Sensitive mapping of recombination hotspots using sequencing-based detection of ssDNA
- Pavel P. Khil1,3,
- Fatima Smagulova2,3,
- Kevin M. Brick1,
- R. Daniel Camerini-Otero1,4 and
- Galina V. Petukhova2,4
- 1Genetics and Biochemistry Branch, National Institute of Diabetes, Digestive and Kidney Diseases, NIH, Bethesda, Maryland 20892, USA;
- 2Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, USA
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↵3 These authors contributed equally to this work.
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 nonspecific double-stranded DNA (dsDNA) background >10-fold. Our method can be extended to other systems where the identification of ssDNA or DSBs is desired.
Footnotes
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↵4 Corresponding authors.
E-mail camerini{at}ncifcrf.gov.
E-mail gpetukhova{at}usuhs.mil.
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[Supplemental material is available for this article.]
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Article published online before print. Article, supplemental material, and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.130583.111.
- Received August 12, 2011.
- Accepted February 23, 2012.
This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 3.0 Unported License), as described at http://creativecommons.org/licenses/by-nc/3.0/.
