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

Nature Protocols volume 11, pages 853–871 (2016)Cite this article

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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.

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Figure 1: Step-by-step overview of HTGTS methods.
Figure 2: Primer design and enzyme cutting site strategies for LAM-HTGTS.
Figure 3: Flowchart of the bioinformatic pipeline for translocation junction identification.
Figure 4: Representative smear of amplified and Illumina sequence–tagged products.
Figure 5: Universal bait detection of off-targets for designed VEGFA gRNA.

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Acknowledgements

We thank members of the Alt laboratory for discussions about improving LAM-HTGTS, and we thank Z. Herbert from the Molecular Biology Core Facilities at Dana-Farber Cancer Institute for discussions on transitioning HTGTS to Illumina Miseq. This work is supported by National Institutes of Health Grant nos. R01AI020047 and R01AI077595 to F.W.A. R.L.F. was supported by the National Institutes of Health National Research Service Award no. T32AI007512. J.H. is supported by a Robertson Foundation/Cancer Research Institute Irvington Fellowship. F.W.A. is an investigator of the Howard Hughes Medical Institute.

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Author notes

  1. Jiazhi Hu and Robin M Meyers: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Genetics, Program in Cellular and Molecular Medicine, Boston Children's Hospital, Harvard Medical School, Boston, Massachusetts, USA

    Jiazhi Hu, Robin M Meyers, Junchao Dong, Rohit A Panchakshari, Frederick W Alt & Richard L Frock

  2. Howard Hughes Medical Institute, Boston, Massachusetts, USA,

    Frederick W Alt

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  1. Jiazhi Hu
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Contributions

J.H., R.M.M., F.W.A. and R.L.F. wrote the manuscript with additional comments from J.D. and R.A.P. J.H. and R.L.F. designed and experimentally developed the LAM-HTGTS approach and R.M.M. wrote the translocation pipeline program. J.H., R.L.F., J.D. and R.A.P. performed experiments quoted in the manuscript that used the LAM-HTGTS approach, and all authors analyzed data that contributed to the development and/or application of the approach.

Corresponding authors

Correspondence to Frederick W Alt or Richard L Frock.

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Competing interests

A patent application has been filed relating to the current LAM-HTGTS method (International Application No. PCT/US2015/061758).

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Hu, J., Meyers, R., Dong, J. et al. Detecting DNA double-stranded breaks in mammalian genomes by linear amplification–mediated high-throughput genome-wide translocation sequencing. Nat Protoc 11, 853–871 (2016). https://doi.org/10.1038/nprot.2016.043

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