Sequencing-based detection of ssDNA

Tuesday, October 25, 2011 — Poster Session II
Noon – 2:00 p.m.	Natcher Conference Center	NIDDK	GEN/GENOM-8

Authors

• P Khil
• F Smagulova
• K Brick
• R-D Camerini-Otero
• G Petukhova

Abstract

Single-stranded DNA(ssDNA) is formed in many fundamental biological processes, such as DNA recombination and DNA replication, and in cancer, where ssDNA intermediates could be associated with genomic rearrangements. Genome-wide detection of ssDNAs remains difficult, however. Here we report a novel method -Asymmetric ChIP-Seq with Kinetic Enrichment (ASKE-ChIP) - that specifically detects protein-bound ssDNA. ASKE-ChIP consists of two parts. One is a computational framework for the specific detection of ssDNA and dsDNA derived fragments in a sequencing library. The second part of ASKE-ChIP is a sequencing library preparation protocol that efficiently removes dsDNA-derived fragments. One example of a biological process where ssDNA intermediates are naturally formed is meiosis. Following DSB formation by SPO11, kilobase-long ssDNA filaments covered with DMC1 and RAD51 are formed around DSBs. We used ASKE-ChIP to map meiotic DSB hotspots in mouse. Compared to standard ChIP-Seq, the non-specific dsDNA background is reduced more than ten-fold. From a practical standpoint, ASKE-ChIP allows generation of a representative DSB hotspots map using one lane of Illumina sequencing from a library prepared from wild-type mice. Although we primarily used ASKE-ChIP to map meiotic DSB hotspots in mouse, a straightforward extension of this approach to other applications is possible.

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