DNA breaks. Ends get joined.
Our research is centered on understanding the fundamental and coordinate processes that repair our genome when double-stranded DNA ends become exposed. We employ a multi-functional DNA junction mapping platform designed to infer the genome-wide impacts of modulating DNA integrity.
High-Throughput Genome-Wide Translocation Sequencing (HTGTS)
Our group focuses on understanding how different sources of DNA double-stranded breaks (DSBs) are repaired and how additional DSBs can lead to the formation of Intrachromosomal deletions and inversions and Interchromosomal translocations. To study this, we employ a highly-sensitive translocation sequencing technology, HTGTS, to locate and measure cellular DSB patterns genome-wide. This is achieved by following the fate of a known "bait" DSB broken end and mapping the genome-wide position of the repaired "prey" DSB broken end at single-nucleotide resolution.
Creating HTGTS libraries from genomic DNA harboring translocations involves linear amplification-mediated (LAM)-PCR using a single biotinylated bait primer, enrichment of ssDNA products, ligation of adapter sequence to unknown prey sequences--stabilized by bridging N-nucleotide overhangs--, and standard PCR to incorporate specific tags for Illumina sequencing. Sequence reads are aligned to optimally define bait/prey sequences.
Data replotted from Frock et al., 2015 Nature Biotechnology
This technology has been used to identify recurrent DSBs generated from Cas9/gRNA, TALEN, and other ectopically expressed endonuclease on- and off-targeting, antigen receptor locus V(D)J recombination in developing B and T lymphocytes, B cell immunoglobulin heavy chain class switch recombination, and fragile/replication-stalled sites. HTGTS can also detect and measure infrequent wide-spread DSBs generated from, for example, ionizing radiation.
Our interests include further developing this technology to more comprehensively measure DSB repair outcomes and to define novel repair factors/pathways. This approach will, in part, involve generating novel molecular and bioinformatic tools to model and to potentially drive specific repair outcomes. For more information about the technology, see Hu et al., 2016 Nature Protocols.