Cargando…

COBRA-Seq: Sensitive and Quantitative Methylome Profiling

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-s...

Descripción completa

Detalles Bibliográficos
Autores principales: Varinli, Hilal, Statham, Aaron L., Clark, Susan J., Molloy, Peter L., Ross, Jason P.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4690032/
https://www.ncbi.nlm.nih.gov/pubmed/26512698
http://dx.doi.org/10.3390/genes6041140
_version_ 1782406938637631488
author Varinli, Hilal
Statham, Aaron L.
Clark, Susan J.
Molloy, Peter L.
Ross, Jason P.
author_facet Varinli, Hilal
Statham, Aaron L.
Clark, Susan J.
Molloy, Peter L.
Ross, Jason P.
author_sort Varinli, Hilal
collection PubMed
description 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 μg) 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.
format Online
Article
Text
id pubmed-4690032
institution National Center for Biotechnology Information
language English
publishDate 2015
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-46900322015-12-30 COBRA-Seq: Sensitive and Quantitative Methylome Profiling Varinli, Hilal Statham, Aaron L. Clark, Susan J. Molloy, Peter L. Ross, Jason P. Genes (Basel) Article 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 μg) 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. MDPI 2015-10-23 /pmc/articles/PMC4690032/ /pubmed/26512698 http://dx.doi.org/10.3390/genes6041140 Text en © 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Varinli, Hilal
Statham, Aaron L.
Clark, Susan J.
Molloy, Peter L.
Ross, Jason P.
COBRA-Seq: Sensitive and Quantitative Methylome Profiling
title COBRA-Seq: Sensitive and Quantitative Methylome Profiling
title_full COBRA-Seq: Sensitive and Quantitative Methylome Profiling
title_fullStr COBRA-Seq: Sensitive and Quantitative Methylome Profiling
title_full_unstemmed COBRA-Seq: Sensitive and Quantitative Methylome Profiling
title_short COBRA-Seq: Sensitive and Quantitative Methylome Profiling
title_sort cobra-seq: sensitive and quantitative methylome profiling
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4690032/
https://www.ncbi.nlm.nih.gov/pubmed/26512698
http://dx.doi.org/10.3390/genes6041140
work_keys_str_mv AT varinlihilal cobraseqsensitiveandquantitativemethylomeprofiling
AT stathamaaronl cobraseqsensitiveandquantitativemethylomeprofiling
AT clarksusanj cobraseqsensitiveandquantitativemethylomeprofiling
AT molloypeterl cobraseqsensitiveandquantitativemethylomeprofiling
AT rossjasonp cobraseqsensitiveandquantitativemethylomeprofiling