Abstract
BackgroundMethylation of cytosine in genomic DNA is a well-characterized epigenetic modification involved in many cellular processes and diseases. Whole-genome bisulfite sequencing (WGBS), such as MethylC-seq and post-bisulfite adaptor tagging sequencing (PBAT-seq), uses the power of high-throughput DNA sequencers and provides genome-wide DNA methylation profiles at single-base resolution. However, the accuracy and consistency of WGBS outputs in relation to the operating conditions of high-throughput sequencers have not been explored.ResultsWe have used the Illumina HiSeq platform for our PBAT-based WGBS, and found that different versions of HiSeq Control Software (HCS) and Real-Time Analysis (RTA) installed on the system provided different global CpG methylation levels (approximately 5% overall difference) for the same libraries. This problem was reproduced multiple times with different WGBS libraries and likely to be associated with the low sequence diversity of bisulfite-converted DNA. We found that HCS was the major determinant in the observed differences. To determine which version of HCS is most suitable for WGBS, we used substrates with predetermined CpG methylation levels, and found that HCS v2.0.5 is the best among the examined versions. HCS v2.0.12 showed the poorest performance and provided artificially lower CpG methylation levels when 5-methylcytosine is read as guanine (first read of PBAT-seq and second read of MethylC-seq). In addition, paired-end sequencing of low diversity libraries using HCS v2.2.38 or the latest HCS v2.2.58 was greatly affected by cluster densities.ConclusionsSoftware updates in the Illumina HiSeq platform can affect the outputs from low-diversity sequencing libraries such as WGBS libraries. More recent versions are not necessarily the better, and HCS v2.0.5 is currently the best for WGBS among the examined HCS versions. Thus, together with other experimental conditions, special care has to be taken on this point when CpG methylation levels are to be compared between different samples by WGBS.
Highlights
Methylation of cytosine in genomic DNA is a well-characterized epigenetic modification involved in many cellular processes and diseases
Different HiSeq Control Software (HCS) and Real-Time Analysis (RTA) versions provide different CpG methylation levels Whole-genome bisulfite sequencing (WGBS) relies on bisulfite conversion of unmethylated C, but not 5mC, to uracil
In the course of our WGBS study of early postnatal mouse spermatogonia [13], we realized that the global CpG methylation level determined by PBAT-seq of the same library significantly changed upon HCS and RTA updates of the HiSeq sequencer
Summary
Methylation of cytosine in genomic DNA is a well-characterized epigenetic modification involved in many cellular processes and diseases. The first complete methylome maps were constructed by MethylC-seq [6,7,8], and the PBAT method was developed for performing amplification-free WGBS of a nanogram quantity of DNA [4]. With this method, methylome maps were constructed for human and mouse cells [9,10,11,12,13,14,15] as well as plant and fungal cells [4, 16]. Low sequence diversity samples, including bisulfite-converted DNAs, are obviously not the best substrates for HiSeq sequencing [18]
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