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Enhanced Methylation Analysis by Recovery of Unsequenceable Fragments
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2016 (English)In: PLoS ONE, E-ISSN 1932-6203, Vol. 11, no 3, article id e0152322Article in journal (Refereed) Published
Abstract [en]

Bisulfite sequencing is a valuable tool for mapping the position of 5-methylcytosine in the genome at single base resolution. However, the associated chemical treatment causes strand scission, which depletes the number of sequenceable DNA fragments in a library and thus necessitates PCR amplification. The AT-rich nature of the library generated from bisulfite treatment adversely affects this amplification, resulting in the introduction of major biases that can confound methylation analysis. Here, we report a method that enables more accurate methylation analysis, by rebuilding bisulfite-damaged components of a DNA library. This recovery after bisulfite treatment (ReBuilT) approach enables PCR-free bisulfite sequencing from low nanogram quantities of genomic DNA. We apply the ReBuilT method for the first whole methylome analysis of the highly AT-rich genome of Plasmodium berghei. Side-by-side comparison to a commercial protocol involving amplification demonstrates a substantial improvement in uniformity of coverage and reduction of sequence context bias. Our method will be widely applicable for quantitative methylation analysis, even for technically challenging genomes, and where limited sample DNA is available.

Place, publisher, year, edition, pages
Public Library of Science , 2016. Vol. 11, no 3, article id e0152322
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Cell and Molecular Biology
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URN: urn:nbn:se:umu:diva-165848DOI: 10.1371/journal.pone.0152322ISI: 000373121800057PubMedID: 27031619Scopus ID: 2-s2.0-84977646307OAI: oai:DiVA.org:umu-165848DiVA, id: diva2:1379368
Available from: 2019-12-17 Created: 2019-12-17 Last updated: 2019-12-20Bibliographically approved

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