Abstract
BackgroundDNA methylation has emerged as an important regulator of development and disease, necessitating the design of more efficient and cost-effective methods for detecting and quantifying this epigenetic modification. Next-generation sequencing (NGS) techniques offer single base resolution of CpG methylation levels with high statistical significance, but are also high cost if performed genome-wide. Here, we describe a simplified targeted bisulfite sequencing approach in which DNA sequencing libraries are prepared following sodium bisulfite conversion and two rounds of PCR for target enrichment and sample barcoding, termed BisPCR2.ResultsWe have applied the BisPCR2 technique to validate differential methylation at several type 2 diabetes risk loci identified in genome-wide studies of human islets. We confirmed some previous findings while not others, in addition to identifying novel differentially methylated CpGs at these genes of interest, due to the much higher depth of sequencing coverage in BisPCR2 compared to prior array-based approaches.ConclusionThis study presents a robust, efficient, and cost-effective technique for targeted bisulfite NGS, and illustrates its utility by reanalysis of prior findings from genome-wide studies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0020-x) contains supplementary material, which is available to authorized users.
Highlights
DNA methylation has emerged as an important regulator of development and disease, necessitating the design of more efficient and cost-effective methods for detecting and quantifying this epigenetic modification
Many of the studies linking DNA methylation to disease have been prompted by the observation that only a small fraction of the inherited risk of these complex disorders can be explained by genetic variation, as determined by genomewide association studies (GWAS) [12, 13]
The BisPCR2 method for targeted bisulfite sequencing In order to simplify targeted bisulfite Next-generation sequencing (NGS), we developed a polymerase chain reaction (PCR)-based method for library preparation, termed BisPCR2 (Fig. 1)
Summary
DNA methylation has emerged as an important regulator of development and disease, necessitating the design of more efficient and cost-effective methods for detecting and quantifying this epigenetic modification. Next-generation sequencing (NGS) techniques offer single base resolution of CpG methylation levels with high statistical significance, but are high cost if performed genome-wide. Results: We have applied the BisPCR2 technique to validate differential methylation at several type 2 diabetes risk loci identified in genome-wide studies of human islets. We confirmed some previous findings while not others, in addition to identifying novel differentially methylated CpGs at these genes of interest, due to the much higher depth of sequencing coverage in BisPCR2 compared to prior array-based approaches. This chemical modification does not alter the DNA sequence, but rather regulates transcriptional programs to direct processes such as cellular differentiation, genomic imprinting, and X-chromosome inactivation, while promoting genomic stability [1,2,3,4]. DNA methylation, along with other epigenetic alterations, may provide the link between environmental factors or intrauterine exposure and complex disorders
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