dc.contributor.author | Olova, N | en_US |
dc.contributor.author | Krueger, F | en_US |
dc.contributor.author | Andrews, S | en_US |
dc.contributor.author | Oxley, D | en_US |
dc.contributor.author | Berrens, RV | en_US |
dc.contributor.author | Branco, MR | en_US |
dc.contributor.author | Reik, W | en_US |
dc.date.accessioned | 2018-03-26T07:58:17Z | |
dc.date.available | 2018-02-14 | en_US |
dc.date.issued | 2018-03-15 | en_US |
dc.date.submitted | 2018-03-20T10:00:58.581Z | |
dc.identifier.uri | http://qmro.qmul.ac.uk/xmlui/handle/123456789/36083 | |
dc.description.abstract | BACKGROUND: Whole-genome bisulfite sequencing (WGBS) is becoming an increasingly accessible technique, used widely for both fundamental and disease-oriented research. Library preparation methods benefit from a variety of available kits, polymerases and bisulfite conversion protocols. Although some steps in the procedure, such as PCR amplification, are known to introduce biases, a systematic evaluation of biases in WGBS strategies is missing. RESULTS: We perform a comparative analysis of several commonly used pre- and post-bisulfite WGBS library preparation protocols for their performance and quality of sequencing outputs. Our results show that bisulfite conversion per se is the main trigger of pronounced sequencing biases, and PCR amplification builds on these underlying artefacts. The majority of standard library preparation methods yield a significantly biased sequence output and overestimate global methylation. Importantly, both absolute and relative methylation levels at specific genomic regions vary substantially between methods, with clear implications for DNA methylation studies. CONCLUSIONS: We show that amplification-free library preparation is the least biased approach for WGBS. In protocols with amplification, the choice of bisulfite conversion protocol or polymerase can significantly minimize artefacts. To aid with the quality assessment of existing WGBS datasets, we have integrated a bias diagnostic tool in the Bismark package and offer several approaches for consideration during the preparation and analysis of WGBS datasets. | en_US |
dc.description.sponsorship | This work was supported by the Biotechnology and Biological Sciences Research
Council (CASE studentship to N.O., BB/K010867/1 to W.R.), Wellcome
Trust (095645/Z/11/Z to W.R.), EU EpiGeneSys (257082 to W.R.) and EU
BLUEPRINT (282510 to W.R.); Babraham Institute/Cambridge European
Trust scholarship to N.O.; M.R.B. is a Sir Henry Dale Fellow (101225/Z/
13/Z), jointly funded by the Wellcome Trust and the Royal Society. | en_US |
dc.format.extent | 33 - ? | en_US |
dc.language | eng | en_US |
dc.language.iso | en | en_US |
dc.relation.ispartof | Genome Biol | en_US |
dc.rights | s This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated | |
dc.subject | Artefacts | en_US |
dc.subject | Biases | en_US |
dc.subject | Bisulfite conversion | en_US |
dc.subject | DNA methylation | en_US |
dc.subject | Degradation | en_US |
dc.subject | GC skew | en_US |
dc.subject | NGS | en_US |
dc.subject | Polymerase | en_US |
dc.subject | WGBS | en_US |
dc.subject | Artifacts | en_US |
dc.subject | DNA Methylation | en_US |
dc.subject | Gene Library | en_US |
dc.subject | Polymerase Chain Reaction | en_US |
dc.subject | Sulfites | en_US |
dc.subject | Whole Genome Sequencing | en_US |
dc.title | Comparison of whole-genome bisulfite sequencing library preparation strategies identifies sources of biases affecting DNA methylation data. | en_US |
dc.type | Article | |
dc.rights.holder | © The Author(s). 2018. | |
dc.identifier.doi | 10.1186/s13059-018-1408-2 | en_US |
pubs.author-url | https://www.ncbi.nlm.nih.gov/pubmed/29544553 | en_US |
pubs.issue | 1 | en_US |
pubs.notes | No embargo | en_US |
pubs.publication-status | Published online | en_US |
pubs.volume | 19 | en_US |
dcterms.dateAccepted | 2018-02-14 | en_US |