Enhanced Methylation Analysis by Recovery of Unsequenceable Fragments
McInroy, Gordon R
van, Delft Pieter
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McInroy, G. R., Beraldi, D., Raiber, E., Modrzynska, K., van, D. P., Billker, O., & Balasubramanian, S. (2016). Enhanced Methylation Analysis by Recovery of Unsequenceable Fragments. PLOS ONE, 11 (e0152322)https://doi.org/10.1371/journal.pone.0152322
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.
DNA methylation, genomic libraries, cytosine, Plasmodium, mammalian genomics, methylation, DNA libraries, polymerase chain reaction
GRM is supported by funding from Trinity College Cambridge and Herchel Smith. DB is supported by funding from the Wellcome Trust and Herchel Smith. EAR is a Herchel Smith Fellow. PVD is a Marie Curie Fellow of the European Union (FP7-PEOPLE-2013-IEF/624885). The Balasubramanian lab is supported by a Senior Investigator Award from the Wellcome Trust (099232/Z/12/Z to SB) and by core funding from Cancer Research UK.
Wellcome Trust (099232/Z/12/Z)
External DOI: https://doi.org/10.1371/journal.pone.0152322
This record's URL: https://www.repository.cam.ac.uk/handle/1810/254869
Attribution 2.0 UK: England & Wales
Licence URL: http://creativecommons.org/licenses/by/2.0/uk/
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