NET-Seq
HTGTS
HTGTS was developed to study translocation mechanisms in mammalian cells. This approach is particularly suitable for studying for AID-dependent IgH class-switching (HTGTS-Rep-seq) and CRISPR/Cas9 genome modifications. In HTGTS-Rep-seq, genomic DNA from B-cell populations is sonicated and linearly amplified with a biotinylated primer that anneals downstream of a J segment. The biotin-labeled single-stranded DNA products are enriched with streptavidin beads, and the 3’ ends are ligated to a bridge adaptor containing a 6-nucleotide UMI.
Pros:
- Higher efficiency compared to whole-genome sequencing
Cons:
- Underestimates the frequency of DSBs 1
- Limited by chromatin accessibility 2
- HTGTS: Chiarle R., Zhang Y., Frock R. L., et al. Genome-wide translocation sequencing reveals mechanisms of chromosome breaks and rearrangements in B cells. Cell. 2011;147:107-119
- HTGTS-Rep-Seq: Lin S. G., Ba Z., Du Z., Zhang Y., Hu J. and Alt F. W. Highly sensitive and unbiased approach for elucidating antibody repertoires. Proc Natl Acad Sci U S A. 2016;113:7846-7851
- HTGTS CRISPR/Cas9: Mei Y., Wang Y., Chen H., Sun Z. S. and Ju X. D. Recent Progress in CRISPR/Cas9 Technology. J Genet Genomics. 2016;43:63-75
- 1. Hu J., Meyers R. M., Dong J., Panchakshari R. A., Alt F. W. and Frock R. L. Detecting DNA double-stranded breaks in mammalian genomes by linear amplification-mediated high-throughput genome-wide translocation sequencing. Nat Protoc. 2016;11:853-871
- 2. Kim D., Bae S., Park J., et al. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods. 2015;12:237-243, 231 p following 243
- Schwer B., Wei P. C., Chang A. N., et al. Transcription-associated processes cause DNA double-strand breaks and translocations in neural stem/progenitor cells. Proc Natl Acad Sci U S A. 2016;113:2258-2263
- Frock R. L., Hu J., Meyers R. M., Ho Y. J., Kii E. and Alt F. W. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nat Biotechnol. 2015;33:179-186