Sensitive mapping of recombination hotspots using sequencing-based detection of ssDNA
- Pavel P Khil1,
- Fatima Smagulova2,
- Kevin M Brick1,
- R. Daniel Camerini-Otero1 and
- Galina V Petukhova2,3
- 1 National Institute of Diabetes and Digestive and Kidney Diseases;
- 2 Uniformed Services University of the Health Sciences
- ↵* Corresponding author; email: gpetukhova{at}usuhs.mil
Abstract
Meiotic DNA double stranded breaks (DSBs) initiate genetic recombination in discrete areas of the genome called recombination hotspots. DSBs can be directly mapped using chromatin immunoprecipitation followed by sequencing (ChIP-Seq). Nevertheless, the genome-wide mapping of recombination hotspots in mammals is still a challenge due to the low frequency of recombination, high heterogeneity of the germ cell population and the relatively low efficiency of ChIP. To overcome these limitations we have developed a novel method single-stranded DNA (ssDNA) sequencing (SSDS) that specifically detects protein-bound single-stranded DNA at DSB ends. SSDS comprises a computational framework for the specific detection of ssDNA-derived reads in a sequencing library and a new library preparation procedure for the enrichment of fragments originating from ssDNA. The use of our technique reduces the non-specific dsDNA background more than ten-fold. Our method can be extended to other systems where the identification of ssDNA or DSBs is desired.
- Received August 12, 2011.
- Accepted February 23, 2012.
- Copyright © 2012, Cold Spring Harbor Laboratory Press
