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COBRA-Seq: Sensitive and Quantitative Methylome Profiling

Abstract

Combined Bisulfite Restriction Analysis (COBRA) quantifies DNA methylation at a specific locus. It does so via digestion of PCR amplicons produced from bisulfite-treated DNA, using a restriction enzyme that contains a cytosine within its recognition sequence, such as TaqI. Here, we introduce COBRA-seq, a genome wide reduced methylome method that requires minimal DNA input (0.1-1.0 mg) and can either use PCR or linear amplification to amplify the sequencing library. Variants of COBRA-seq can be used to explore CpG-depleted as well as CpG-rich regions in vertebrate DNA. The choice of enzyme influences enrichment for specific genomic features, such as CpG-rich promoters and CpG islands, or enrichment for less CpG dense regions such as enhancers. COBRA-seq coupled with linear amplification has the additional advantage of reduced PCR bias by producing full length fragments at high abundance. Unlike other reduced representative methylome methods, COBRA-seq has great flexibility in the choice of enzyme and can be multiplexed and tuned, to reduce sequencing costs and to interrogate different numbers of sites. Moreover, COBRA-seq is applicable to non-model organisms without the reference genome and compatible with the investigation of non-CpG methylation by using restriction enzymes containing CpA, CpT, and CpC in their recognition site.

Type Journal
ISBN 2073-4425 (Electronic) 2073-4425 (Linking)
Authors Varinli, H.; Statham, A. L.; Clark, S. J.; Molloy, P. L.; Ross, J. P.;
Responsible Garvan Author Aaron Statham
Publisher Name Genes
Published Date 2015-01-01
Published Volume 6
Published Issue 4
Published Pages 1140-63
Status Published in-print
URL link to publisher's version http://www.ncbi.nlm.nih.gov/pubmed/26512698
OpenAccess link to author's accepted manuscript version https://publications.gimr.garvan.org.au/open-access/13332