOI 10.1371/journal.pone.0021211

View details for Web of Science ID 000292033700046

View details for PubMedID 21731674

View details for PubMedCentralID PMC3120839
Dynamic Chromatin Localization of Sirt6 Shapes Stress- and Aging-Related Transcriptional Networks PLOS GENETICS Kawahara, T. L., Rapicavoli, N. A., Wu, A. R., Qu, K., Quake, S. R., Chang, H. Y. 2011; 7 (6)
Abstract

The sirtuin Sirt6 is a NAD-dependent histone deacetylase that is implicated in gene regulation and lifespan control. Sirt6 can interact with the stress-responsive transcription factor NF-?B and regulate some NF-?B target genes, but the full scope of Sirt6 target genes as well as dynamics of Sirt6 occupancy on chromatin are not known. Here we map Sirt6 occupancy on mouse promoters genome-wide and show that Sirt6 occupancy is highly dynamic in response to TNF-?. More than half of Sirt6 target genes are only revealed upon stress-signaling. The majority of genes bound by NF-?B subunit RelA recruit Sirt6, and dynamic Sirt6 relocalization is largely driven in a RelA-dependent manner. Integrative analysis with global gene expression patterns in wild-type, Sirt6-/-, and double Sirt6-/- RelA-/- cells reveals the epistatic relationships between Sirt6 and RelA in shaping diverse temporal patterns of gene expression. Genes under the direct joint control of Sirt6 and RelA include several with prominent roles in cell senescence and organismal aging. These data suggest dynamic chromatin relocalization of Sirt6 as a key output of NF-?B signaling in stress response and aging.

View details for DOI 10.1371/journal.pgen.1002153

View details for PubMedID 21738489
Universal noninvasive detection of solid organ transplant rejection PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Snyder, T. M., Khush, K. K., Valantine, H. A., Quake, S. R. 2011; 108 (15): 6229-6234
Abstract

It is challenging to monitor the health of transplanted organs, particularly with respect to rejection by the host immune system. Because transplanted organs have genomes that are distinct from the recipient's genome, we used high throughput shotgun sequencing to develop a universal noninvasive approach to monitoring organ health. We analyzed cell-free DNA circulating in the blood of heart transplant recipients and observed significantly increased levels of cell-free DNA from the donor genome at times when an endomyocardial biopsy independently established the presence of acute cellular rejection in these heart transplant recipients. Our results demonstrate that cell-free DNA can be used to detect an organ-specific signature that correlates with rejection, and this measurement can be made on any combination of donor and recipient. This noninvasive test holds promise for replacing the endomyocardial biopsy in heart transplant recipients and may be applicable to other solid organ transplants.

View details for DOI 10.1073/pnas.1013924108

View details for Web of Science ID 000289413600060

View details for PubMedID 21444804

View details for PubMedCentralID PMC3076856
MOLECULAR AUTOPSY FOR SUDDEN CARDIAC DEATH USING WHOLE GENOME SEQUENCING 60th Annual Scientific Session and Expo of the American-College-of-Cardiology (ACC) / I2 Summit / ACCF/Herman K. Gold Young Investigator's Award in Molecular and Cellular Cardiology Dewey, F. E., Wheeler, M. T., Cordero, S., Perez, M. V., Pavlovic, A., Pushkarev, D., Freeman, J. V., Quake, S. R., Ashley, E. A. ELSEVIER SCIENCE INC. 2011: E1159?E1159
View details for Web of Science ID 000291695101162
Single-cell genomics NATURE METHODS Kalisky, T., Quake, S. R. 2011; 8 (4): 311-314
Abstract

Methods for genomic analysis at single-cell resolution enable new understanding of complex biological phenomena. Single-cell techniques, ranging from flow cytometry and microfluidics to PCR and sequencing, are used to understand the cellular composition of complex tissues, find new microbial species and perform genome-wide haplotyping.

View details for DOI 10.1038/nmeth0411-308

View details for Web of Science ID 000288940300017

View details for PubMedID 21451520
Heart Transplants Are Genome Transplants: Universal Noninvasive Detection of Solid Organ Transplant Rejection 31st Annual Meeting and Scientific Sessions of the International-Society-for-Heart-and-Lung-Transplantation (ISHLT) Khush, K. K., Snyder, T. M., Quake, S. R., Valantine, H. A. ELSEVIER SCIENCE INC. 2011: S186?S187
View details for Web of Science ID 000288924300552
Determinism and stochasticity during maturation of the zebrafish antibody repertoire PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Jiang, N., Weinstein, J. A., Penland, L., White, R. A., Fisher, D. S., Quake, S. R. 2011; 108 (13): 5348-5353
Abstract

It is thought that the adaptive immune system of immature organisms follows a more deterministic program of antibody creation than is found in adults. We used high-throughput sequencing to characterize the diversifying antibody repertoire in zebrafish over five developmental time points. We found that the immune system begins in a highly stereotyped state with preferential use of a small number of V (variable) D (diverse) J (joining) gene segment combinations, but that this stereotypy decreases dramatically as the zebrafish mature, with many of the top VDJ combinations observed in 2-wk-old zebrafish virtually disappearing by 1 mo. However, we discovered that, in the primary repertoire, there are strong correlations in VDJ use that increase with zebrafish maturity, suggesting that VDJ recombination involves a level of deterministic programming that is unexpected. This stereotypy is masked by the complex diversification processes of antibody maturation; the variation and lack of correlation in full repertoires between individuals appears to be derived from randomness in clonal expansion during the affinity maturation process. These data provide a window into the mechanisms of VDJ recombination and diversity creation and allow us to better understand how the adaptive immune system achieves diversity.

View details for DOI 10.1073/pnas.1014277108

View details for Web of Science ID 000288894800043

View details for PubMedID 21393572

View details for PubMedCentralID PMC3069157
Digital MDA for enumeration of total nucleic acid contamination NUCLEIC ACIDS RESEARCH Blainey, P. C., Quake, S. R. 2011; 39 (4)
Abstract

Multiple displacement amplification (MDA) is an isothermal, sequence-independent method for the amplification of high molecular weight DNA that is driven by ?29 DNA polymerase (DNAP). Here we report digital MDA (dMDA), an ultrasensitive method for quantifying nucleic acid fragments of unknown sequence. We use the new assay to show that our custom ?29 DNAP preparation is free of contamination at the limit of detection of the dMDA assay (1 contaminating molecule per assay microliter). Contamination in commercially available preparations is also investigated. The results of the dMDA assay provide strong evidence that the so-called 'template-independent' MDA background can be attributed to high-molecular weight contaminants and is not primer-derived in the commercial kits tested. dMDA is orders of magnitude more sensitive than PCR-based techniques for detection of microbial genomic DNA fragments and opens up new possibilities for the ultrasensitive quantification of DNA fragments in a wide variety of application areas using MDA chemistry and off-the-shelf hardware developed for digital PCR.

View details for DOI 10.1093/nar/gkq1074

View details for Web of Science ID 000288019400001

View details for PubMedID 21071419

View details for PubMedCentralID PMC3045575
High-throughput single-molecule optofluidic analysis NATURE METHODS Kim, S., Streets, A. M., Lin, R. R., Quake, S. R., Weiss, S., Majumdar, D. S. 2011; 8 (3): 242-U83
Abstract

We describe a high-throughput, automated single-molecule measurement system, equipped with microfluidics. The microfluidic mixing device has integrated valves and pumps to accurately accomplish titration of biomolecules with picoliter resolution. We demonstrate that the approach enabled rapid sampling of biomolecule conformational landscape and of enzymatic activity, in the form of transcription by Escherichia coli RNA polymerase, as a function of the chemical environment.

View details for DOI 10.1038/NMETH.1569

View details for Web of Science ID 000287734800016

View details for PubMedID 21297618

View details for PubMedCentralID PMC3075913
COMMUNITY CORNER Opening the Pandora's box of prenatal genetic testing NATURE MEDICINE Wright, C., Quake, S. R., Bianchi, D., Wald, N. J. 2011; 17 (3): 250-251
View details for DOI 10.1038/nm0311-250

View details for Web of Science ID 000288070000017

View details for PubMedID 21383717
Genome of a Low-Salinity Ammonia-Oxidizing Archaeon Determined by Single-Cell and Metagenomic Analysis PLOS ONE Blainey, P. C., Mosier, A. C., Potanina, A., Francis, C. A., Quake, S. R. 2011; 6 (2)
Abstract

Ammonia-oxidizing archaea (AOA) are thought to be among the most abundant microorganisms on Earth and may significantly impact the global nitrogen and carbon cycles. We sequenced the genome of AOA in an enrichment culture from low-salinity sediments in San Francisco Bay using single-cell and metagenomic genome sequence data. Five single cells were isolated inside an integrated microfluidic device using laser tweezers, the cells' genomic DNA was amplified by multiple displacement amplification (MDA) in 50 nL volumes and then sequenced by high-throughput DNA pyrosequencing. This microscopy-based approach to single-cell genomics minimizes contamination and allows correlation of high-resolution cell images with genomic sequences. Statistical properties of coverage across the five single cells, in combination with the contrasting properties of the metagenomic dataset allowed the assembly of a high-quality draft genome. The genome of this AOA, which we designate Candidatus Nitrosoarchaeum limnia SFB1, is ?1.77 Mb with >2100 genes and a G+C content of 32%. Across the entire genome, the average nucleotide identity to Nitrosopumilus maritimus, the only AOA in pure culture, is ?70%, suggesting this AOA represents a new genus of Crenarchaeota. Phylogenetically, the 16S rRNA and ammonia monooxygenase subunit A (amoA) genes of this AOA are most closely related to sequences reported from a wide variety of freshwater ecosystems. Like N. maritimus, the low-salinity AOA genome appears to have an ammonia oxidation pathway distinct from ammonia oxidizing bacteria (AOB). In contrast to other described AOA, these low-salinity AOA appear to be motile, based on the presence of numerous motility- and chemotaxis-associated genes in the genome. This genome data will be used to inform targeted physiological and metabolic studies of this novel group of AOA, which may ultimately advance our understanding of AOA metabolism and their impacts on the global carbon and nitrogen cycles.

View details for DOI 10.1371/journal.pone.0016626

View details for Web of Science ID 000287656600009

View details for PubMedID 21364937

View details for PubMedCentralID PMC3043068
Mapping Virus-Host Protein Interactions using the Ping Microfluidics Platform 55th Annual Meeting of the Biophysical-Society Gerber, D., Einav, S., Quake, S. R. CELL PRESS. 2011: 368?68
View details for Web of Science ID 000306288603130
Whole-genome molecular haplotyping of single cells NATURE BIOTECHNOLOGY Fan, H. C., Wang, J., Potanina, A., Quake, S. R. 2011; 29 (1): 51-?
Abstract

Conventional experimental methods of studying the human genome are limited by the inability to independently study the combination of alleles, or haplotype, on each of the homologous copies of the chromosomes. We developed a microfluidic device capable of separating and amplifying homologous copies of each chromosome from a single human metaphase cell. Single-nucleotide polymorphism (SNP) array analysis of amplified DNA enabled us to achieve completely deterministic, whole-genome, personal haplotypes of four individuals, including a HapMap trio with European ancestry (CEU) and an unrelated European individual. The phases of alleles were determined at ?99.8% accuracy for up to ?96% of all assayed SNPs. We demonstrate several practical applications, including direct observation of recombination events in a family trio, deterministic phasing of deletions in individuals and direct measurement of the human leukocyte antigen haplotypes of an individual. Our approach has potential applications in personal genomics, single-cell genomics and statistical genetics.

View details for DOI 10.1038/nbt.1739

View details for Web of Science ID 000286048900020

View details for PubMedID 21170043
Digital PCR provides absolute quantitation of viral load for an occult RNA virus J Virol Methods White, R. A., Quake, S. R., Curr, K. 2011
High-Throughput Single Molecule Optofluidic Analysis Nature Methods Kim, S., Streets, A. M., Lin, R. R., Quake, S. R., Weiss, S., Majumdar, D, S. 2011; 3 (8): 242-245
Dynamic Chromatin Localization of Sirt6 Shapes Stress - and aging - related Transcriptional Networks PLoS Genet Kawahara, T. L., Rapicavoli, N. A., Wu, A. R., Qu, K., Quake, S. R., Chang, H, Y. 2011; 6 (7)
Genomic Analysis at the Single-Cell Level ANNUAL REVIEW GENETICS, VOL 45 Kalisky, T., Blainey, P., Quake, S. R. 2011; 45: 431-445
Abstract

Studying complex biological systems such as a developing embryo, a tumor, or a microbial ecosystem often involves understanding the behavior and heterogeneity of the individual cells that constitute the system and their interactions. In this review, we discuss a variety of approaches to single-cell genomic analysis.

View details for DOI 10.1146/annurev-genet-102209-163607

View details for Web of Science ID 000299299600019

View details for PubMedID 21942365
Biocompatibility and Reduced Drug Absorption of Sol-Gel-Treated Poly(dimethyl siloxane) for Microfluidic Cell Culture Applications ANALYTICAL CHEMISTRY Gomez-Sjoberg, R., Leyrat, A. A., Houseman, B. T., Shokat, K., Quake, S. R. 2010; 82 (21): 8954-8960
Abstract

Poly(dimethyl siloxane) (PDMS)-based microfluidic devices are now commonly used for a wide variety of biological experiments, including cell culture assays. However, the porous, hydrophobic polymer matrix of PDMS rapidly absorbs small hydrophobic molecules, including hormones and most small-molecule drugs. This makes it challenging to perform experiments that require such substances in PDMS microfluidic devices. This study presents evidence that a sol-gel treatment of PDMS that fills the polymer matrix with silica nanoparticles is effective at reducing the absorption of drugs into the material while preserving its biocompatibility, transparency, and oxygen permeability. We show that the absorption of two anticancer drugs, camptothecin and a kinase inhibitor, is reduced to such an extent that on-chip microfluidic cell culture experiments can recapitulate the results obtained off-chip.

View details for DOI 10.1021/ac101870s

View details for Web of Science ID 000283531200031

View details for PubMedCentralID PMC3032040
Variability in G-Protein-Coupled Signaling Studied with Microfluidic Devices BIOPHYSICAL JOURNAL Bao, X. R., Fraser, I. D., Wall, E. A., Quake, S. R., Simon, M. I. 2010; 99 (8): 2414-2422
Abstract

Different cells, even those that are genetically identical, can respond differently to identical stimuli, but the precise source of this variability remains obscure. To study this problem, we built a microfluidic experimental system which can track responses of individual cells across multiple stimulations. We used this system to determine that amplitude variation in G-protein-activated calcium release in RAW264.7 macrophages is generally extrinsic, i.e., they arise from long-lived variations between cells and not from stochastic activation of signaling components. In the case of responses linked to P2Y family purine receptors, we estimate that approximately one-third of the observed variability in calcium release is receptor-specific. We further demonstrate that the signaling apparatus downstream of P2Y6 receptor activation is moderately saturable. These observations will be useful in constructing and constraining single-cell models of G protein-coupled calcium dynamics.

View details for DOI 10.1016/j.bpj.2010.08.043

View details for Web of Science ID 000283412500007

View details for PubMedID 20959081

View details for PubMedCentralID PMC2955501
De novo identification and biophysical characterization of transcription-factor binding sites with microfluidic affinity analysis NATURE BIOTECHNOLOGY Fordyce, P. M., Gerber, D., Tran, D., Zheng, J., Li, H., DeRisi, J. L., Quake, S. R. 2010; 28 (9): 970-976
Abstract

Gene expression is regulated in part by protein transcription factors that bind target regulatory DNA sequences. Predicting DNA binding sites and affinities from transcription factor sequence or structure is difficult; therefore, experimental data are required to link transcription factors to target sequences. We present a microfluidics-based approach for de novo discovery and quantitative biophysical characterization of DNA target sequences. We validated our technique by measuring sequence preferences for 28 Saccharomyces cerevisiae transcription factors with a variety of DNA-binding domains, including several that have proven difficult to study by other techniques. For each transcription factor, we measured relative binding affinities to oligonucleotides covering all possible 8-bp DNA sequences to create a comprehensive map of sequence preferences; for four transcription factors, we also determined absolute affinities. We expect that these data and future use of this technique will provide information essential for understanding transcription factor specificity, improving identification of regulatory sites and reconstructing regulatory interactions.

View details for DOI 10.1038/nbt.1675

View details for Web of Science ID 000281719100024

View details for PubMedID 20802496

View details for PubMedCentralID PMC2937095
Analysis of the Size Distributions of Fetal and Maternal Cell-Free DNA by Paired-End Sequencing CLINICAL CHEMISTRY Fan, H. C., Blumenfeld, Y. J., Chitkara, U., Hudgins, L., Quake, S. R. 2010; 56 (8): 1279-1286
Abstract

Noninvasive prenatal diagnosis with cell-free DNA in maternal plasma is challenging because only a small portion of the DNA sample is derived from the fetus. A few previous studies provided size-range estimates of maternal and fetal DNA, but direct measurement of the size distributions is difficult because of the small quantity of cell-free DNA.We used high-throughput paired-end sequencing to directly measure the size distributions of maternal and fetal DNA in cell-free maternal plasma collected from 3 typical diploid and 4 aneuploid male pregnancies. As a control, restriction fragments of lambda DNA were also sequenced.Cell-free DNA had a dominant peak at approximately 162 bp and a minor peak at approximately 340 bp. Chromosome Y sequences were rarely longer than 250 bp but were present in sizes of <150 bp at a larger proportion compared with the rest of the sequences. Selective analysis of the shortest fragments generally increased the fetal DNA fraction but did not necessarily increase the sensitivity of aneuploidy detection, owing to the reduction in the number of DNA molecules being counted. Restriction fragments of lambda DNA with sizes between 60 bp and 120 bp were preferentially sequenced, indicating that the shotgun sequencing work flow introduced a bias toward shorter fragments.Our results confirm that fetal DNA is shorter than maternal DNA. The enrichment of fetal DNA by size selection, however, may not provide a dramatic increase in sensitivity for assays that rely on length measurement in situ because of a trade-off between the fetal DNA fraction and the number of molecules being counted.

View details for DOI 10.1373/clinchem.2010.144188

View details for Web of Science ID 000280501400016

View details for PubMedID 20558635
Single-cell NF-kappa B dynamics reveal digital activation and analogue information processing NATURE Tay, S., Hughey, J. J., Lee, T. K., Lipniacki, T., Quake, S. R., Covert, M. W. 2010; 466 (7303): 267-U149
Abstract

Cells operate in dynamic environments using extraordinary communication capabilities that emerge from the interactions of genetic circuitry. The mammalian immune response is a striking example of the coordination of different cell types. Cell-to-cell communication is primarily mediated by signalling molecules that form spatiotemporal concentration gradients, requiring cells to respond to a wide range of signal intensities. Here we use high-throughput microfluidic cell culture and fluorescence microscopy, quantitative gene expression analysis and mathematical modelling to investigate how single mammalian cells respond to different concentrations of the signalling molecule tumour-necrosis factor (TNF)-alpha, and relay information to the gene expression programs by means of the transcription factor nuclear factor (NF)-kappaB. We measured NF-kappaB activity in thousands of live cells under TNF-alpha doses covering four orders of magnitude. We find, in contrast to population-level studies with bulk assays, that the activation is heterogeneous and is a digital process at the single-cell level with fewer cells responding at lower doses. Cells also encode a subtle set of analogue parameters to modulate the outcome; these parameters include NF-kappaB peak intensity, response time and number of oscillations. We developed a stochastic mathematical model that reproduces both the digital and analogue dynamics as well as most gene expression profiles at all measured conditions, constituting a broadly applicable model for TNF-alpha-induced NF-kappaB signalling in various types of cells. These results highlight the value of high-throughput quantitative measurements with single-cell resolution in understanding how biological systems operate.

View details for DOI 10.1038/nature09145

View details for Web of Science ID 000279580800043

View details for PubMedID 20581820

View details for PubMedCentralID PMC3105528
Sensitivity of Noninvasive Prenatal Detection of Fetal Aneuploidy from Maternal Plasma Using Shotgun Sequencing Is Limited Only by Counting Statistics PLOS ONE Fan, H. C., Quake, S. R. 2010; 5 (5)
Abstract

We recently demonstrated noninvasive detection of fetal aneuploidy by shotgun sequencing cell-free DNA in maternal plasma using next-generation high throughput sequencer. However, GC bias introduced by the sequencer placed a practical limit on the sensitivity of aneuploidy detection. In this study, we describe a method to computationally remove GC bias in short read sequencing data by applying weight to each sequenced read based on local genomic GC content. We show that sensitivity is limited only by counting statistics and that sensitivity can be increased to arbitrary precision in sample containing arbitrarily small fraction of fetal DNA simply by sequencing more DNA molecules. High throughput shotgun sequencing of maternal plasma DNA should therefore enable noninvasive diagnosis of any type of fetal aneuploidy.

View details for DOI 10.1371/journal.pone.0010439

View details for Web of Science ID 000277240300015

View details for PubMedID 20454671

View details for PubMedCentralID PMC2862719
Clinical assessment incorporating a personal genome LANCET Ashley, E. A., Butte, A. J., Wheeler, M. T., Chen, R., Klein, T. E., Dewey, F. E., Dudley, J. T., Ormond, K. E., Pavlovic, A., Morgan, A. A., Pushkarev, D., Neff, N. F., Hudgins, L., Gong, L., Hodges, L. M., Berlin, D. S., Thorn, C. F., Sangkuhl, K., Hebert, J. M., Woon, M., Sagreiya, H., Whaley, R., Knowles, J. W., Chou, M. F., Thakuria, J. V., Rosenbaum, A. M., Zaranek, A. W., Church, G. M., Greely, H. T., Quake, S. R., Altman, R. B. 2010; 375 (9725): 1525-1535
Abstract

The cost of genomic information has fallen steeply, but the clinical translation of genetic risk estimates remains unclear. We aimed to undertake an integrated analysis of a complete human genome in a clinical context.We assessed a patient with a family history of vascular disease and early sudden death. Clinical assessment included analysis of this patient's full genome sequence, risk prediction for coronary artery disease, screening for causes of sudden cardiac death, and genetic counselling. Genetic analysis included the development of novel methods for the integration of whole genome and clinical risk. Disease and risk analysis focused on prediction of genetic risk of variants associated with mendelian disease, recognised drug responses, and pathogenicity for novel variants. We queried disease-specific mutation databases and pharmacogenomics databases to identify genes and mutations with known associations with disease and drug response. We estimated post-test probabilities of disease by applying likelihood ratios derived from integration of multiple common variants to age-appropriate and sex-appropriate pre-test probabilities. We also accounted for gene-environment interactions and conditionally dependent risks.Analysis of 2.6 million single nucleotide polymorphisms and 752 copy number variations showed increased genetic risk for myocardial infarction, type 2 diabetes, and some cancers. We discovered rare variants in three genes that are clinically associated with sudden cardiac death-TMEM43, DSP, and MYBPC3. A variant in LPA was consistent with a family history of coronary artery disease. The patient had a heterozygous null mutation in CYP2C19 suggesting probable clopidogrel resistance, several variants associated with a positive response to lipid-lowering therapy, and variants in CYP4F2 and VKORC1 that suggest he might have a low initial dosing requirement for warfarin. Many variants of uncertain importance were reported.Although challenges remain, our results suggest that whole-genome sequencing can yield useful and clinically relevant information for individual patients.National Institute of General Medical Sciences; National Heart, Lung And Blood Institute; National Human Genome Research Institute; Howard Hughes Medical Institute; National Library of Medicine, Lucile Packard Foundation for Children's Health; Hewlett Packard Foundation; Breetwor Family Foundation.

View details for Web of Science ID 000277655100025

View details for PubMedID 20435227
A microfluidic oligonucleotide synthesizer NUCLEIC ACIDS RESEARCH Lee, C., Snyder, T. M., Quake, S. R. 2010; 38 (8): 2514-2521
Abstract

De novo gene and genome synthesis enables the design of any sequence without the requirement of a pre-existing template as in traditional genetic engineering methods. The ability to mass produce synthetic genes holds great potential for biological research, but widespread availability of de novo DNA constructs is currently hampered by their high cost. In this work, we describe a microfluidic platform for parallel solid phase synthesis of oligonucleotides that can greatly reduce the cost of gene synthesis by reducing reagent consumption (by 100-fold) while maintaining a approximately 100 pmol synthesis scale so there is no need for amplification before assembly. Sixteen oligonucleotides were synthesized in parallel on this platform and then successfully used in a ligation-mediated assembly method to generate DNA constructs approximately 200 bp in length.

View details for DOI 10.1093/nar/gkq092

View details for Web of Science ID 000277238900002

View details for PubMedID 20176572

View details for PubMedCentralID PMC2860119
Ostwald Ripening of Clusters during Protein Crystallization PHYSICAL REVIEW LETTERS Streets, A. M., Quake, S. R. 2010; 104 (17)
Abstract

Contrary to classical nucleation theory, protein crystals can nucleate via a two-step process in which the molecular arrangement of the ordered solid phase is preceded by nucleation of a dense amorphous phase. We study the growth of these precrystalline clusters in lysozyme using a combination of dynamic light scattering, optical microscopy, and microfluidics. Clusters display Ostwald ripening growth kinetics but deviate from this trend after nucleation of the crystal phase. This behavior arises from the metastable relationship between clusters and the ordered solid and is explained numerically using a population balance model.

View details for DOI 10.1103/PhysRevLett.104.178102

View details for Web of Science ID 000277210600053

View details for PubMedID 20482145
Microtechnology in the Clinical Laboratory: Will It Solve Analytical Problems, and When Will It Make an Impact? CLINICAL CHEMISTRY Wilding, P., Verpoorte, S., Northrup, A., Yager, P., Quake, S., Landers, J. 2010; 56 (4): 508-514
View details for DOI 10.1373/clinchem.2009.138719

View details for Web of Science ID 000277059900004

View details for PubMedID 20167692
Single molecule localization on self-assembled, semi-ordered nanoparticle arrays Stoltenberg, R. M., Schwartz, J. J., Quake, S. R., Bao, Z. AMER CHEMICAL SOC. 2010
View details for Web of Science ID 000208189304608
Static control logic for microfluidic devices using pressure-gain valves NATURE PHYSICS Weaver, J. A., Melin, J., Stark, D., Quake, S. R., Horowitz, M. A. 2010; 6 (3): 218-223
View details for DOI 10.1038/NPHYS1513

View details for Web of Science ID 000275024000024
Colloidal lenses allow high-temperature single-molecule imaging and improve fluorophore photostability NATURE NANOTECHNOLOGY Schwartz, J. J., Stavrakis, s., Quake, S. R. 2010; 5 (2): 127-132
Abstract

Although single-molecule fluorescence spectroscopy was first demonstrated at near-absolute zero temperatures (1.8 K), the field has since advanced to include room-temperature observations, largely owing to the use of objective lenses with high numerical aperture, brighter fluorophores and more sensitive detectors. This has opened the door for many chemical and biological systems to be studied at native temperatures at the single-molecule level both in vitro and in vivo. However, it is difficult to study systems and phenomena at temperatures above 37 degrees C, because the index-matching fluids used with high-numerical-aperture objective lenses can conduct heat from the sample to the lens, and sustained exposure to high temperatures can cause the lens to fail. Here, we report that TiO(2) colloids with diameters of 2 microm and a high refractive index can act as lenses that are capable of single-molecule imaging at 70 degrees C when placed in immediate proximity to an emitting molecule. The optical system is completed by a low-numerical-aperture optic that can have a long working distance and an air interface, which allows the sample to be independently heated. Colloidal lenses were used for parallel imaging of surface-immobilized single fluorophores and for real-time single-molecule measurements of mesophilic and thermophilic enzymes at 70 degrees C. Fluorophores in close proximity to TiO(2) also showed a 40% increase in photostability due to a reduction of the excited-state lifetime.

View details for DOI 10.1038/NNANO.2009.452

View details for Web of Science ID 000275058500013

View details for PubMedID 20023643
Biocompatibility and Reduced Drug Absorption of Sol-Gel-Treated Poly(dimethyl siloxane) for Microfluidic Cell Culture Applications. Analytical chemistry Gomez-Sjoberg, R., Leyrat, A. A., Houseman, B. T., Shokat, K., Quake, S. R. 2010
Abstract

Poly(dimethyl siloxane) (PDMS)-based microfluidic devices are now commonly used for a wide variety of biological experiments, including cell culture assays. However, the porous, hydrophobic polymer matrix of PDMS rapidly absorbs small hydrophobic molecules, including hormones and most small-molecule drugs. This makes it challenging to perform experiments that require such substances in PDMS microfluidic devices. This study presents evidence that a sol-gel treatment of PDMS that fills the polymer matrix with silica nanoparticles is effective at reducing the absorption of drugs into the material while preserving its biocompatibility, transparency, and oxygen permeability. We show that the absorption of two anticancer drugs, camptothecin and a kinase inhibitor, is reduced to such an extent that on-chip microfluidic cell culture experiments can recapitulate the results obtained off-chip.

View details for PubMedID 20936785

View details for PubMedCentralID PMC3032040
Niocompatibility and Reduced Drug Absorption of Sol-Gel-Treated Poly(dimethyl siloxane) for Microfluidic Cell Culture Applications Ana Chem. Bao, X. R., Fraser, I. D., Wall, E. A., Quake, S. R., Simon, M. I. 2010
Clinical assessment incorportating a personal genome Lancet Ashley, E. A., Butte, A. J., Wheeler, M. T., Chen, R., Klein, T. E., Dewey, F. E., Quake, S. 2010; 9725 (375): 1525:1535
Variability in G-Protein-Coupled Signaling Studied with Microfluidic Devices Biophysical Journal Bao, X. R., Fraser, I. D., Wall, E. A., Quake, S. R., Simon, M. I. 2010; 99: 2414?2422
Single-cell NF-kappaB dynamics reveal digital activation and analogue information processing. Nature. Tay, S., Hughey, J. J., Lee, T. K., Lipniacki, T., Quake, S. R., Covert, M, W. 2010; 7303 (466): 267-71
Whole Genome Molecular Haplotyping of Single Cells Nature Biotechnology. Fan, H. C., Wang, J., Potanina, A., Quake, S, R. 2010
Comprehensive maternal-fetal pharmacogenomics - a novel pharmacogenomic thumbprint 30th Annual Clinical Meeting of the Society-for-Maternal-Fetal-Medicine Blumenfeld, Y., Fan, C., Sangkuhl, K., Altman, R., El-Sayed, Y., Quake, S. MOSBY-ELSEVIER. 2009: S254?S254
View details for Web of Science ID 000279559500695
Single molecule measurement of the "speed limit'' of DNA polymerase PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Schwartz, J. J., Quake, S. R. 2009; 106 (48): 20294-20299
Abstract

Although DNA replication is often imagined as a regular and continuous process, the DNA polymerase enzyme is a complicated machine and can pause upon encountering physical and chemical barriers. We used single molecule measurements to make a detailed characterization of this behavior as a function of the template's secondary structure and the sequence context. Strand displacement replication through a DNA hairpin by single DNA polymerase molecules was measured in real time with near single base resolution and physiological concentrations of nucleotides. These data enabled the measurement of the intrinsic "speed limit" of DNA polymerase by separating the burst synthesis rate from pausing events. The strand displacement burst synthesis rate for Escherichia coli DNA Polymerase I (KF) was found to be an order of magnitude faster than the reported bulk strand displacement rate, a discrepancy that can be accounted for by to sequence specific pausing. The ability to follow trajectories of single molecules revealed that the burst synthesis rate is also highly stochastic and varies up to 50-fold from molecule to molecule. Surprisingly, our results allow a unified explanation of strand displacement and single strand primer extension synthesis rates.

View details for DOI 10.1073/pnas.0907404106

View details for Web of Science ID 000272254400031

View details for PubMedID 19906998

View details for PubMedCentralID PMC2787106
Digital PCR provides sensitive and absolute calibration for high throughput sequencing (vol 10, pg 541, 2009) BMC GENOMICS White, R. A., Blainey, P. C., Fan, H. C., Quake, S. R. 2009; 10
View details for DOI 10.1186/1471-2164-10-541

View details for Web of Science ID 000272358000001
Experimental determination of the evolvability of a transcription factor PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Maerkl, S. J., Quake, S. R. 2009; 106 (44): 18650-18655
Abstract

Sequence-specific binding of a transcription factor to DNA is the central event in any transcriptional regulatory network. However, relatively little is known about the evolutionary plasticity of transcription factors. For example, the exact functional consequence of an amino acid substitution on the DNA-binding specificity of most transcription factors is currently not predictable. Furthermore, although the major structural families of transcription factors have been identified, the detailed DNA-binding repertoires within most families have not been characterized. We studied the sequence recognition code and evolvability of the basic helix-loop-helix transcription factor family by creating all possible 95 single-point mutations of five DNA-contacting residues of Max, a human helix-loop-helix transcription factor and measured the detailed DNA-binding repertoire of each mutant. Our results show that the sequence-specific repertoire of Max accessible through single-point mutations is extremely limited, and we are able to predict 92% of the naturally occurring diversity at these positions. All naturally occurring basic regions were also found to be accessible through functional intermediates. Finally, we observed a set of amino acids that are functional in vitro but are not found to be used naturally, indicating that functionality alone is not sufficient for selection.

View details for DOI 10.1073/pnas.0907688106

View details for Web of Science ID 000271429800044

View details for PubMedID 19841254

View details for PubMedCentralID PMC2765923
Single-molecule sequencing of an individual human genome NATURE BIOTECHNOLOGY Pushkarev, D., Neff, N. F., Quake, S. R. 2009; 27 (9): 847-U101
Abstract

Recent advances in high-throughput DNA sequencing technologies have enabled order-of-magnitude improvements in both cost and throughput. Here we report the use of single-molecule methods to sequence an individual human genome. We aligned billions of 24- to 70-bp reads (32 bp average) to approximately 90% of the National Center for Biotechnology Information (NCBI) reference genome, with 28x average coverage. Our results were obtained on one sequencing instrument by a single operator with four data collection runs. Single-molecule sequencing enabled analysis of human genomic information without the need for cloning, amplification or ligation. We determined approximately 2.8 million single nucleotide polymorphisms (SNPs) with a false-positive rate of less than 1% as validated by Sanger sequencing and 99.8% concordance with SNP genotyping arrays. We identified 752 regions of copy number variation by analyzing coverage depth alone and validated 27 of these using digital PCR. This milestone should allow widespread application of genome sequencing to many aspects of genetics and human health, including personal genomics.

View details for DOI 10.1038/nbt.1561

View details for Web of Science ID 000269751400027

View details for PubMedID 19668243
Highly parallel measurements of interaction kinetic constants with a microfabricated optomechanical device APPLIED PHYSICS LETTERS Bates, S. R., Quake, S. R. 2009; 95 (7)
View details for DOI 10.1063/1.3211382

View details for Web of Science ID 000269288300081
Automated microfluidic chromatin immunoprecipitation from 2,000 cells LAB ON A CHIP Wu, A. R., Hiatt, J. B., Lu, R., Attema, J. L., Lobo, N. A., Weissman, I. L., Clarke, M. F., Quake, S. R. 2009; 9 (10): 1365-1370
Abstract

Chromatin immunoprecipitation (ChIP) is a powerful assay used to probe DNA-protein interactions. Traditional methods of implementing this assay are lengthy, cumbersome and require a large number of cells, making it difficult to study rare cell types such as certain cancer and stem cells. We have designed a microfluidic device to perform sensitive ChIP analysis on low cell numbers in a rapid, automated fashion while preserving the specificity of the assay. Comparing ChIP results for two modified histone protein targets, we showed our automated microfluidic ChIP (AutoChIP) from 2,000 cells to be comparable to that of conventional ChIP methods using 50,000-500,000 cells. This technology may provide a solution to the need for a high sensitivity, rapid, and automated ChIP assay, and in doing so facilitate the use of ChIP for many interesting and valuable applications.

View details for DOI 10.1039/b819648f

View details for Web of Science ID 000268227400008

View details for PubMedID 19417902
High-Throughput Sequencing of the Zebrafish Antibody Repertoire SCIENCE Weinstein, J. A., Jiang, N., White, R. A., Fisher, D. S., Quake, S. R. 2009; 324 (5928): 807-810
Abstract

Despite tremendous progress in understanding the nature of the immune system, the full diversity of an organism's antibody repertoire is unknown. We used high-throughput sequencing of the variable domain of the antibody heavy chain from 14 zebrafish to analyze VDJ usage and antibody sequence. Zebrafish were found to use between 50 and 86% of all possible VDJ combinations and shared a similar frequency distribution, with some correlation of VDJ patterns between individuals. Zebrafish antibodies retained a few thousand unique heavy chains that also exhibited a shared frequency distribution. We found evidence of convergence, in which different individuals made the same antibody. This approach provides insight into the breadth of the expressed antibody repertoire and immunological diversity at the level of an individual organism.

View details for DOI 10.1126/science.1170020

View details for Web of Science ID 000265832400053

View details for PubMedID 19423829

View details for PubMedCentralID PMC3086368
Microfluidic digital PCR enables rapid prenatal diagnosis of fetal aneuploidy 29th Annual Meeting of the Society-for-Maternal-Fetal-Medicine Fan, H. C., Blumenfeld, Y. J., El-Sayed, Y. Y., Chueh, J., Quake, S. R. MOSBY-ELSEVIER. 2009
Abstract

The purpose of this study was to demonstrate that digital polymerase chain reaction (PCR) enables rapid, allele independent molecular detection of fetal aneuploidy.Twenty-four amniocentesis and 16 chorionic villus samples were used for microfluidic digital PCR analysis. Three thousand and sixty PCR reactions were performed for each of the target chromosomes (X, Y, 13, 18, and 21), and the number of single molecule amplifications was compared to a reference. The difference between target and reference chromosome counts was used to determine the ploidy of each of the target chromosomes.Digital PCR accurately identified all cases of fetal trisomy (3 cases of trisomy 21, 3 cases of trisomy 18, and 2 cases of triosmy 13) in the 40 specimens analyzed. The remaining specimens were determined to have normal ploidy for the chromosomes tested.Microfluidic digital PCR allows detection of fetal chromosomal aneuploidy utilizing uncultured amniocytes and chorionic villus tissue in less than 6 hours.

View details for DOI 10.1016/j.ajog.2009.03.002

View details for Web of Science ID 000265253800029

View details for PubMedID 19375573
Association of reactive oxygen species levels and radioresistance in cancer stem cells NATURE Diehn, M., Cho, R. W., Lobo, N. A., Kalisky, T., Dorie, M. J., Kulp, A. N., Qian, D., Lam, J. S., Ailles, L. E., Wong, M., Joshua, B., Kaplan, M. J., Wapnir, I., Dirbas, F. M., Somlo, G., Garberoglio, C., Paz, B., Shen, J., Lau, S. K., Quake, S. R., Brown, J. M., Weissman, I. L., Clarke, M. F. 2009; 458 (7239): 780-U123
Abstract

The metabolism of oxygen, although central to life, produces reactive oxygen species (ROS) that have been implicated in processes as diverse as cancer, cardiovascular disease and ageing. It has recently been shown that central nervous system stem cells and haematopoietic stem cells and early progenitors contain lower levels of ROS than their more mature progeny, and that these differences are critical for maintaining stem cell function. We proposed that epithelial tissue stem cells and their cancer stem cell (CSC) counterparts may also share this property. Here we show that normal mammary epithelial stem cells contain lower concentrations of ROS than their more mature progeny cells. Notably, subsets of CSCs in some human and murine breast tumours contain lower ROS levels than corresponding non-tumorigenic cells (NTCs). Consistent with ROS being critical mediators of ionizing-radiation-induced cell killing, CSCs in these tumours develop less DNA damage and are preferentially spared after irradiation compared to NTCs. Lower ROS levels in CSCs are associated with increased expression of free radical scavenging systems. Pharmacological depletion of ROS scavengers in CSCs markedly decreases their clonogenicity and results in radiosensitization. These results indicate that, similar to normal tissue stem cells, subsets of CSCs in some tumours contain lower ROS levels and enhanced ROS defences compared to their non-tumorigenic progeny, which may contribute to tumour radioresistance.

View details for DOI 10.1038/nature07733

View details for PubMedID 19194462
In Vitro Embryo Culture in Defined, Sub-microliter Volumes DEVELOPMENTAL DYNAMICS Melin, J., Lee, A., Foygel, K., Leong, D. E., Quake, S. R., Yao, M. W. 2009; 238 (4): 950-955
Abstract

The high attrition rate of in vitro human embryo culture presents a major obstacle in the treatment of clinical infertility by in vitro fertilization (IVF). Physical and genetic requirements are not well understood for human or mouse preimplantation embryo development. Group culture is an established requirement for optimal embryo development in the mouse model. However, conventional microdrop culture limitations hinder investigations of the effects of physical parameters on in vitro embryo development. We report a microfluidics platform that enables embryo culture in precisely defined, sub-microliter volumes (5-500 nl) which cannot be investigated using conventional methods. Groups of two embryos per microfluidic well successfully developed to the blastocyst stage, at a rate of over 80%, which is comparable to those cultured in 20-microl microdrops. This system can be used to dissect physical requirements of in vitro single or group embryo culture, and be made highly parallel to increase experimental throughput.

View details for DOI 10.1002/dvdy.21918

View details for Web of Science ID 000264921200015

View details for PubMedID 19301395

View details for PubMedCentralID PMC2678198
Digital PCR provides sensitive and absolute calibration for high throughput sequencing BMC GENOMICS White, R. A., Blainey, P. C., Fan, H. C., Quake, S. R. 2009; 10
Abstract

Next-generation DNA sequencing on the 454, Solexa, and SOLiD platforms requires absolute calibration of the number of molecules to be sequenced. This requirement has two unfavorable consequences. First, large amounts of sample-typically micrograms-are needed for library preparation, thereby limiting the scope of samples which can be sequenced. For many applications, including metagenomics and the sequencing of ancient, forensic, and clinical samples, the quantity of input DNA can be critically limiting. Second, each library requires a titration sequencing run, thereby increasing the cost and lowering the throughput of sequencing.We demonstrate the use of digital PCR to accurately quantify 454 and Solexa sequencing libraries, enabling the preparation of sequencing libraries from nanogram quantities of input material while eliminating costly and time-consuming titration runs of the sequencer. We successfully sequenced low-nanogram scale bacterial and mammalian DNA samples on the 454 FLX and Solexa DNA sequencing platforms. This study is the first to definitively demonstrate the successful sequencing of picogram quantities of input DNA on the 454 platform, reducing the sample requirement more than 1000-fold without pre-amplification and the associated bias and reduction in library depth.The digital PCR assay allows absolute quantification of sequencing libraries, eliminates uncertainties associated with the construction and application of standard curves to PCR-based quantification, and with a coefficient of variation close to 10%, is sufficiently precise to enable direct sequencing without titration runs.

View details for DOI 10.1186/1471-2164-10-116

View details for Web of Science ID 000265791900001

View details for PubMedID 19298667

View details for PubMedCentralID PMC2667538
An in vitro microfluidic approach to generating protein-interaction networks NATURE METHODS Gerber, D., Maerkl, S. J., Quake, S. R. 2009; 6 (1): 71-74
Abstract

We developed an in vitro protein expression and interaction analysis platform based on a highly parallel and sensitive microfluidic affinity assay, and used it for 14,792 on-chip experiments, which exhaustively measured the protein-protein interactions of 43 Streptococcus pneumoniae proteins in quadruplicate. The resulting network of 157 interactions was denser than expected based on known networks. Analysis of the network revealed previously undescribed physical interactions among members of some biochemical pathways.

View details for DOI 10.1038/NMETH.1289

View details for Web of Science ID 000262370200027

View details for PubMedID 19098921
DIGITAL PCR ENABLES RAPID PRENATAL DIAGNOSIS OF FETAL ANEUPLOIDY 29th Annual Meeting of the Society-for-Maternal-Fetal-Medicine Fan, C., Blumenfeld, Y., Chueh, J., El-Sayed, Y., Quake, S. MOSBY-ELSEVIER. 2008: S30?S30
View details for DOI 10.1016/j.ajog.2008.09.089

View details for Web of Science ID 000262205700066
Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Fan, H. C., Blumenfeld, Y. J., Chitkara, U., Hudgins, L., Quake, S. R. 2008; 105 (42): 16266-16271
Abstract

We directly sequenced cell-free DNA with high-throughput shotgun sequencing technology from plasma of pregnant women, obtaining, on average, 5 million sequence tags per patient sample. This enabled us to measure the over- and underrepresentation of chromosomes from an aneuploid fetus. The sequencing approach is polymorphism-independent and therefore universally applicable for the noninvasive detection of fetal aneuploidy. Using this method, we successfully identified all nine cases of trisomy 21 (Down syndrome), two cases of trisomy 18 (Edward syndrome), and one case of trisomy 13 (Patau syndrome) in a cohort of 18 normal and aneuploid pregnancies; trisomy was detected at gestational ages as early as the 14th week. Direct sequencing also allowed us to study the characteristics of cell-free plasma DNA, and we found evidence that this DNA is enriched for sequences from nucleosomes.

View details for DOI 10.1073/pnas.0808319105

View details for Web of Science ID 000260597400037

View details for PubMedID 18838674

View details for PubMedCentralID PMC2562413
PHARMACOLOGICAL INHIBITORS OF A NEW HEPATITIS C TARGET-RNA BINDING BY NS4B-DISCOVERED BY MICROFLUIDIC AFFINITY ANALYSIS 59th Annual Meeting of the American-Association-for-the-Study-of-Liver-Diseases Einav, S., Gerber, D., Bryson, P. D., Sklan, E., Elazar, M., Maerkl, S., Dvory, H. S., Machlin, E., Gu, W., Quake, S., Glenn, J. S. WILEY-BLACKWELL. 2008: 356A?356A
View details for Web of Science ID 000259757400105
Discovery of a hepatitis C target and its pharmacological inhibitors by microfluidic affinity analysis NATURE BIOTECHNOLOGY Einav, S., Gerber, D., Bryson, P. D., Sklan, E. H., Elazar, M., Maerkl, S. J., Glenn, J. S., Quake, S. R. 2008; 26 (9): 1019-1027
Abstract

More effective therapies are urgently needed against hepatitis C virus (HCV), a major cause of viral hepatitis. We used in vitro protein expression and microfluidic affinity analysis to study RNA binding by the HCV transmembrane protein NS4B, which plays an essential role in HCV RNA replication. We show that HCV NS4B binds RNA and that this binding is specific for the 3' terminus of the negative strand of the viral genome with a dissociation constant (Kd) of approximately 3.4 nM. A high-throughput microfluidic screen of a compound library identified 18 compounds that substantially inhibited binding of RNA by NS4B. One of these compounds, clemizole hydrochloride, was found to inhibit HCV RNA replication in cell culture that was mediated by its suppression of NS4B's RNA binding, with little toxicity for the host cell. These results yield new insight into the HCV life cycle and provide a candidate compound for pharmaceutical development.

View details for DOI 10.1038/nbt.1490

View details for PubMedID 18758449
ANYL 350-Highly automated microfluidic system for cell biology 236th National Meeting of the American-Chemical-Society Gomez-Sjoeberg, R., Leyrat, A. A., Quake, S. R. AMER CHEMICAL SOC. 2008
View details for Web of Science ID 000270256301171
Single-molecule DNA sequencing of a viral genome SCIENCE Harris, T. D., Buzby, P. R., Babcock, H., Beer, E., Bowers, J., Braslavsky, I., Causey, M., Colonell, J., DiMeo, J., Efcavitch, J. W., Giladi, E., Gill, J., Healy, J., Jarosz, M., Lapen, D., Moulton, K., Quake, S. R., Steinmann, K., Thayer, E., Tyurina, A., Ward, R., Weiss, H., Xie, Z. 2008; 320 (5872): 106-109
Abstract

The full promise of human genomics will be realized only when the genomes of thousands of individuals can be sequenced for comparative analysis. A reference sequence enables the use of short read length. We report an amplification-free method for determining the nucleotide sequence of more than 280,000 individual DNA molecules simultaneously. A DNA polymerase adds labeled nucleotides to surface-immobilized primer-template duplexes in stepwise fashion, and the asynchronous growth of individual DNA molecules was monitored by fluorescence imaging. Read lengths of >25 bases and equivalent phred software program quality scores approaching 30 were achieved. We used this method to sequence the M13 virus to an average depth of >150x and with 100% coverage; thus, we resequenced the M13 genome with high-sensitivity mutation detection. This demonstrates a strategy for high-throughput low-cost resequencing.

View details for DOI 10.1126/science.1150427

View details for Web of Science ID 000254633000042

View details for PubMedID 18388294
A synthetic Escherichia coli predator-prey ecosystem MOLECULAR SYSTEMS BIOLOGY Balagadde, F. K., Song, H., Ozaki, J., Collins, C. H., Barnet, M., Arnold, F. H., Quake, S. R., You, L. 2008; 4
Abstract

We have constructed a synthetic ecosystem consisting of two Escherichia coli populations, which communicate bi-directionally through quorum sensing and regulate each other's gene expression and survival via engineered gene circuits. Our synthetic ecosystem resembles canonical predator-prey systems in terms of logic and dynamics. The predator cells kill the prey by inducing expression of a killer protein in the prey, while the prey rescue the predators by eliciting expression of an antidote protein in the predator. Extinction, coexistence and oscillatory dynamics of the predator and prey populations are possible depending on the operating conditions as experimentally validated by long-term culturing of the system in microchemostats. A simple mathematical model is developed to capture these system dynamics. Coherent interplay between experiments and mathematical analysis enables exploration of the dynamics of interacting populations in a predictable manner.

View details for DOI 10.1038/msb.2008.24

View details for Web of Science ID 000255551300007

View details for PubMedID 18414488

View details for PubMedCentralID PMC2387235
A microfluidic processor for gene expression profiling of single human embryonic stem cells LAB ON A CHIP Zhong, J. F., Chen, Y., Marcus, J. S., Scherer, A., Quake, S. R., Taylor, C. R., Weiner, L. P. 2008; 8 (1): 68-74
Abstract

The gene expression of human embryonic stem cells (hESC) is a critical aspect for understanding the normal and pathological development of human cells and tissues. Current bulk gene expression assays rely on RNA extracted from cell and tissue samples with various degree of cellular heterogeneity. These 'cell population averaging' data are difficult to interpret, especially for the purpose of understanding the regulatory relationship of genes in the earliest phases of development and differentiation of individual cells. Here, we report a microfluidic approach that can extract total mRNA from individual single-cells and synthesize cDNA on the same device with high mRNA-to-cDNA efficiency. This feature makes large-scale single-cell gene expression profiling possible. Using this microfluidic device, we measured the absolute numbers of mRNA molecules of three genes (B2M, Nodal and Fzd4) in a single hESC. Our results indicate that gene expression data measured from cDNA of a cell population is not a good representation of the expression levels in individual single cells. Within the G0/G1 phase pluripotent hESC population, some individual cells did not express all of the 3 interrogated genes in detectable levels. Consequently, the relative expression levels, which are broadly used in gene expression studies, are very different between measurements from population cDNA and single-cell cDNA. The results underscore the importance of discrete single-cell analysis, and the advantages of a microfluidic approach in stem cell gene expression studies.

View details for DOI 10.1039/b712116d

View details for Web of Science ID 000251771000017

View details for PubMedID 18094763
Transcriptional instability is not a universal attribute of aging AGING CELL Warren, L. A., Rossi, D. J., Schiebinger, G. R., Weissman, I. L., Kim, S. K., Quake, S. R. 2007; 6 (6): 775-782
Abstract

It has been proposed that cumulative somatic mutations contribute to the aging process by disrupting the transcriptional networks that regulate cell structure and function. Experimental support for this model emerged from a recent study of cardiomyocytes that showed a dramatic increase in the transcriptional heterogeneity of these long-lived postmitotic cells with age. To determine if regulatory instability is a hallmark of aging in renewing tissues, we evaluated gene expression noise in four hematopoietic cell types: stem cells, granulocytes, na�ve B cells and na�ve T cells. We used flow cytometry to purify phenotypically equivalent cells from young and old mice, and applied multiplexed quantitative reverse transcription-polymerase chain reaction to measure the copy number of six different mRNA transcripts in 324 individual cells. There was a trend toward higher transcript levels in cells isolated from old animals, but no significant increase in transcriptional heterogeneity with age was found in the surveyed populations. Flow cytometric analysis of membrane protein expression also indicated that cell-to-cell variability was unaffected by age. We conclude that large-scale regulatory destabilization is not a universal concomitant of aging, and may be of significance as an aging mechanism primarily in nonrenewing tissues.

View details for DOI 10.1111/j.1474-9726.2007.00337.x

View details for Web of Science ID 000250938400007

View details for PubMedID 17925006
Versatile, fully automated, microfluidic cell culture system ANALYTICAL CHEMISTRY Gomez-Sjoeberg, R., Leyrat, A. A., Pirone, D. M., Chen, C. S., Quake, S. R. 2007; 79 (22): 8557-8563
Abstract

There is increasing demand for automated and quantitative cell culture technology, driven both by the intense activity in stem cell biology and by the emergence of systems biology. We built a fully automated cell culture screening system based on a microfluidic chip that creates arbitrary culture media formulations in 96 independent culture chambers and maintains cell viability for weeks. Individual culture conditions are customized in terms of cell seeding density, composition of culture medium, and feeding schedule, and each chamber is imaged with time-lapse microscopy. Using this device, we perform the first quantitative measurements of the influence of transient stimulation schedules on the proliferation, osteogenic differentiation, and motility of human primary mesenchymal stem cells.

View details for DOI 10.1021/ac071311w

View details for Web of Science ID 000250937500017

View details for PubMedID 17953452
Detection of aneuploidy with digital polymerase chain reaction ANALYTICAL CHEMISTRY Fan, H. C., Quake, S. R. 2007; 79 (19): 7576-7579
Abstract

The widespread use of genetic testing in high-risk pregnancies has created strong interest in rapid and accurate molecular diagnostics for common chromosomal aneuploidies. We show here that digital polymerase chain reaction (dPCR) can be used for accurate measurement of trisomy 21 (Down syndrome), the most common human aneuploidy. dPCR is generally applicable to any aneuploidy, does not depend on allelic distribution or gender, and is able to detect signals in the presence of mosaics or contaminating maternal DNA.

View details for DOI 10.1021/ac0709394

View details for Web of Science ID 000249871000052

View details for PubMedID 17715994
Nanoliter reactors improve multiple displacement amplification of genomes from single cells PLOS GENETICS Marcy, Y., Ishoey, T., Lasken, R. S., Stockwell, T. B., Walenz, B. P., Halpern, A. L., Beeson, K. Y., Goldberg, S. M., Quake, S. R. 2007; 3 (9): 1702-1708
Abstract

Since only a small fraction of environmental bacteria are amenable to laboratory culture, there is great interest in genomic sequencing directly from single cells. Sufficient DNA for sequencing can be obtained from one cell by the Multiple Displacement Amplification (MDA) method, thereby eliminating the need to develop culture methods. Here we used a microfluidic device to isolate individual Escherichia coli and amplify genomic DNA by MDA in 60-nl reactions. Our results confirm a report that reduced MDA reaction volume lowers nonspecific synthesis that can result from contaminant DNA templates and unfavourable interaction between primers. The quality of the genome amplification was assessed by qPCR and compared favourably to single-cell amplifications performed in standard 50-microl volumes. Amplification bias was greatly reduced in nanoliter volumes, thereby providing a more even representation of all sequences. Single-cell amplicons from both microliter and nanoliter volumes provided high-quality sequence data by high-throughput pyrosequencing, thereby demonstrating a straightforward route to sequencing genomes from single cells.

View details for DOI 10.1371/journal.pgen.0030155

View details for Web of Science ID 000249767800013

View details for PubMedID 17892324

View details for PubMedCentralID PMC1988849
High density single molecule surface patterning with colloidal epitaxy APPLIED PHYSICS LETTERS Schwartz, J. J., Quakea, S. R. 2007; 91 (8)
View details for DOI 10.1063/1.2772762

View details for Web of Science ID 000248984800110
Dissecting biological "dark matter" with single-cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Marcy, Y., Ouverney, C., Bik, E. M., Loesekann, T., Ivanova, N., Martin, H. G., Szeto, E., Platt, D., Hugenholtz, P., Relman, D. A., Quake, S. R. 2007; 104 (29): 11889-11894
Abstract

We have developed a microfluidic device that allows the isolation and genome amplification of individual microbial cells, thereby enabling organism-level genomic analysis of complex microbial ecosystems without the need for culture. This device was used to perform a directed survey of the human subgingival crevice and to isolate bacteria having rod-like morphology. Several isolated microbes had a 16S rRNA sequence that placed them in candidate phylum TM7, which has no cultivated or sequenced members. Genome amplification from individual TM7 cells allowed us to sequence and assemble >1,000 genes, providing insight into the physiology of members of this phylum. This approach enables single-cell genetic analysis of any uncultivated minority member of a microbial community.

View details for DOI 10.1073/pnas.0704662104

View details for Web of Science ID 000248199200007

View details for PubMedID 17620602

View details for PubMedCentralID PMC1924555
Lola regulates Drosophila olfactory projection neuron identity and targeting specificity NEURAL DEVELOPMENT Spletter, M. L., Liu, J., Liu, J., Su, H., Giniger, E., Komiyama, T., Quake, S., Luo, L. 2007; 2
Abstract

Precise connections of neural circuits can be specified by genetic programming. In the Drosophila olfactory system, projection neurons (PNs) send dendrites to single glomeruli in the antenna lobe (AL) based upon lineage and birth order and send axons with stereotyped terminations to higher olfactory centers. These decisions are likely specified by a PN-intrinsic transcriptional code that regulates the expression of cell-surface molecules to instruct wiring specificity.We find that the loss of longitudinals lacking (lola), which encodes a BTB-Zn-finger transcription factor with 20 predicted splice isoforms, results in wiring defects in both axons and dendrites of all lineages of PNs. RNA in situ hybridization and quantitative RT-PCR suggest that most if not all lola isoforms are expressed in all PNs, but different isoforms are expressed at widely varying levels. Overexpression of individual lola isoforms fails to rescue the lola null phenotypes and causes additional phenotypes. Loss of lola also results in ectopic expression of Gal4 drivers in multiple cell types and in the loss of transcription factor gene lim1 expression in ventral PNs.Our results indicate that lola is required for wiring of axons and dendrites of most PN classes, and suggest a need for its molecular diversity. Expression pattern changes of Gal4 drivers in lola-/- clones imply that lola normally represses the expression of these regulatory elements in a subset of the cells surrounding the AL. We propose that Lola functions as a general transcription factor that regulates the expression of multiple genes ultimately controlling PN identity and wiring specificity.

View details for DOI 10.1186/1749-8104-2-14

View details for Web of Science ID 000258981200001

View details for PubMedID 17634136

View details for PubMedCentralID PMC1947980
At the interface of physics and biology BIOTECHNIQUES Quake, S. 2007; 43 (1): 19-19
View details for Web of Science ID 000248207100005

View details for PubMedID 17695250
Crystal structure of a hyperactive Escherichia coli glycerol kinase mutant Gly230 -> Asp obtained using microfluidic crystallization devices BIOCHEMISTRY Anderson, M. J., DeLaBarre, B., Raghunathan, A., Palsson, B. O., Brunger, A. T., Quake, S. R. 2007; 46 (19): 5722-5731
Abstract

The crystal structure of an Escherichia coli glycerol kinase mutant Gly230 --> Asp (GKG230D) was determined to 2.0 A resolution using a microfluidics based crystallization platform. The crystallization strategy involved a suite of microfluidic devices that characterized the solubility trends of GKG230D, performed nanoliter volume free interface diffusion crystallization experiments, and produced diffraction-quality crystals for in situ data collection. GKG230D displays increased enzymatic activity and decreased allosteric regulation by the glycolytic pathway intermediate fructose 1,6-bisphosphate (FBP) compared to wild-type GK (GKWT). Structural analysis revealed that the decreased allosteric regulation is a result of the altered FBP binding loop conformations in GKG230D that interfere with the wild-type FBP binding site. The altered FBP binding loop conformations in GKG230D are supported through a series of intramolecular loop interactions. The appearance of Asp230 in the FBP binding loops also repositions the wild-type FBP binding residues away from the FBP binding site. Light scattering analysis confirmed GKG230D is a dimer and is resistant to tetramer formation in the presence of FBP, whereas GKWT dimers are converted into putatively inactive tetramers in the presence of FBP. GKG230D also provides the first structural evidence for multiple GK monomer conformations in the presence of glycerol and in the absence of a nucleotide substrate and verifies that glycerol binding is not responsible for locking GK into the closed conformation necessary for GK activity.

View details for DOI 10.1021/bi700096p

View details for Web of Science ID 000246283600010

View details for PubMedID 17441732
Experimentally validated quantitative linear model for the device physics of elastomeric microfluidic valves JOURNAL OF APPLIED PHYSICS Kartalov, E. P., Scherer, A., Quake, S. R., Taylor, C. R., French Anderson, W. 2007; 101 (6)
Abstract

A systematic experimental study and theoretical modeling of the device physics of polydimethylsiloxane "pushdown" microfluidic valves are presented. The phase space is charted by 1587 dimension combinations and encompasses 45-295 ?m lateral dimensions, 16-39 ?m membrane thickness, and 1-28 psi closing pressure. Three linear models are developed and tested against the empirical data, and then combined into a fourth-power-polynomial superposition. The experimentally validated final model offers a useful quantitative prediction for a valve's properties as a function of its dimensions. Typical valves (80-150 ?m width) are shown to behave like thin springs.

View details for DOI 10.1063/1.2511688

View details for Web of Science ID 000245317700148

View details for PubMedCentralID PMC2670093
A systems approach to measuring the binding energy landscapes of transcription factors SCIENCE Maerkl, S. J., Quake, S. R. 2007; 315 (5809): 233-237
Abstract

A major goal of systems biology is to predict the function of biological networks. Although network topologies have been successfully determined in many cases, the quantitative parameters governing these networks generally have not. Measuring affinities of molecular interactions in high-throughput format remains problematic, especially for transient and low-affinity interactions. We describe a high-throughput microfluidic platform that measures such properties on the basis of mechanical trapping of molecular interactions. With this platform we characterized DNA binding energy landscapes for four eukaryotic transcription factors; these landscapes were used to test basic assumptions about transcription factor binding and to predict their in vivo function.

View details for DOI 10.1126/science.1131007

View details for Web of Science ID 000243407400045

View details for PubMedID 17218526
Microfluidic large-scale integration: The evolution of design rules for biological automation ANNUAL REVIEW OF BIOPHYSICS AND BIOMOLECULAR STRUCTURE Melin, J., Quake, S. R. 2007; 36: 213-231
Abstract

Microfluidic large-scale integration (mLSI) refers to the development of microfluidic chips with thousands of integrated micromechanical valves and control components. This technology is utilized in many areas of biology and chemistry and is a candidate to replace today's conventional automation paradigm, which consists of fluid-handling robots. We review the basic development of mLSI and then discuss design principles of mLSI to assess the capabilities and limitations of the current state of the art and to facilitate the application of mLSI to areas of biology. Many design and practical issues, including economies of scale, parallelization strategies, multiplexing, and multistep biochemical processing, are discussed. Several microfluidic components used as building blocks to create effective, complex, and highly integrated microfluidic networks are also highlighted.

View details for DOI 10.1146/annurev.biophys.36.040306.132646

View details for Web of Science ID 000247773000011

View details for PubMedID 17269901
Dissecting biological "dark matter" with single cell genetic analysis of rare and uncultivated TM7 microbes from the human mouth Marcy, Y., Ouverney, C., Blik, E. M., Losekann, T., Ivanova, N., Martin, H. G., Quake, S. 2007
View details for DOI 10.1073/pnas.0704662104
Lola regulates Drosophila olfactory projection meuron identity and targeting specificity Neural. Develop. Spletter, M. L., Liu, J., Liu, J., Su, H., Giniger, E., Komiyama, T., Quake, S. 2007; 14 (2)
Crystal Structure of a Hyperactive Escherichia coli Gylcerol Kinase Mutand Gly230Asp Obtained Using Microfluidic Crystallization Devices Biochemistry Anderson, M. J., DeLaBarre, B., Raghunathan, A., Palsson, B. O., Brunger, A. T., Quake, S, R. 2007; 46: 5277-5731
High density single molecule surface patterning with colloidal epitaxy Appl. Phys. Lett. Schwartz, J. J., Quake, S, R. 2007; 91: 83902
The Digital Array Response Curve Preprint. Warren, L. A., Weinstein, J. A., Quake, S, R. 2007
Solvent resistant microfluidic DNA synthesizer LAB ON A CHIP Huang, Y., Castrataro, P., Lee, C., Quake, S. R. 2007; 7 (1): 24-26
Abstract

We fabricated a microfluidic DNA synthesizer out of perfluoropolyether (PFPE), an elastomer with excellent chemical compatibility which makes it possible to perform organic chemical reactions, and synthesized 20-mer oligonucleotides on chip.

View details for DOI 10.1039/b613923j

View details for Web of Science ID 000244616200005

View details for PubMedID 17180201
Fluorescence near-field microscopy of DNA at sub-10 nm resolution PHYSICAL REVIEW LETTERS Ma, Z., Gerton, J. M., Wade, L. A., Quake, S. R. 2006; 97 (26)
Abstract

We demonstrate apertureless near-field microscopy of single molecules at sub-10 nm resolution. With a novel phase filter, near-field images of single organic fluorophores were obtained with approximately sixfold improvement in the signal-to-noise ratio. The improvement allowed pairs of molecules separated by approximately 15 nm to be reliably and repeatedly resolved, thus demonstrating the first true Rayleigh resolution test for near-field images of single molecules. The potential of this technique for biological applications was demonstrated with an experiment that measured the helical rise of A-form DNA.

View details for DOI 10.1103/PhysRevLett.97.260801

View details for Web of Science ID 000243167300006

View details for PubMedID 17280412
Microfluidic digital PCR enables multigene analysis of individual environmental bacteria SCIENCE Ottesen, E. A., Hong, J. W., Quake, S. R., Leadbetter, J. R. 2006; 314 (5804): 1464-1467
Abstract

Gene inventory and metagenomic techniques have allowed rapid exploration of bacterial diversity and the potential physiologies present within microbial communities. However, it remains nontrivial to discover the identities of environmental bacteria carrying two or more genes of interest. We have used microfluidic digital polymerase chain reaction (PCR) to amplify and analyze multiple, different genes obtained from single bacterial cells harvested from nature. A gene encoding a key enzyme involved in the mutualistic symbiosis occurring between termites and their gut microbiota was used as an experimental hook to discover the previously unknown ribosomal RNA-based species identity of several symbionts. The ability to systematically identify bacteria carrying a particular gene and to link any two or more genes of interest to single species residing in complex ecosystems opens up new opportunities for research on the environment.

View details for DOI 10.1126/science.1131370

View details for Web of Science ID 000242406100045

View details for PubMedID 17138901
Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Warren, L., Bryder, D., Weissman, I. L., Quake, S. R. 2006; 103 (47): 17807-17812
Abstract

We report here a systematic, quantitative population analysis of transcription factor expression within developmental progenitors, made possible by a microfluidic chip-based "digital RT-PCR" assay that can count template molecules in cDNA samples prepared from single cells. In a survey encompassing five classes of early hematopoietic precursor, we found markedly heterogeneous expression of the transcription factor PU.1 in hematopoietic stem cells and divergent patterns of PU.1 expression within flk2- and flk2+ common myeloid progenitors. The survey also revealed significant differences in the level of the housekeeping transcript GAPDH across the surveyed populations, which demonstrates caveats of normalizing expression data to endogenous controls and underscores the need to put gene measurement on an absolute, copy-per-cell basis.

View details for DOI 10.1073/pnas.0608512103

View details for Web of Science ID 000242464900042

View details for PubMedID 17098862

View details for PubMedCentralID PMC1693828
Phase knowledge enables rational screens for protein crystallization PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Anderson, M. J., Hansen, C. L., Quake, S. R. 2006; 103 (45): 16746-16751
Abstract

We show that knowledge of the phase behavior of a protein allows one to create a rational screen that increases the success rate of crystallizing challenging proteins. The strategy is based on using microfluidics to perform large numbers of protein solubility experiments across many different chemical conditions to identify reagents for crystallization experiments. Phase diagrams were generated for the identified reagents and used to design customized crystallization screens for every protein. This strategy was applied with a 75% success rate to the crystallization of 12 diverse proteins, most of which failed to crystallize when using traditional techniques. The overall diffraction success rate was 33%, about double what was achieved with conventional automation in large-scale protein structure consortia. The higher diffraction success rates are achieved by designing customized crystallization screens using the phase behavior information for each target. The identification of reagents based on an understanding of protein solubility and the use of phase diagrams in the design of individualized crystallization screens therefore promotes high crystallization rates and the production of diffraction-quality crystals.

View details for DOI 10.1073/pnas.0605293103

View details for Web of Science ID 000241969500024

View details for PubMedID 17075056

View details for PubMedCentralID PMC1636526
Biological large scale integration Quake, S. AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC. 2006: S379?S379
View details for Web of Science ID 000241506401456
Developing optofluidic technology through the fusion of microfluidics and optics NATURE Psaltis, D., Quake, S. R., Yang, C. 2006; 442 (7101): 381-386
Abstract

We describe devices in which optics and fluidics are used synergistically to synthesize novel functionalities. Fluidic replacement or modification leads to reconfigurable optical systems, whereas the implementation of optics through the microfluidic toolkit gives highly compact and integrated devices. We categorize optofluidics according to three broad categories of interactions: fluid-solid interfaces, purely fluidic interfaces and colloidal suspensions. We describe examples of optofluidic devices in each category.

View details for DOI 10.1038/nature05060

View details for Web of Science ID 000239278900030

View details for PubMedID 16871205
Molecular biology on a microfluidic chip JOURNAL OF PHYSICS-CONDENSED MATTER Hong, J. W., Chen, Y., Anderson, W. F., Quake, S. R. 2006; 18 (18): S691-S701
View details for DOI 10.1088/0953-8984/18/18/S14

View details for Web of Science ID 000237968200015
The biological frontier of physics PHYSICS TODAY Phillips, R., Quake, S. R. 2006; 59 (5): 38-43
View details for Web of Science ID 000237264200018
Microfluidic single-cell mRNA isolation and analysis ANALYTICAL CHEMISTRY Marcus, J. S., Anderson, W. F., Quake, S. R. 2006; 78 (9): 3084-3089
Abstract

Single-cell gene expression analysis holds great promise for studying diverse biological systems, but methodology to process these precious samples in a reproducible, quantitative, and parallel fashion remains challenging. Here, we utilize microfluidics to isolate picogram and subpicogram mRNA templates, as well as to synthesize cDNA from these templates. We demonstrate single-cell mRNA isolation and cDNA synthesis, provide quantitative calibrations for each step in the process, and measure gene expression in individual cells. The techniques presented here form the foundation for highly parallel single-cell gene expression studies.

View details for DOI 10.1021/ac0519460

View details for Web of Science ID 000237456400031

View details for PubMedID 16642997
Microfabricated rubber microscope using soft solid immersion lenses APPLIED PHYSICS LETTERS Gambin, Y., Legrand, O., Quake, S. R. 2006; 88 (17)
View details for DOI 10.1063/1.2194477

View details for Web of Science ID 000237136600097
Microfluidic platform for production of 18F-radiolabeled PET imaging probes 231st National Meeting of the American-Chemical-Society Sui, G., Lee, C., Elizarov, A., Satyamurthy, N., Heath, J. R., Phelps, M. E., Quake, S. R., Tseng, H. AMER CHEMICAL SOC. 2006
View details for Web of Science ID 000238125907256
A microfluidic device for kinetic optimization of protein crystallization and in situ structure determination JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Hansen, C. L., Classen, S., Berger, J. M., Quake, S. R. 2006; 128 (10): 3142-3143
Abstract

The unprecedented economies of scale and unique mass transport properties of microfluidic devices made them viable nano-volume protein crystallization screening platforms. However, realizing the full potential of microfluidic crystallization requires complementary technologies for crystal optimization and harvesting. In this paper, we report a microfluidic device which provides a link between chip-based nanoliter volume crystallization screening and structure analysis through "kinetic optimization" of crystallization reactions and in situ structure determination. Kinetic optimization through systematic variation of reactor geometry and actuation of micromechanical valves is used to screen a large ensemble of kinetic trajectories that are not practical with conventional techniques. Using this device, we demonstrate control over crystal quality, reliable scale-up from nanoliter volume reactions, facile harvesting and cryoprotectant screening, and protein structure determination at atomic resolution from data collected in-chip.

View details for DOI 10.1021/ja0576637

View details for Web of Science ID 000236035100018

View details for PubMedID 16522084
Anomalous vibrational dispersion in holographically trapped colloidal arrays PHYSICAL REVIEW LETTERS Polin, M., Grier, D. G., Quake, S. R. 2006; 96 (8)
Abstract

Colloidal spheres localized in an array of harmonic wells form a thermally excited, viscously damped dynamical system capable of supporting propagating elastic waves. Experimentally realized with micrometer-scale polystyrene spheres localized in a line of holographic optical traps, the hydrodynamically coupled array's behavior is quantitatively explained by a model based on the Oseen superposition approximation. The spheres' purely dissipative coupling is predicted to mediate a crossover to a regime of underdamped propagating elastic waves with uniformly negative group velocities that has yet to be verified experimentally.

View details for DOI 10.1103/PhysRevLett.96.088101

View details for Web of Science ID 000235736200074

View details for PubMedID 16606228
Parallel picoliter RT-PCR assays using microfluidics ANALYTICAL CHEMISTRY Marcus, J. S., Anderson, W. F., Quake, S. R. 2006; 78 (3): 956-958
Abstract

The development of microfluidic tools for high-throughput nucleic acid analysis has become a burgeoning area of research in the post-genome era. Here, we have developed a microfluidic chip to perform 72 parallel 450-pL RT-PCRs. We took advantage of Taqman hydrolysis probe chemistry to detect RNA templates as low as 34 copies. The device and method presented here may enable highly parallel single cell gene expression analysis.

View details for DOI 10.1021/ac0513865

View details for Web of Science ID 000235195200056

View details for PubMedID 16448074
Enhanced signals and fast nucleic acid hybridization by microfluidic chaotic mixing ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Liu, J., Williams, B. A., Gwirtz, R. M., Wold, B. J., Quake, S. 2006; 45 (22): 3618-3623
View details for DOI 10.1002/anie.200503830

View details for Web of Science ID 000238068000009

View details for PubMedID 16639763
A microfluidic device for kinetic optimization of protein crystallization and in situ structure determination J. Am. Chem. Soc. Hansen, C. L., Classen, S., Berger, J. M., Quake, S, R. 2006; 10 (128): 3142-3
Microfluidic Digital PCR Enables Multigene Analysis of Individual Environmental Bacteria Science. Ottesen, E. A., Hong, J. W., Quake, S. R., Leadbetter, J, R. 2006; 314: 1464-67
Transcription factor profiling in individual hematopoietic progenitors by digital RT-PCR Warren, L., Bryder, D., Weissman, I. L., Quake, S, R. 2006
View details for DOI 10.1073/pnas.0608512103
Parallel Picoliter rt-PCR Assays Using Microfluidics. Anal. Chem. Marcus, J. S., erson, W. F., Quake, S, R. 2006; 3 (78): 956-8
Microfluidic Single-Cell mRNA Isolation and Analysis Anal. Chem. Marcus, J. S., erson, W. F., Quake, S, R. 2006
View details for DOI 10.1021/ac0518506
Anomalous Vibrational Dispersion in Holographically Trapped Colloidal Arrays Phys. Rev. Lett. Polin, M., Grier, D. G., Quake, S, R. 2006; 8 (96): 88101
Molecular Biology on a Microfluidic Chip J. Cond. Matt. Phys . Hong, J. W., Chen, Y., Anderson, W. F., Quake, S, R. 2006; 18 (18): S691-S701
Microfabricated rubber microscope using soft solid immersion lenses Appl. Phys. Lett. Gambin, Y., Legrand, O., Quake, S, R. 2006; 17 (88): 174102
Fluorescence Near-Field Microscopy of DNA at Sub-10nm Resolution Phys. Rev. Lett. Ma, Z., Gerton, J. M., Wade, L. A., Quake, S, R. 2006; 26 (97): 260801
Effects of a Modified Dye-Labeled Nucleotide Spacer Arm on Incorporation Thermophilic DNA Polymerases Nucleosides, Nucleotides & Nucleic Acids. Lacenere, C. J., Garg, N. K., Stoltz, B. M., Quake, S, R. 2006; 25: 9-15
High-throughput multi-antigen microfluidic fluorescence immunoassays BIOTECHNIQUES Kartalov, E. P., Zhong, J. F., Scherer, A., Quake, S. R., Taylor, C. R., Anderson, W. F. 2006; 40 (1): 85-90
Abstract

Here we describe the development of a high-throughput multi-antigen microfluidic fluorescence immunoassay system. A 100-chamber polydimethylsiloxane (PDMS) chip performs up to 5 tests for each of 10 samples. In this particular study system, the specificity of detection was demonstrated, and calibration curves were produced for C-reactive protein (CRP), prostate-specific antigen (PSA), ferritin, and vascular endothelial growth factor (VEGF). The measurements show sensitivity at and below clinically normal levels (with a signal-to-noise ratio >8 at as low as 10 pM antigen concentration). The chip uses 100 nL per sample for all tests. The developed system is an important step toward derivative immunoassay applications in scientific research and "point-of-care" testing in medicine.

View details for DOI 10.2144/000112071

View details for Web of Science ID 000234806900012

View details for PubMedID 16454045
Effects of a modified dye-labeled nucleotide spacer arm on incorporation by thermophilic DNA polymerases NUCLEOSIDES NUCLEOTIDES & NUCLEIC ACIDS Lacenere, C. J., Garg, N. K., Stoltz, B. M., Quake, S. R. 2006; 25 (1): 9-15
Abstract

The ability of eight commercially available thermophilic DNA polymerases to sequentially incorporate fluorescently labeled nucleotides sequentially was analyzed by a gel based primer extension assay. Cy5-dUTP or a variant nucleotide in which the linker had been lengthened by 14 atoms between the dye and the nucleobase were compared. We found that the Cy5-dUTP with a longer linker resulted in longer primer extension lengths. Furthermore, some of the assayed polymerases are capable of extending the primer to the full or near full length of 30 nucleotides using dye-labeled nucleotides exclusively.

View details for DOI 10.1080/15257770500377714

View details for Web of Science ID 000234388200002

View details for PubMedID 16440981
Multistep synthesis of a radiolabeled imaging probe using integrated microfluidics SCIENCE Lee, C. C., Sui, G. D., Elizarov, A., Shu, C. Y., SHIN, Y. S., Dooley, A. N., Huang, J., Daridon, A., Wyatt, P., Stout, D., Kolb, H. C., Witte, O. N., Satyamurthy, N., Heath, J. R., Phelps, M. E., Quake, S. R., Tseng, H. R. 2005; 310 (5755): 1793-1796
Abstract

Microreactor technology has shown potential for optimizing synthetic efficiency, particularly in preparing sensitive compounds. We achieved the synthesis of an [(18)F]fluoride-radiolabeled molecular imaging probe, 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG), in an integrated microfluidic device. Five sequential processes-[18F]fluoride concentration, water evaporation, radiofluorination, solvent exchange, and hydrolytic deprotection-proceeded with high radio-chemical yield and purity and with shorter synthesis time relative to conventional automated synthesis. Multiple doses of [18F]FDG for positron emission tomography imaging studies in mice were prepared. These results, which constitute a proof of principle for automated multistep syntheses at the nanogram to microgram scale, could be generalized to a range of radiolabeled substrates.

View details for DOI 10.1126/science.1118919

View details for Web of Science ID 000234093600040

View details for PubMedID 16357255
Multi-step preparation of PET imaging probes in integrated microfluidic circuits 230th National Meeting of the American-Chemical-Society Sui, G., Lee, C., Satyamurthy, N., Heath, J. R., Phelps, M. E., Quake, S. R., Tseng, H. AMER CHEMICAL SOC. 2005: U2757?U2758
View details for Web of Science ID 000236797305437
Polydimethylsiloxane based microfluidic diode JOURNAL OF MICROMECHANICS AND MICROENGINEERING Adams, M. L., Johnston, M. L., Scherer, A., Quake, S. R. 2005; 15 (8): 1517-1521
View details for DOI 10.1088/0960-1317/15/8/020

View details for Web of Science ID 000231512500020
Long-term monitoring of bacteria undergoing programmed population control in a microchemostat SCIENCE Balagadde, F. K., You, L. C., Hansen, C. L., Arnold, F. H., Quake, S. R. 2005; 309 (5731): 137-140
Abstract

Using an active approach to preventing biofilm formation, we implemented a microfluidic bioreactor that enables long-term culture and monitoring of extremely small populations of bacteria with single-cell resolution. We used this device to observe the dynamics of Escherichia coli carrying a synthetic "population control" circuit that regulates cell density through a feedback mechanism based on quorum sensing. The microfluidic bioreactor enabled long-term monitoring of unnatural behavior programmed by the synthetic circuit, which included sustained oscillations in cell density and associated morphological changes, over hundreds of hours.

View details for DOI 10.1126/science.1109173

View details for Web of Science ID 000230212800077

View details for PubMedID 15994559
Microfluidics: Fluid physics at the nanoliter scale REVIEWS OF MODERN PHYSICS Squires, T. M., Quake, S. R. 2005; 77 (3): 977-1026
View details for Web of Science ID 000232443800008
A ligand-free solid-supported system for Sonogashira couplings: applications in nucleoside chemistry CHEMICAL COMMUNICATIONS Garg, N. K., Woodroofe, C. C., Lacenere, C. J., Quake, S. R., Stoltz, B. M. 2005: 4551-4553
Abstract

A mild heterogeneous, ligand-free protocol for Sonogashira and Heck couplings has been developed and used to access several biologically important deoxynucleoside derivatives in a facile manner.

View details for DOI 10.1039/b505737j

View details for Web of Science ID 000231809200014

View details for PubMedID 16158111
Spectrographic microfluidic memory 3rd International Conference on Microchannels and Minichannels Erickson, D., Li, B., Adleman, J. R., Vyawahare, S., Quake, S., Psaltis, D. AMER SOC MECHANICAL ENGINEERS. 2005: 563?568
View details for Web of Science ID 000243025800078
An integrated microfluidic blood sampler for determination of blood input function in quantitative mouse microPET studies. Nuclear Science Symposium/Medical Imaging Conference Wu, H., Sui, G., Lee, C., Ladno, W., Quake, S. R., Tseng, H., Huang, S. IEEE. 2005: 1658?1661
View details for Web of Science ID 000241851902100
Long-term monitoring of bacteria undergoing programmed population control in a microchemostat. Science Balagadde, F. K., You, L., Hansen, C. L., Arnold, F. H., Quake, S, R. 2005; 5731 (309): 137-40
Microfluidics: Fluid physics at the nanoliter scale Rev. Mod. Phys. Squires, T. M., Quake, S, R. 2005; 3 (7): 977-1026
Multistep Synthesis of a Radiolabeled Imaging Probe Using Integrated Microfluidics Science. Lee, C. C., Sui, G., Elizarov, A., Shu, C. J., Shin, Y. S., Dooley, A. N., Quake, S. 2005: 1793-1796.
Programmed population control by cell-cell communication in microfluidic chemostats 49th Annual Meeting of the Biophysical-Society Balagadde, F. K., You, L. C., Arnold, F. H., Quake, S. R. CELL PRESS. 2005: 519A?519A
View details for Web of Science ID 000226378502536
Tip-enhanced fluorescence microscopy at 10 nanometer resolution PHYSICAL REVIEW LETTERS Gerton, J. M., Wade, L. A., Lessard, G. A., Ma, Z., Quake, S. R. 2004; 93 (18)
Abstract

We demonstrate unambiguously that the field enhancement near the apex of a laser-illuminated silicon tip decays according to a power law that is moderated by a single parameter characterizing the tip sharpness. Oscillating the probe in intermittent contact with a semiconductor nanocrystal strongly modulates the fluorescence excitation rate, providing robust optical contrast and enabling excellent background rejection. Laterally encoded demodulation yields images with <10 nm spatial resolution, consistent with independent measurements of tip sharpness.

View details for DOI 10.1103/PhysRevLett.93.180801

View details for Web of Science ID 000224799500014

View details for PubMedID 15525147
Systematic investigation of protein phase behavior with a microfluidic formulator PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hansen, C. L., Sommer, M. O., Quake, S. R. 2004; 101 (40): 14431-14436
Abstract

We demonstrated a microfluidic device for rapidly generating complex mixtures of 32 stock reagents in a 5-nl reactor. This "formulation chip" is fully automated and allows thousands of experiments to be performed in a single day with minimal reagent consumption. It was applied to systematically study the phase behavior of the protein xylanase over a large and complex chemical space. For each chemical formulation that demonstrated a pronounced effect on solubility, the protein phase behavior was completely mapped in the chip, generating a set of empirical phase diagrams. This ab initio phase information was used to devise a rational crystallization screen that resulted in 72-fold improvement in successful crystallization hits compared with conventional sparse matrix screens. This formulations tool allows a physics-based approach to protein crystallization that may prove useful in structural genomics efforts.

View details for DOI 10.1073/pnas.0405847101

View details for Web of Science ID 000224369600024

View details for PubMedID 15452343
Nanometer-scale fluorescence resonance optical waveguides NANO LETTERS Vyawahare, S., Eyal, S., Mathews, K. D., Quake, S. R. 2004; 4 (6): 1035-1039
View details for DOI 10.1021/nl049660i

View details for Web of Science ID 000221978700006
Microfluidic device reads up to four consecutive base pairs in DNA sequencing-by-synthesis NUCLEIC ACIDS RESEARCH Kartalov, E. P., Quake, S. R. 2004; 32 (9): 2873-2879
Abstract

We have developed the first fully integrated microfluidic system for DNA sequencing-by-synthesis. Using this chip and fluorescence detection, we have reliably sequenced up to 4 consecutive bps. The described sequencer can be integrated with other microfluidic components on the same chip to produce true lab-on-a-chip technology. The surface chemistry that was designed to anchor the DNA to elastomeric microchannels is useful in a broad range of studies and applications.

View details for DOI 10.1093/nar/gkh613

View details for Web of Science ID 000221746900031

View details for PubMedID 15155856
A nanoliter-scale nucleic acid processor with parallel architecture NATURE BIOTECHNOLOGY Hong, J. W., Studer, V., Hang, G., Anderson, W. F., Quake, S. R. 2004; 22 (4): 435-439
Abstract

The purification of nucleic acids from microbial and mammalian cells is a crucial step in many biological and medical applications. We have developed microfluidic chips for automated nucleic acid purification from small numbers of bacterial or mammalian cells. All processes, such as cell isolation, cell lysis, DNA or mRNA purification, and recovery, were carried out on a single microfluidic chip in nanoliter volumes without any pre- or postsample treatment. Measurable amounts of mRNA were extracted in an automated fashion from as little as a single mammalian cell and recovered from the chip. These microfluidic chips are capable of processing different samples in parallel, thereby illustrating how highly parallel microfluidic architectures can be constructed to perform integrated batch-processing functionalities for biological and medical applications.

View details for DOI 10.1038/nbt951

View details for Web of Science ID 000220610100032

View details for PubMedID 15024389
Correlating AFM probe morphology to image resolution for single-wall carbon nanotube tips NANO LETTERS Wade, L. A., Shapiro, I. R., Ma, Z. Y., Quake, S. R., Collier, C. P. 2004; 4 (4): 725-731
View details for DOI 10.1021/nl049976q

View details for Web of Science ID 000220857800037
A microfluidic rectifier: Anisotropic flow resistance at low Reynolds numbers PHYSICAL REVIEW LETTERS Groisman, A., Quake, S. R. 2004; 92 (9)
Abstract

It is one of the basic concepts of Newtonian fluid dynamics that at low Reynolds number (Re) the Navier-Stokes equation is linear and flows are reversible. In microfluidic devices, where Re is essentially always low, this implies that flow resistance in microchannels is isotropic. Here we present a microfluidic rectifier: a microscopic channel of a special shape whose flow resistance is strongly anisotropic, differing by up to a factor of 2 for opposite flow directions. Its nonlinear operation at arbitrary small Re is due to non-Newtonian elastic properties of the working fluid, which is a 0.01% aqueous solution of a high molecular weight polymer. The rectifier works as a dynamic valve and may find applications in microfluidic pumps and other integrated devices.

View details for DOI 10.1103/PhysRevLett.92.094501

View details for Web of Science ID 000220055400017

View details for PubMedID 15089471
Solvent-resistant photocurable liquid fluoropolymers for microfluidic device fabrication [corrected]. Journal of the American Chemical Society Rolland, J. P., van Dam, R. M., Schorzman, D. A., Quake, S. R., DeSimone, J. M. 2004; 126 (8): 2322-2323
Abstract

We report the first fabrication of a solvent-compatible microfluidic device based on photocurable "Liquid Teflon" materials. The materials are highly fluorinated functionalized perfluoropolyethers (PFPEs) that have liquidlike viscosities that can be cured into tough, highly durable elastomers that exhibit the remarkable chemical resistance of fluoropolymers such as Teflon. Poly(dimethylsiloxane) (PDMS) elastomers have rapidly become the material of choice for many recent microfluidic device applications. Despite the advantages of PDMS in relation to microfluidics technology, the material suffers from a serious drawback in that it swells in most organic solvents. The swelling of PDMS-based devices in organic solvents greatly disrupts the micrometer-sized features and makes it impossible for fluids to flow inside the channels. Our approach to this problem has been to replace PDMS with photocurable perfluoropolyethers. Device fabrication and valve actuation were accomplished using established procedures for PDMS devices. The additional advantage of photocuring allows fabrication time to be decreased from several hours to a matter of minutes. The PFPE-based device exhibited mechanical properties similar to those of Sylgard 184 before and after curing as well as remarkable resistance to organic solvents. This work has the potential to expand the field of microfluidics to many novel applications.

View details for PubMedID 14982433
Solvent-resistant photocurable "liquid teflon" for microfluidic device fabrication JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Rolland, J. P., van Dam, R. M., Schorzman, D. A., Quake, S. R., DeSimone, J. M. 2004; 126 (8): 2322-2323
View details for DOI 10.1021/ja031657y

View details for Web of Science ID 000189279700032
Correlating AFM Probe Morphology to Image Resolution for Single-Wall Carbon Nanotube Tips Nano Lett. Wade, L. A., Shaprio, I. R., Ma, Z. 2004; 4 (4): 725-731
Scaling properties of a low-actuation pressure microfluidic valve JOURNAL OF APPLIED PHYSICS Studer, V., Hang, G., Pandolfi, A., Ortiz, M., Anderson, W. F., Quake, S. R. 2004; 95 (1): 393-398
View details for DOI 10.1063/1.1629781

View details for Web of Science ID 000187341900062
Scaling properties of a low-actuation pressure microfluidic valve J. Appl. Phys. Studer, V., Hang, G., Pandolfi, A., Ortiz, M., Anderson, W. F., Quake, S, R. 2004; 1 (95): 393-398
Solvent-resistant photocurable liquid fluoropolymers for microfluidic device fabrication J. Am. Chem. Soc. Rolland, J. P., Van Dam, R. M., Schorzman, D. A., Quake, S. R., DeSimone, J, M. 2004; 8 (126): 2322-3
Nanometer-scale Fluorescence Resonance Optical Waveguides Nano Lett. Vyawahare, S., Eyal, S., Mathews, S., Quake, K., D. 2004; 6 (4): 1035-1039
Behavior of complex knots in single DNA molecules PHYSICAL REVIEW LETTERS Bao, X. R., Lee, H. J., Quake, S. R. 2003; 91 (26)
Abstract

We used optical tweezers to tie individual DNA molecules in knots. Although these knots become highly localized under tension, they remain surprisingly mobile and undergo thermal diffusion with classical random walk statistics. The diffusion constants of knots with different complexities correlate with theoretical calculations of knot sizes. We show that this correlation can be explained by a simple hydrodynamical model of "self-reptation" of the knot along a polymer.

View details for DOI 10.1103/PhysRevLett.91.265506

View details for Web of Science ID 000187719300038

View details for PubMedID 14754067
Generation of uniform photonic balls by template-assisted colloidal crystallization 5th International Topical Conference on Optical Probes of Conjugated Polymers, Organic and Inorganic Nanostructure Yi, G. R., Jeon, S. J., Thorsen, T., Manoharan, V. N., Quake, S. R., Pine, D. J., Yang, S. M. ELSEVIER SCIENCE SA. 2003: 803?6
View details for DOI 10.1016/S0379-6779(03)00246-7

View details for Web of Science ID 000185303600061
Microfabricated fountain pens for high-density DNA arrays GENOME RESEARCH Reese, M. O., van Dam, R. M., Scherer, A., Quake, S. R. 2003; 13 (10): 2348-2352
Abstract

We used photolithographic microfabrication techniques to create very small stainless steel fountain pens that were installed in place of conventional pens on a microarray spotter. Because of the small feature size produced by the microfabricated pens, we were able to print arrays with up to 25,000 spots/cm2, significantly higher than can be achieved by other deposition methods. This feature density is sufficiently large that a standard microscope slide can contain multiple replicates of every gene in a complex organism such as a mouse or human. We tested carryover during array printing with dye solution, labeled DNA, and hybridized DNA, and we found it to be indistinguishable from background. Hybridization also showed good sequence specificity to printed oligonucleotides. In addition to improved slide capacity, the microfabrication process offers the possibility of low-cost mass-produced pens and the flexibility to include novel pen features that cannot be machined with conventional techniques.

View details for DOI 10.1101/gr.623903

View details for Web of Science ID 000185876400017

View details for PubMedID 12975313
Integrated nanoliter systems NATURE BIOTECHNOLOGY Hong, J. W., Quake, S. R. 2003; 21 (10): 1179-1183
Abstract

Microfluidic chip platforms for manipulating liquid volumes in the nanoliter range are slowly inching their way into mainstream genomic and proteomic research. The principal challenge faced by these technologies is the need for high-throughput processing of increasingly smaller volumes, with ever higher degrees of parallelization. Significant advances have been made over the past few years in addressing these needs through electrokinetic manipulation, vesicle encapsulation and mechanical valve approaches. These strategies allow levels of integration density and platform complexity that promise to make them into serious alternatives to current robotic systems.

View details for DOI 10.1038/nbt871

View details for Web of Science ID 000185647200029

View details for PubMedID 14520403
Microfluidics in structural biology: smaller, faster... better CURRENT OPINION IN STRUCTURAL BIOLOGY Hansen, C., Quake, S. R. 2003; 13 (5): 538-544
Abstract

Microfluidic technologies promise unprecedented savings in cost and time through the integration of complex chemical and biological assays on a microfabricated chip. Recent advances are making elements of this vision a reality, facilitating the first large-scale integration of microfluidic plumbing with biological assays. The power of miniaturization lies not only in achieving an economy of scale, but also in exploiting the unusual physics of fluid flow and mass transport on small length scales to realize precise and efficient assays that are not accessible with macroscopic tools. Diverse applications ranging from time-resolved studies of protein folding to highly efficient protein crystal growth suggest that microfluidics may become an indispensable tool in biology.

View details for DOI 10.1016/j.sbi.2003.09.010

View details for Web of Science ID 000186317900002

View details for PubMedID 14568607
Solving the "world-to-chip" interface problem with a microfluidic matrix ANALYTICAL CHEMISTRY Liu, J., Hansen, C., Quake, S. R. 2003; 75 (18): 4718-4723
Abstract

We report an effective solution to the macroscopic/microfluidic interface issue and demonstrate how microfluidics can achieve impressive economies of scale in reducing the complexity of pipetting operations. Using an N x N microfluidic matrix with N = 20, we performed N2 = 400 distinct PCR reactions with only 2N + 1 = 41 pipetting steps, compared with the 3N2 = 1200 steps required with conventional fluid handling. Each vertex of the matrix has a 3-nL reactor, and a single 2-microL aliquot of polymerase is amortized over all 400 independent reactions, thus dramatically reducing sample overhead and minimizing reagent consumption. Beyond PCR, the matrix chip provides a general method to perform chemical and biological experiments with precious reagents in a highly automated fashion.

View details for DOI 10.1021/ac0346407

View details for Web of Science ID 000185439300002

View details for PubMedID 14674446
Generation of uniform colloidal assemblies in soft microfluidic devices ADVANCED MATERIALS Yi, G. R., Thorsen, T., Manoharan, V. N., Hwang, M. J., Jeon, S. J., Pine, D. J., Quake, S. R., Yang, S. M. 2003; 15 (15): 1300-?
View details for DOI 10.1002/adma.200304890

View details for Web of Science ID 000184798700017
Microfluidic memory and control devices SCIENCE Groisman, A., Enzelberger, M., Quake, S. R. 2003; 300 (5621): 955-958
Abstract

We demonstrate microscopic fluidic control and memory elements through the use of an aqueous viscoelastic polymer solution as a working fluid. By exploiting the fluid's non-Newtonian rheological properties, we were able to demonstrate both a flux stabilizer and a bistable flip-flop memory. These circuit elements are analogous to their solid-state electronic counterparts and could be used as components of control systems for integrated microfluidic devices. Such miniaturized fluidic circuits are insensitive to electromagnetic interference and may also find medical applications for implanted drug-delivery devices.

View details for Web of Science ID 000182719800049

View details for PubMedID 12738857
Sequence information can be obtained from single DNA molecules PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Braslavsky, I., Hebert, B., Kartalov, E., Quake, S. R. 2003; 100 (7): 3960-3964
Abstract

The completion of the human genome draft has taken several years and is only the beginning of a period in which large amounts of DNA and RNA sequence information will be required from many individuals and species. Conventional sequencing technology has limitations in cost, speed, and sensitivity, with the result that the demand for sequence information far outstrips current capacity. There have been several proposals to address these issues by developing the ability to sequence single DNA molecules, but none have been experimentally demonstrated. Here we report the use of DNA polymerase to obtain sequence information from single DNA molecules by using fluorescence microscopy. We monitored repeated incorporation of fluorescently labeled nucleotides into individual DNA strands with single base resolution, allowing the determination of sequence fingerprints up to 5 bp in length. These experiments show that one can study the activity of DNA polymerase at the single molecule level with single base resolution and a high degree of parallelization, thus providing the foundation for a practical single molecule sequencing technology.

View details for DOI 10.1073/pnas.0230489100

View details for Web of Science ID 000182058400078

View details for PubMedID 12651960
Microfluidic integration on detector arrays for absorption and fluorescence micro-spectrometers SENSORS AND ACTUATORS A-PHYSICAL Adams, M. L., Enzelberger, M., Quake, S., Scherer, A. 2003; 104 (1): 25-31
View details for DOI 10.1016/S0924-4247(02)00477-6

View details for Web of Science ID 000181236100005
Polyelectrolyte surface interface for single-molecule fluorescence studies of DNA polymerase BIOTECHNIQUES Kartalov, E. P., Unger, M. A., Quake, S. R. 2003; 34 (3): 505-?
Abstract

We report the use of polyelectrolyte multilayers in a stable robust surface chemistry for specific anchoring of DNA to glass. The nonspecific binding of fluorescently tagged nucleotides is suppressed down to the single-molecule level, and DNA polymerase is active on the anchored DNA template. This surface-chemistry platform can be used for single-molecule studies of DNA and DNA polymerase and may be more broadly applicable for other situations in which it is important to have specific biomolecular surface chemistry with extremely low nonspecific binding.

View details for Web of Science ID 000181471500014

View details for PubMedID 12661156
Quantifying double-strand breaks and clustered damages in DNA by single-molecule laser fluorescence sizing BIOPHYSICAL JOURNAL Filippova, E. M., Monteleone, D. C., Trunk, J. G., Sutherland, B. M., Quake, S. R., Sutherland, J. C. 2003; 84 (2): 1281-1290
Abstract

Fluorescence from a single DNA molecule passing through a laser beam is proportional to the size (contour length) of the molecule, and molecules of different sizes can be counted with equal efficiencies. Single-molecule fluorescence can thus determine the average length of the molecules in a sample and hence the frequency of double-strand breaks induced by various treatments. Ionizing radiation-induced frank double-strand breaks can thus be quantified by single-molecule sizing. Moreover, multiple classes of clustered damages involving damaged bases and abasic sites, alone or in combination with frank single-strand breaks, can be quantified by converting them to double-strand breaks by chemical or enzymatic treatments. For a given size range of DNA molecules, single-molecule sizing is as or more sensitive than gel electrophoresis, and requires several orders-of-magnitude less DNA to determine damage levels.

View details for Web of Science ID 000183123700052

View details for PubMedID 12547808
Generation of uniform photonic balls by template-assisted colloidal crystallization Synth. Mat. Yi, G. R., Jeon, S. J., Thorsen, T., Manoharan, V. N., Quake, S. R., Pine, D. J. 2003; 3 (139): 803-806
Single-molecule fluorescence and force microscopy employing carbon nanotubes Nanotechnology Conference and Trade Show (Nanotech 2003) Wade, L. A., Shapiro, I., Ma, Z. Y., Quake, S. R., Collier, C. P. COMPUTATIONAL PUBLICATIONS. 2003: 317?320
View details for Web of Science ID 000223047000085
Microfluidics in structural biology: smaller, faster ... better. Curr Opin Struct Biol. Hansen, C., Quake, S, R. 2003; 5 (13): 538-44
Single Molecule Fluorescence and Force Microscopy Employing Carbon Nanotubes Nanotech Wade, L. A., Shapiro, I., Ma, Z., Quake, S. R., Collier, C, P. 2003; 3: 317-320
Generation of uniform colloidal assemblies in soft microfluidic devices Adv. Mat. Yi, G. R., Thorsen, T., Manoharan, V. N., Hwang, M. J., Jeon, S. J., Pine, D. J., Quake, S. 2003; 15 (15): 1300+
A robust and scalable microfluidic metering method that allows protein crystal growth by free interface diffusion PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hansen, C. L., Skordalakes, E., Berger, J. M., Quake, S. R. 2002; 99 (26): 16531-16536
Abstract

Producing robust and scalable fluid metering in a microfluidic device is a challenging problem. We developed a scheme for metering fluids on the picoliter scale that is scalable to highly integrated parallel architectures and is independent of the properties of the working fluid. We demonstrated the power of this method by fabricating and testing a microfluidic chip for rapid screening of protein crystallization conditions, a major hurdle in structural biology efforts. The chip has 480 active valves and performs 144 parallel reactions, each of which uses only 10 nl of protein sample. The properties of microfluidic mixing allow an efficient kinetic trajectory for crystallization, and the microfluidic device outperforms conventional techniques by detecting more crystallization conditions while using 2 orders of magnitude less protein sample. We demonstrate that diffraction-quality crystals may be grown and harvested from such nanoliter-volume reactions.

View details for Web of Science ID 000180101600008

View details for PubMedID 12486223
Number, density, and surface/cytoplasmic distribution of GABA transporters at presynaptic structures of knock-in mice carrying GABA transporter subtype 1-green fluorescent protein fusions JOURNAL OF NEUROSCIENCE Chiu, C. S., Jensen, K., Sokolova, I., Wang, D., Li, M., Deshpande, P., Davidson, N., Mody, I., Quick, M. W., Quake, S. R., Lester, H. A. 2002; 22 (23): 10251-10266
Abstract

GABA transporter subtype 1 (GAT1) molecules were counted near GABAergic synapses, to a resolution of approximately 0.5 microm. Fusions between GAT1 and green fluorescent protein (GFP) were tested in heterologous expression systems, and a construct was selected that shows function, expression level, and trafficking similar to that of wild-type (WT) GAT1. A strain of knock-in mice was constructed that expresses this mGAT1-GFP fusion in place of the WT GAT1 gene. The pattern of fluorescence in brain slices agreed with previous immunocytochemical observations. [3H]GABA uptake, synaptic electrophysiology, and subcellular localization of the mGAT1-GFP construct were also compared with WT mice. Quantitative fluorescence microscopy was used to measure the density of mGAT1-GFP at presynaptic structures in CNS preparations from the knock-in mice. Fluorescence measurements were calibrated with transparent beads and gels that have known GFP densities. Surface biotinylation defined the fraction of transporters on the surface versus those in the nearby cytoplasm. The data show that the presynaptic boutons of GABAergic interneurons in cerebellum and hippocampus have a membrane density of 800-1300 GAT1 molecules per square micrometer, and the axons that connect boutons have a linear density of 640 GAT1 molecules per micrometer. A cerebellar basket cell bouton, a pinceau surrounding a Purkinje cell axon, and a cortical chandelier cell cartridge carry 9000, 7.8 million, and 430,000 GAT1 molecules, respectively; 61-63% of these molecules are on the surface membrane. In cultures from hippocampus, the set of fluorescent cells equals the set of GABAergic interneurons. Knock-in mice carrying GFP fusions of membrane proteins provide quantitative data required for understanding the details of synaptic transmission in living neurons.

View details for Web of Science ID 000179458100022

View details for PubMedID 12451126
Microfluidic large-scale integration SCIENCE Thorsen, T., Maerkl, S. J., Quake, S. R. 2002; 298 (5593): 580-584
Abstract

We developed high-density microfluidic chips that contain plumbing networks with thousands of micromechanical valves and hundreds of individually addressable chambers. These fluidic devices are analogous to electronic integrated circuits fabricated using large-scale integration. A key component of these networks is the fluidic multiplexor, which is a combinatorial array of binary valve patterns that exponentially increases the processing power of a network by allowing complex fluid manipulations with a minimal number of inputs. We used these integrated microfluidic networks to construct the microfluidic analog of a comparator array and a microfluidic memory storage device whose behavior resembles random-access memory.

View details for DOI 10.1126/science.1076996

View details for Web of Science ID 000178634800035

View details for PubMedID 12351675
Significance and statistical errors in the analysis of DNA microarray data PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Brody, J. P., Williams, B. A., Wold, B. J., Quake, S. R. 2002; 99 (20): 12975-12978
Abstract

DNA microarrays are important devices for high throughput measurements of gene expression, but no rational foundation has been established for understanding the sources of within-chip statistical error. We designed a specialized chip and protocol to investigate the distribution and magnitude of within-chip errors and discovered that, as expected from theoretical expectations, measurement errors follow a Lorentzian-like distribution, which explains the widely observed but unexplained ill-reproducibility in microarray data. Using this specially designed chip, we examined a data set of repeated measurements to extract estimates of the distribution and magnitude of statistical errors in DNA microarray measurements. Using the common "ratio of medians" method, we find that the measurements follow a Lorentzian-like distribution, which is problematic for subsequent analysis. We show that a method of analysis dubbed "median of ratios" yields a more Gaussian-like distribution of errors. Finally, we show that the bootstrap algorithm can be used to extract the best estimates of the error in the measurement. Quantifying the statistical error in such measurements has important applications for estimating significance levels, clustering algorithms, and process optimization.

View details for DOI 10.1073/pnas.162468199

View details for Web of Science ID 000178391700087

View details for PubMedID 12235357
Identification and confirmation of a module of coexpressed genes GENOME RESEARCH Thompson, H. G., Harris, J. W., Wold, B. J., Quake, S. R., Brody, J. P. 2002; 12 (10): 1517-1522
Abstract

We synthesize a large gene expression data set using dbEST and UniGene. We use guilt-by-association (GBA) to analyze this data set and identify coexpressed genes. One module, or group of genes, was found to be coexpressed mainly in tissue extracted from breast and ovarian cancers, but also found in tissue from lung cancers, brain cancers, and bone marrow. This module contains at least six members that are believed to be involved in either transcritional regulation (PDEF, H2AFO, NUCKS) or the ubiquitin proteasome pathway (PSMD7, SQSTM1, FLJ10111). We confirm these observations of coexpression by real-time RT-PCR analysis of mRNA extracted from four model breast epithelial cell lines.

View details for DOI 10.1101/gr.418402

View details for Web of Science ID 000178396400006

View details for PubMedID 12368243
Velocity-independent microfluidic flow cytometry ELECTROPHORESIS Eyal, S., Quake, S. R. 2002; 23 (16): 2653-2657
Abstract

Pressure-driven flow in microfluidic channels is characterized by a distribution of velocities. This distribution makes it difficult to implement conventional flow cytometry data analysis. We have demonstrated a method to measure velocity as an independent parameter when performing microfluidic flow cytometry. This method allows velocity-independent analysis of particles such as beads or cells, and allows flow cytometry analysis of extended objects, such as long DNA molecules. It allows accurate flow cytometry in transient and nonuniform flows. This general measurement method could be used in the future to measure the velocity of particles in a variety of existing microfluidic devices without the need for changes in their design.

View details for Web of Science ID 000177888500013

View details for PubMedID 12210169
An integrated microfabricated cell sorter ANALYTICAL CHEMISTRY Fu, A. Y., Chou, H. P., Spence, C., Arnold, F. H., Quake, S. R. 2002; 74 (11): 2451-2457
Abstract

We have developed an integrated microfabricated cell sorter using multilayer soft lithography. This integrated cell sorter is incorporated with various microfluidic functionalities, including peristaltic pumps, dampers, switch valves, and input and output wells, to perform cell sorting in a coordinated and automated fashion. The active volume of an actuated valve on this integrated cell sorter can be as small as 1 pL, and the volume of optical interrogation is approximately 100 fL. Different algorithms of cell manipulation, including cell trapping, were implemented in these devices. We have also demonstrated sorting and recovery of Escherichia coli cells on the chip.

View details for DOI 10.1021/ac0255330

View details for Web of Science ID 000175995400008

View details for PubMedID 12069222
A nanoliter rotary device for polymerase chain reaction ELECTROPHORESIS Liu, J., Enzelberger, M., Quake, S. 2002; 23 (10): 1531-1536
Abstract

Polymerase chain reaction (PCR) has revolutionized a variety of assays in biotechnology. The ability to implement PCR in disposable and reliable microfluidic chips will facilitate its use in applications such as rapid medical diagnostics, food control testing, and biological weapons detection. We fabricated a microfluidic chip with integrated heaters and plumbing in which various forms of PCR have been successfully demonstrated. The device uses only 12 nL of sample, one of the smallest sample volumes demonstrated to date. Minimizing the sample volume allows low power consumption, reduced reagent costs, and ultimately more rapid thermal cycling.

View details for Web of Science ID 000175992800021

View details for PubMedID 12116165
Gene expression analysis with universal n-mer arrays GENOME RESEARCH van Dam, R. M., Quake, S. R. 2002; 12 (1): 145-152
Abstract

Gene expression profiling is one of the many applications that have benefited from the massively parallel nucleic acid detection capability of DNA microarrays. Current expression arrays, however, are expensive and inflexible. They are custom-designed for each organism and they do not offer the possibility of incorporating updated genomic information without production of a new chip. One possible solution is the development of a universal chip, consisting of all 4n possible DNA sequences of length n. Studying different organisms or new genes would simply require modifications to the hybridization pattern analysis software. The key problem is to find a value of n that is large enough to afford sufficient specificity, yet is small enough for practical fabrication and readout. We developed an analytical model, supported by computer-assisted calculation with yeast and mouse transcript data, to argue that it is both practical and useful to fabricate n-mer arrays with 10 < or = n < or = 16.

View details for Web of Science ID 000173064900015

View details for PubMedID 11779839
A wireless modular monitoring system for civil structures 20th IMAC Conference on Structural Dynamics Lynch, J. P., Law, K. H., Kiremidjian, A. S., Kenny, T., Carryer, E. SOC EXPERIMENTAL MECHANICS INC. 2002: 1?6
View details for Web of Science ID 000176646000001
On-chip absorption and fluorescence spectroscopy with polydimethylsiloxane (PDMS) microfluidic flow channels 2nd Annual International IEEE/EMBS Conference on Microtechnologies in Medicine and Biology Adams, M. L., Quake, S., Scherer, A. IEEE. 2002: 369?373
View details for Web of Science ID 000176123400076
Microfluidic Large Scale Integration Science Thorsen, T., Maerkl, S. J., Quake, S. R. 2002; 298: 580-584
Fundamental approach for optoelectronic and microfluidic integration for miniaturizing spectroscopic devices Adams, M., DeRose, G., Quake, S. R., Scherer, A, . 2002
Velocity Independent Microfluidic Flow Cytometry Electrophoresis Eyal, S., Quake, S. R. 2002; 23: 2653-2657
A Microfabricated Rotary Pump BIOMEDICAL MICRODEVICES Chou, H., Unger, M. A., Quake, S. R. 2001; 3 (4): 323-330
View details for Web of Science ID 000209018800009
Dynamic pattern formation in a vesicle-generating microfluidic device PHYSICAL REVIEW LETTERS Thorsen, T., Roberts, R. W., Arnold, F. H., Quake, S. R. 2001; 86 (18): 4163-4166
Abstract

Spatiotemporal pattern formation occurs in a variety of nonequilibrium physical and chemical systems. Here we show that a microfluidic device designed to produce reverse micelles can generate complex, ordered patterns as it is continuously operated far from thermodynamic equilibrium. Flow in a microfluidic system is usually simple-viscous effects dominate and the low Reynolds number leads to laminar flow. Self-assembly of the vesicles into patterns depends on channel geometry and relative fluid pressures, enabling the production of motifs ranging from monodisperse droplets to helices and ribbons.

View details for Web of Science ID 000168525900060

View details for PubMedID 11328121
Single-molecule measurements calibrate green fluorescent protein surface densities on transparent beads for use with 'knock-in' animals and other expression systems JOURNAL OF NEUROSCIENCE METHODS Chiu, C. S., Kartalov, E., Unger, M., Quake, S., Lester, H. A. 2001; 105 (1): 55-63
Abstract

Quantitative aspects of synaptic transmission can be studied by inserting green fluorescent protein (GFP) moieties into the genes encoding membrane proteins. To provide calibrations for measurements on synapses expressing such proteins, we developed methods to quantify histidine-tagged GFP molecules (His6-GFP) bound to Ni-NTA moieties on transparent beads (80-120 microm diameter) over a density range comprising nearly four orders of magnitude (to 30000 GFP/microm2). The procedures employ commonly available Hg lamps, fluorescent microscopes, and CCD cameras. Two independent routes are employed: (1) single-molecule fluorescence measurements are made at the lowest GFP densities, providing an absolute calibration for macroscopic signals at higher GFP densities; (2) known numbers of His6-GFP molecules are coupled quantitatively to the beads. Each of the two independent routes provides linear data over the measured density range, and the two independent methods agree with root mean square (rms) deviation of 11-21% over this range. These satisfactory results are obtained on two separate microscope systems. The data can be corrected for bleaching rates, which are linear with light intensity and become appreciable at intensities > approximately 1 W/cm2. If a suitable GFP-tagged protein can be chosen and incorporated into a 'knock-in' animal, the density of the protein can be measured with an absolute accuracy on the order of 20%.

View details for Web of Science ID 000166873400006

View details for PubMedID 11166366
A Microfabricated Rotary Pump Biomedical Microdevices Chou, H. P., Unger, M. A., Quake, S. R. 2001; 3: 323-330
Single molecule measurements calibrate green fluorescent protein surface densities on transparent beads for use in "knock-in" animals and other expression systems Journal of Neuroscience Methods Chiu, C. S., Kartalov, E., Unger, M. A., Quake, S. R., Lester, H. 2001; 105: 55-63
From micro- to nanofabrication with soft materials SCIENCE Quake, S. R., Scherer, A. 2000; 290 (5496): 1536-1540
Abstract

Soft materials are finding applications in areas ranging from microfluidic device technology to nanofabrication. We review recent work in these areas, discuss the motivation for device fabrication with soft materials, and describe applications of soft materials. In particular, we discuss active microfluidic devices for cell sorting and biochemical assays, replication-molded optics with subdiffraction limit features, and nanometer-scale resonators and wires formed from single-molecule DNA templates as examples of how the special properties of soft materials address outstanding problems in device fabrication.

View details for Web of Science ID 000165446200039

View details for PubMedID 11090344
Femtonewton force spectroscopy of single extended DNA molecules PHYSICAL REVIEW LETTERS Meiners, J. C., Quake, S. R. 2000; 84 (21): 5014-5017
Abstract

We studied the thermal fluctuations of single DNA molecules with a novel optical tweezer based force spectroscopy technique. This technique combines femtonewton sensitivity with millisecond time resolution, surpassing the sensitivity of previous force measurements in aqueous solution with comparable bandwidth by a hundredfold. Our data resolve long-standing questions concerning internal hydrodynamics of the polymer and anisotropy in the molecular relaxation times and friction coefficients. The dynamics at high extension show interesting nonlinear behavior.

View details for Web of Science ID 000087114400058

View details for PubMedID 10990855
Monolithic microfabricated valves and pumps by multilayer soft lithography SCIENCE Unger, M. A., Chou, H. P., Thorsen, T., Scherer, A., Quake, S. R. 2000; 288 (5463): 113-116
Abstract

Soft lithography is an alternative to silicon-based micromachining that uses replica molding of nontraditional elastomeric materials to fabricate stamps and microfluidic channels. We describe here an extension to the soft lithography paradigm, multilayer soft lithography, with which devices consisting of multiple layers may be fabricated from soft materials. We used this technique to build active microfluidic systems containing on-off valves, switching valves, and pumps entirely out of elastomer. The softness of these materials allows the device areas to be reduced by more than two orders of magnitude compared with silicon-based devices. The other advantages of soft lithography, such as rapid prototyping, ease of fabrication, and biocompatibility, are retained.

View details for Web of Science ID 000086387700044

View details for PubMedID 10753110
An apertureless near-field microscope for fluorescence imaging APPLIED PHYSICS LETTERS Yang, T. J., Lessard, G. A., Quake, S. R. 2000; 76 (3): 378-380
View details for Web of Science ID 000084675100043
The fluorescence apertureless near field microscope... A step towards imaging information in DNA 2nd Conference on Scanning and Force Microscopies for Biomedical Applications Quake, S. R., Yang, T. J., Lessard, G. A., Unger, M., Kartalov, E. SPIE-INT SOC OPTICAL ENGINEERING. 2000: 199?204
View details for Web of Science ID 000087779800024
Integrated Elastomer Fluidic Lab on a Chip - Surface Patterning and DNA diagnostics Chou, H. P., Unger, M. A., Scherer, A., Quake, S. R. 2000
From Micro to Nano Fabrication with Soft Materials Science Quake, S. R., Scherer, A. 2000; 290: 1536-40
Fluorescence apertureless near-field microscope: a step towards imaging information in DNA Quake, S. R., Yang, T. J., Lessard, G. A., Unger, A., Kartalov, E. 2000
An Apertureless Near-Field Microscope for Fluorescence Imaging Applied Physics Letters Yang, T. J., Lessard, G. A., Quake, S. R. 2000; 76: 378-380
Single-molecule fluorescence observed with mercury lamp illumination BIOTECHNIQUES Unger, M., Kartalov, E., Chiu, C. S., Lester, H. A., Quake, S. R. 1999; 27 (5): 1008-?
Abstract

We demonstrate that it is possible to observe single fluorescent molecules using a standard fluorescence microscope with mercury lamp excitation and an inexpensive cooled charge-coupled device (CCD) camera. With this equipment, we have been able to observe single molecules of tetramethyl-rhodamine, rhodamine 6G, fluorescein isothiocyanate and green fluorescent protein. Immobilized molecules were observed both in air and in aqueous solution.

View details for Web of Science ID 000087218400022

View details for PubMedID 10572649
A microfabricated fluorescence-activated cell sorter NATURE BIOTECHNOLOGY Fu, A. Y., Spence, C., Scherer, A., Arnold, F. H., Quake, S. R. 1999; 17 (11): 1109-1111
Abstract

We have demonstrated a disposable microfabricated fluorescence-activated cell sorter (microFACS) for sorting various biological entities. Compared with conventional FACS machines, the microFACS provides higher sensitivity, no cross-contamination, and lower cost. We have used microFACS chips to obtain substantial enrichment of micron-sized fluorescent bead populations of differing colors. Furthermore, we have separated Escherichia coli cells expressing green fluorescent protein from a background of nonfluorescent E. coli cells and shown that the bacteria are viable after extraction from the sorting device. These sorters can function as stand-alone devices or as components of an integrated microanalytical chip.

View details for Web of Science ID 000083428000030

View details for PubMedID 10545919
Direct measurement of hydrodynamic cross correlations between two particles in an external potential PHYSICAL REVIEW LETTERS Meiners, J. C., Quake, S. R. 1999; 82 (10): 2211-2214
View details for Web of Science ID 000078959700051
A microfabricated device for sizing and sorting DNA molecules PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chou, H. P., Spence, C., Scherer, A., Quake, S. 1999; 96 (1): 11-13
Abstract

We have demonstrated a microfabricated single-molecule DNA sizing device. This device does not depend on mobility to measure molecule size, is 100 times faster than pulsed-field gel electrophoresis, and has a resolution that improves with increasing DNA length. It also requires a million times less sample than pulsed-field gel electrophoresis and has comparable resolution for large molecules. Here we describe the fabrication and use of the single-molecule DNA sizing device for sizing and sorting DNA restriction digests and ladders spanning 2-200 kbp.

View details for Web of Science ID 000078004400004

View details for PubMedID 9874762
A self-assembled microlensing rotational probe APPLIED PHYSICS LETTERS Brody, J. P., Quake, S. R. 1999; 74 (1): 144-146
View details for Web of Science ID 000077791800049
Single Molecule Fluorescence Observed with Mercury Lamp Illumination Biotechniques Unger, M. A., Kartalov, E., Chiu, C. S., Lester, H., Quake, S. R. 1999; 27: 1008-1013
A scanning apertureless fluorescence microscope Conference on Scanning and Force Microscopies for Biomedical Applications Lessard, G. A., Yang, T. J., Barritault, P., Quake, S. R. SPIE-INT SOC OPTICAL ENGINEERING. 1999: 158?165
View details for Web of Science ID 000081436700019
A Direct Measurement of Hydrodynamic Cross Correlations Between Two Particles in an External Potential Phys Rev Lett Meiners, J. C., Quake, S. R. 1999; 82: 2211-2214
A Self-Assembled Microlensing Rotational Probe Appl Phys Lett. Brody, J. P., Quake, S. R. 1999; 74 (1)
A Scanning Apertureless Fluorescence Microscope Lessard, G., Yang, T. J., Barritault, P., Quake, S. R. 1999
Dynamic Properties of an Extended Polymer in Solution Phys Rev Lett Hatfield, J. W., Quake, S. R. 1999; 82: 3548-51
Disposable Microdevices for DNA Analysis and Cell Sorting Quake, S. R., Chou, H. P., Spence, C., Fu, A. Y., Scherer, A. 1998
Microfabricated devices for sizing DNA and sorting cells Conference on Microfabricated and Nanofabricated Structures and Devices for Biomedical Environmental Applications Chou, H. P., Spence, C., Scherer, A., Quake, S. SPIE - INT SOC OPTICAL ENGINEERING. 1998: 181?187
View details for Web of Science ID 000073452100022
Microfabricated devices for sizing DNA and sorting cells Chou, H. P., Spence, C., Scherer, A., Quake, S. R. 1998
The dynamics of partially extended single molecules of DNA NATURE Quake, S. R., Babcock, H., Chu, S. 1997; 388 (6638): 151-154
Abstract

The behaviour of an isolated polymer floating in a solvent forms the basis of our understanding of polymer dynamics. Classical theories describe the motion of a polymer with linear equations of motion, which yield a set of 'normal modes', analogous to the fundamental frequency and the harmonics of a vibrating violin string. But hydrodynamic interactions make polymer dynamics inherently nonlinear, and the linearizing approximations required for the normal-mode picture have therefore been questioned. Here we test the normal-mode theory by measuring the fluctuations of single molecules of DNA held in a partially extended state with optical tweezers. We find that the motion of the DNA can be described by linearly independent normal modes, and we have experimentally determined the eigenstates of the system. Furthermore, we show that the spectrum of relaxation times obeys a power law.

View details for Web of Science ID A1997XK10900043

View details for PubMedID 9217154
Fast Monte Carlo algorithms for knotted polymers Phys. Rev. E Stat. Phys. Plasmas Fluids Relat. Interdiscip. Topics. Quake, S, R. 1997; 1 (52): 1176-1180
The Zimm model applied to extended single polymers J. Chem Phys. Quake, S, R. 1997; 5 (101): 4307-4311
Topological effects of knots in polymers Phys. Rev. Lett. Quake, S, R. 1997; 24 (73): 3317-3320
Applications of laser cooling and trapping to precision measurements and polymer physics 5th International Symposium on Foundations of Quantum Mechanics in the Light of New Technology - Quantum Coherence and Decoherence (ISQM-Tokyo 95) Young, B., Weitz, M., Hensley, J., Perkins, T., Smith, D., Quake, S., Chu, S. ELSEVIER SCIENCE BV. 1996: 47?52
View details for Web of Science ID A1996BG81E00010
POLYMER EXPERIMENTS ON SINGLE DNA-MOLECULES Chu, S., Quake, S., Perkins, T., Smith, D. AMER CHEMICAL SOC. 1995: 374-PHYS
View details for Web of Science ID A1995QP23301127
Fast Monte Carlo algorithms for knotted polymers. Physical review. E, Statistical physics, plasmas, fluids, and related interdisciplinary topics Quake, S. R. 1995; 52 (1): 1176?80
View details for PubMedID 9963522
RELAXATION OF A SINGLE DNA MOLECULE OBSERVED BY OPTICAL MICROSCOPY SCIENCE Perkins, T. T., Quake, S. R., Smith, D. E., Chu, S. 1994; 264 (5160): 822-826
Abstract

Single molecules of DNA, visualized in video fluorescence microscopy, were stretched to full extension in a flow, and their relaxation was measured when the flow stopped. The molecules, attached by one end to a 1-micrometer bead, were manipulated in an aqueous solution with optical tweezers. Inverse Laplace transformations of the relaxation data yielded spectra of decaying exponentials with distinct peaks, and the longest time component (tau) increased with length (L) as tau approximately L 1.68 +/- 0.10. A rescaling analysis showed that most of the relaxation curves had a universal shape and their characteristic times (lambda t) increased as lambda t approximately L 1.65 +/- 0.13. These results are in qualitative agreement with the theoretical prediction of dynamical scaling.

View details for Web of Science ID A1994NJ94900026

View details for PubMedID 8171336
Topological effects of knots in polymers. Physical review letters Quake, S. R. 1994; 73 (24): 3317?20
View details for PubMedID 10057346
Single molecule measurement of the "speed limit" of DNA polymerase. Schwartz, J. J., Quake, S. R. 2,009

Stephen Quake

Lee Otterson Professor in the School of Engineering and Professor of Bioengineering, of Applied Physics and, by courtesy, of Physics

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