Ultradeep Bisulfite Sequencing Analysis of DNA Methylation Patterns in Multiple Gene Promoters by 454 Sequencing

Abstract

We developed a novel approach for conducting multisample, multigene, ultradeep bisulfite sequencing analysis of DNA methylation patterns in clinical samples. A massively parallel sequencing-by-synthesis method (454 sequencing) was used to directly sequence >100 bisulfite PCR products in a single sequencing run without subcloning. We showed the utility, robustness, and superiority of this approach by analyzing methylation in 25 gene-related CpG rich regions from >40 cases of primary cells, including normal peripheral blood lymphocytes, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), follicular lymphoma (FL), and mantle cell lymphoma (MCL). A total of 294,631 sequences was generated with an average read length of 131 bp. On average, >1,600 individual sequences were generated for each PCR amplicon far beyond the few clones (<20) typically analyzed by traditional bisulfite sequencing. Comprehensive analysis of CpG methylation patterns at a single DNA molecule level using clustering algorithms revealed differential methylation patterns between diseases. A significant increase in methylation was detected in ALL and FL samples compared with CLL and MCL. Furthermore, a progressive spreading of methylation was detected from the periphery toward the center of select CpG islands in the ALL and FL samples. The ultradeep sequencing also allowed simultaneous analysis of genetic and epigenetic data and revealed an association between a single nucleotide polymorphism and the methylation present in the LRP1B promoter. This new generation of methylome sequencing will provide digital profiles of aberrant DNA methylation for individual human cancers and offers a robust method for the epigenetic classification of tumor subtypes. [Cancer Res 2007;67(18):8511-8]

Applications of next generation sequencing in molecular ecology of non-model organisms

“…Sequencing of these regions can then pinpoint the specific methylated nucleotides. In recent years, NGS has been utilized to characterize DNA methylation patterns genome-wide, using an application known as ultra-deep bisulphite DNA sequencing (BSSeq) (Taylor et al, 2007;Cokus et al, 2008). Methylated fractions of genomes can also be sequenced using a combination of NGS and methyl-DNA immunoprecipitation (MeDIP).…”

Prospects for Epigenetic Epidemiology

“…The development of ultra-high-throughput DNA sequencing technologies for the direct sequencing of enriched, methylated DNA fragments or of bisulfite-converted genomic DNA (168,169) will ultimately permit measurement of comprehensive methylation profiles. The dynamic nature of epigenetic markings obviously warrants longitudinal biospecimen sampling where possible.…”

High definition profiling of mammalian DNA methylation by array capture and single molecule bisulfite sequencing

“…For example, direct shotgun bisulfite sequencing provided adequate coverage depth and proved cost-effective for a small genome like Arabidopsis (119 Mbp) (Cokus et al 2008). However, these approaches are currently impractical for routine application in complex mammalian genomes, and simplification of DNA fragment populations (genome partitioning) is still required to boost sampling depth of individual CpG sites (Taylor et al 2007;Meissner et al 2008). This problem becomes compounded as one considers that, within a multicellular organism, there are probably at least as many epigenomic states as there are cell types.…”