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MethylSeq Direct workflow: a fast method for DNA methylation analysis.

The presence or absence of methylation at carbon 5 in the cytosine base (5-meC) of the CpG dinucleotide motif is a key epigenetic regulator of cellular and developmental processes in higher eukaryotes. Large scale NGS analysis of changes in DNA methylation patterns associated with cancer and other pathologies has led to the discovery of many genomic regions and specific loci where CpG methylation influences the cell physiology. These discoveries can be expanded and extended by analyzing discrete methylation state changes in the large number of archived samples. However, retrospective studies using the rich resource of formalin-fixed, paraffin-embedded (FFPE) samples are hampered by the degraded nature of DNA extracted from these samples. Here we present a workflow for DNA methylation analysis method that is compatible with FFPE tissues. We demonstrate the use of a method for bisulfite sequencing, termed the MethylSeq Direct workflow, using the PCR and sequencing reagents of the Applied Biosystems™ BigDye™ Direct Cycle Sequencing Kit to determine the methylation status in the p16 and MGMT gene promoters as examples. FFPE extracted DNA is bisulfite modified using the Thermo Scientific™ EpiJET™ Bisulfite Conversion Kit. PCR primers specific for bisulfite converted sequences are designed using Applied Biosystems™ Methyl Primer Express™ Software. PCR using these M13-tagged primers is then used to amplify the locus of interest. The amplicon is sequenced using BigDye Direct Sequencing Master Mix and purified using the Applied Biosystems™ BigDye XTerminator™ Purification Kit. The sequencing reaction can be analyzed on the Applied Biosystems™ SeqStudio™ Genetic Analyzer, 3500 Genetic Analyzer, or other Applied Biosystems™ CE instrument. The entire workflow for bisulfite conversion, PCR amplification, and DNA sequencing, takes about 7 hours, including about an hour of hands-on time enabling the use of archived FFPE samples for retrospective studies.