title: Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing.
abstract: Unbiased, high-throughput assays for detecting and quantifying DNA double-stranded breaks (DSBs) across the genome in mammalian cells will facilitate basic studies of the mechanisms that generate and repair endogenous DSBs. They will also enable more applied studies, such as those to evaluate the on- and off-target activities of engineered nucleases. Here we describe a linear amplification-mediated high-throughput genome-wide sequencing (LAM-HTGTS) method for the detection of genome-wide 'prey' DSBs via their translocation in cultured mammalian cells to a fixed 'bait' DSB. Bait-prey junctions are cloned directly from isolated genomic DNA using LAM-PCR and unidirectionally ligated to bridge adapters; subsequent PCR steps amplify the single-stranded DNA junction library in preparation for Illumina Miseq paired-end sequencing. A custom bioinformatics pipeline identifies prey sequences that contribute to junctions and maps them across the genome. LAM-HTGTS differs from related approaches because it detects a wide range of broken end structures with nucleotide-level resolution. Familiarity with nucleic acid methods and next-generation sequencing analysis is necessary for library generation and data interpretation. LAM-HTGTS assays are sensitive, reproducible, relatively inexpensive, scalable and straightforward to implement with a turnaround time of <1 week.
authors.affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Jiazhi
authors.initials: J
authors.lastname: Hu
authors.affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Robin M
authors.identifier: http://orcid.org/0000-0002-1802-7231
authors.initials: RM
authors.lastname: Meyers
authors.affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Junchao
authors.initials: J
authors.lastname: Dong
authors.affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Rohit A
authors.initials: RA
authors.lastname: Panchakshari
authors.affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.|Howard Hughes Medical Institute, Boston, Massachusetts, USA.
authors.forename: Frederick W
authors.initials: FW
authors.lastname: Alt
authors.affiliation: Program in Cellular and Molecular Medicine, Boston Children's Hospital, Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Richard L
authors.initials: RL
authors.lastname: Frock
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002875:Chromosomes / Q000235:genetics; D019295:Computational Biology / Q000379:methods; D053903:DNA Breaks, Double-Stranded; D004277:DNA, Single-Stranded; D003851:Deoxyribonucleases / Q000235:genetics; D015723:Gene Library; D016678:Genome; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D008322:Mammals / Q000235:genetics*; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D014178:Translocation, Genetic
chemical_list: D004277:DNA, Single-Stranded; D003851:Deoxyribonucleases