title: 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.
authors.affiliation: CSIRO Food and Nutrition Flagship, North Ryde, New South Wales 1670, Australia. hilal.varinli@csiro.au.|Genomics and Epigenetics Division, Garvan Institute of Medical Research, Darlinghurst, New South Wales 2010, Australia. hilal.varinli@csiro.au.|Department of Biological Sciences, Macquarie University, North Ryde, New South Wales 2109, Australia. hilal.varinli@csiro.au.
authors.forename: Hilal
authors.initials: H
authors.lastname: Varinli
authors.affiliation: Genomics and Epigenetics Division, Garvan Institute of Medical Research, Darlinghurst, New South Wales 2010, Australia. a.statham@garvan.org.au.
authors.forename: Aaron L
authors.initials: AL
authors.lastname: Statham
authors.affiliation: Genomics and Epigenetics Division, Garvan Institute of Medical Research, Darlinghurst, New South Wales 2010, Australia. s.clark@garvan.org.au.|Vincent's Clinical School, Faculty of Medicine, UNSW, New South Wales 2010, Australia. s.clark@garvan.org.au.
authors.forename: Susan J
authors.initials: SJ
authors.lastname: Clark
authors.affiliation: CSIRO Food and Nutrition Flagship, North Ryde, New South Wales 1670, Australia. peter.molloy@csiro.au.
authors.forename: Peter L
authors.initials: PL
authors.lastname: Molloy
authors.affiliation: CSIRO Food and Nutrition Flagship, North Ryde, New South Wales 1670, Australia. jason.ross@csiro.au.
authors.forename: Jason P
authors.initials: JP
authors.lastname: Ross
keywords: CHH; COBRA; DNA methylation; enhancer; next generation sequencing; non CpG; non-model organism; reduced representation; restriction enzymes