Enhanced Methylation Analysis by Recovery of Unsequenceable Fragments

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 amplificatio...

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Bibliographic Details
Published in:PloS one 2016-03, Vol.11 (3), p.e0152322-e0152322
Main Authors: McInroy, Gordon R, Beraldi, Dario, Raiber, Eun-Ang, Modrzynska, Katarzyna, van Delft, Pieter, Billker, Oliver, Balasubramanian, Shankar
Format: Article
Language:English
Subjects:
5-Methylcytosine - metabolism
Amplification
Analysis
Bioinformatics
Biology and life sciences
Bisulfite
Chemical treatment
Chromatography, High Pressure Liquid
Cleavage
CpG Islands
Deoxyribonucleic acid
DNA
DNA - analysis
DNA - chemistry
DNA - metabolism
DNA Methylation
DNA sequencing
Epigenetics
Fragments
Gene expression
Gene Library
Gene mapping
Gene sequencing
Genomes
Malaria
Medical research
Methods
Methylation
Nucleotide sequence
Physical Sciences
Plasmodium
Plasmodium berghei
Plasmodium berghei - genetics
Plasmodium falciparum
Polymerase chain reaction
Recovery
Research and Analysis Methods
Sequence Analysis, DNA - methods
Sulfites - chemistry
Tandem Mass Spectrometry
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0152322