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Genes 2015, 6(4), 1140-1163; doi:10.3390/genes6041140

COBRA-Seq: Sensitive and Quantitative Methylome Profiling

1
CSIRO Food and Nutrition Flagship, North Ryde, New South Wales 1670, Australia
2
Genomics and Epigenetics Division, Garvan Institute of Medical Research, Darlinghurst, New South Wales 2010, Australia
3
Department of Biological Sciences, Macquarie University, North Ryde, New South Wales 2109, Australia
4
Vincent’s Clinical School, Faculty of Medicine, UNSW, New South Wales 2010, Australia
*
Author to whom correspondence should be addressed.
Academic Editors: Jeffrey Craig and Thomas Mikeska
Received: 10 August 2015 / Revised: 22 September 2015 / Accepted: 24 September 2015 / Published: 23 October 2015
(This article belongs to the Special Issue Epigenetic Biomarkers)
View Full-Text   |   Download PDF [3014 KB, uploaded 23 October 2015]   |  

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. View Full-Text
Keywords: COBRA; DNA methylation; reduced representation; non CpG; non-model organism; restriction enzymes; next generation sequencing; enhancer; CHH COBRA; DNA methylation; reduced representation; non CpG; non-model organism; restriction enzymes; next generation sequencing; enhancer; CHH
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Varinli, H.; Statham, A.L.; Clark, S.J.; Molloy, P.L.; Ross, J.P. COBRA-Seq: Sensitive and Quantitative Methylome Profiling. Genes 2015, 6, 1140-1163.

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