Whole Methylome Analysis by Ultra-Deep Sequencing Using Two-Base Encoding

Abstract

Methylation, the addition of methyl groups to cytosine (C), plays an important role in the regulation of gene expression in both normal and dysfunctional cells. During bisulfite conversion and subsequent PCR amplification, unmethylated Cs are converted into thymine (T), while methylated Cs will not be converted. Sequencing of this bisulfite-treated DNA permits the detection of methylation at specific sites. Through the introduction of next-generation sequencing technologies (NGS) simultaneous analysis of methylation motifs in multiple regions provides the opportunity for hypothesis-free study of the entire methylome. Here we present a whole methylome sequencing study that compares two different bisulfite conversion methods (in solution versus in gel), utilizing the high throughput of the SOLiD™ System. Advantages and disadvantages of the two different bisulfite conversion methods for constructing sequencing libraries are discussed. Furthermore, the application of the SOLiD™ bisulfite sequencing to larger and more complex genomes is shown with preliminary in silico created bisulfite converted reads.

Advances in Genome-Wide DNA Methylation Analysis

“…SOLiD technology differs from other high-throughput deep sequencing approaches, as it detects two nucleotides at a time by ligation and predicts one of the four colors associated with those specific two bases. Bromann et al (48) assessed the capabilities of the ABI SOLiD approach for large-scale bisulfite sequencing and generated two libraries (in-solution bisulfite-converted and gel bisulfite-converted library) from Escherichia coli DH10B. These libraries were then amplified on magnetic beads by emulsion PCR (ePCR) using the standard SOLiD protocol, with the exception that additional dATP and dTTP were added to the aqueous ePCR phase to compensate for the low complexity of the bisulfite-converted libraries (48).…”

Next Generation Sequencing: Advances in Characterizing the Methylome

“…Nonetheless, this technology was used by Laurent and colleagues to compare human cell types at three progressive stages of differentiation [41] and by Lister and colleagues to compare human embryonic stem cells and fetal fibroblasts [23]. To date, the only other group to use the shot-gun bisulfite sequencing approach constructed an Escherichia coli methylome using the SOLiD TM platform [39]. Whole-genome bisulfite sequencing provides unbiased coverage of the genome allowing for the interrogation of regions of the genome that are often missed using other methodologies.…”

“…Each run requires 6-8 days and produces 40-50 GB of data with a read length of 50 bp. In addition to genome sequencing, RNA-seq, small RNA sequencing, and ChIP-seq, bisulfite sequencing of a bacterial genome has recently been published using this technology [39]. The main advantages of this technology are increased throughput and increased sequencing accuracy compared to the other sequencing platforms.…”

Genetic and Epigenomic Footprints of Folate

“…The focus of such experiments was primarily on known protein-coding genes. Genome-wide technologies to assess the epigenomics footprints of folate – mostly based on next-generation sequencing - have become available, not just for DNA methylation (Bock et al, 2010; Bormann Chung et al, 2010), but also for histone methylation (Barski et al, 2007; Barski and Zhao, 2009; Mikkelsen et al, 2007), as well as other epigenomics modalities. In addition, new sequencing-based methods (Mortazavi et al, 2008) now make it possible to overcome the prior restriction of expression analyses to known protein-coding genes, and permit genome-wide assessments of the entire transcriptome, including antisense and non-coding RNAs (Kuchen et al, 2010), in relation to folate status.…”