Detecting DNA double-stranded breaks in mammalian genomes by linear amplification–mediated high-throughput genome-wide translocation sequencing

摘要: Unbiased, high-throughput assays for detecting and quantifying DNA double-stranded breaks (DSBs) across the genome in mammalian cells will facilitate basic studies of mechanisms that generate repair endogenous DSBs. They also enable more applied studies, such as those to evaluate on- off-target activities engineered nucleases. Here we describe a linear amplification-mediated genome-wide sequencing (LAM-HTGTS) method detection 'prey' DSBs via their translocation cultured fixed 'bait' DSB. Bait-prey junctions are cloned directly from isolated genomic using LAM-PCR unidirectionally ligated bridge adapters; subsequent PCR steps amplify single-stranded junction library preparation Illumina Miseq paired-end sequencing. A custom bioinformatics pipeline identifies prey sequences contribute maps them genome. LAM-HTGTS differs related approaches because it detects wide range broken end structures with nucleotide-level resolution. Familiarity nucleic acid methods next-generation analysis is necessary generation data interpretation. sensitive, reproducible, relatively inexpensive, scalable straightforward implement turnaround time <1 week.