Stanford Human OligoExome Resource
Stanford Human OligoExome is a scalable oligonucleotide management system for genomic analysis and high-throughput resequencing.
This set of capture oligonucleotides will cover the CCDS exon space and a high proportion of related regulatory regions from the human genome.
The Stanford Human OligoExome management system has been made publicly available at
http://oligoexome.stanford.edu
These capture oligonucleotides utilize a highly multiplexed approach for selectively circularizing and capturing multiple genomic regions using an in-solution method. Combined pools of capture oligonucleotides selectively circularize the genomic DNA target, followed by specific PCR amplification of regions of interest using a universal primer pair common to all of the capture oligonucleotides. Unlike multiplexed PCR methods, selective genomic circularization is capable of efficiently amplifying hundreds of genomic regions simultaneously in multiplex without requiring extensive PCR optimization or producing unwanted side reaction products. Benefits of the selective genomic circularization method are the relative robustness of the technique and low costs of synthesizing standard capture oligonucleotide for selecting genomic targets.
Personnel
- Georges Natsoulis
- Nancy Zhang
- John M. Bell
- Hua Xu
- Jason D. Buenrostro
- Heather Ordonez
- Sue Grimes
- Michael Jensen
- Daniel E. Newburger
- Jacob M. Zahn
- Jochen Kumm
- Hanlee P. Ji
Funding
Technology Approach
For the selective circularization capture assay, genomic DNA is initially subject to restriction enzyme digestion in separate tubes. Pooled capture oligonucleotides with end regions homologous to the genomic targets (two capture arms) are then hybridized to the restriction digested DNA, forcing the genomic DNA of the target into a circular conformation.Addition of a second backbone vector with universal homology to the interior of all oligonucleotides completes the double-stranded DNA circle, after which blunt-end ligation takes place. Uracil-DNA excision mix is added to digest the selector oligonucleotides and linearize the DNA containing the targeted genomic regions. Universal PCR primers specific to the vector oligonucleotides are added and PCR amplification of the captured genomic region is carried out and then sequenced using next-generation DNA sequencers. Two different molecular mechanisms mediate the target-specific circularization. In the first case, the oligonucleotide’s capture arms 1 and 2 recognize the complementary termini of the genomic DNA fragment created by both restriction sites. In the second case, capture arm 1 of the target complementary end-sequence recognizes one fragment terminus and capture arm 2 specifically anneals to the target sequence anywhere within the restriction enzyme fragment. For capture arm 2, the oligonucleotide -genomic DNA hybrid forms a branched structure, referred to as a flap in the absence of a restriction site. This specific DNA flap structure is cleaved by the Taq polymerase, forming ends suitable for ligation of the universal vector and complete genomic circularization as in the first case. This second case provides substantial design flexibility in regards to place capture sequences for any given target.
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