Abstract
BackgroundWhole genome bisulfite sequencing (WGBS) also known as BS-seq has been widely used to measure the methylation of whole genome at single-base resolution. One of the key steps in the assay is converting unmethylated cytosines into thymines (BS conversion). Incomplete conversion of unmethylated cytosines can introduce false positive methylation call. Developing a quick method to evaluate bisulfite conversion ratio (BCR) is benefit for both quality control and data analysis of WGBS.ResultsHere we provide a computational method named “BCREval” to estimate the unconverted rate (UCR) by using telomeric repetitive DNA as native spike-in control. We tested the method by using public WGBS data and found that it is very stable and most of BS conversion assays can achieve> 99.5% efficiency. The non-CpG DNA methylation at telomere fits a binomial model and may result from a random process with very low possibility (the ratio < 0.4%). And the comparison between BCREval and Bismark (Krueger and Andrews, Bioinformatics 27:1571–1572, 2011), a widely used BCR evaluator, suggests that our algorithm is much faster and more efficient than the latter.ConclusionOur method is a simple but robust method to QC and speculates BCR for WGBS experiments to make sure it achieves acceptable level. It is faster and more efficient than current tools and can be easily integrated into presented WGBS pipelines.
Highlights
Whole genome bisulfite sequencing (WGBS) known as BS-seq has been widely used to measure the methylation of whole genome at single-base resolution
The peaks at n = 24~25 indicates that many reads are composed by telomeric repeats completely and suggests the ability of WGBS to capture telomeric DNA
The data show that the G strand original telomeric reads (50% GC contents) are much more than ones from C strand in both FASTQ files, which may result in the GC bias of Polymerase chain reaction (PCR) step (Fig. 1a)
Summary
Whole genome bisulfite sequencing (WGBS) known as BS-seq has been widely used to measure the methylation of whole genome at single-base resolution. DNA methylation, as a fundamental epigenetics modification, plays critical roles in various biology processes including embryonic development, pluripotency maintenance, genomic imprinting, gene expression regulation, and genomic stability maintenance. WGBS is the only one to identify all the C information and global pattern has become the standard profiling method in major epigenome consortiums such as NIH Roadmap [3], ENCODE [4], Blueprint [5] and IHEC [6] In this assay, genomic DNA is purified and sheared into fragments and treated with bisulfite, a chemical that converts unmethylated cytosine but not. The methylation states of cytosines are determined by searching T-C mismatches between sequences obtained and the reference genome
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