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Title: Methylation analysis by direct sequencing of PCR products
P91
Lewin, Jörn; Schmitt, Armin O.; Genc, Bülent; Hildmann, Thomas; Novic, Karen L.; Beck, Stephan

joern.lewin@epigenomics.com
Epigenomics AG, Wellcome Trust Sanger Institute

Methylation of DNA is known to play a major role in the regulation of gene expression and can, for example, be used for tissue classification. Sequencing of bisulphite treated DNA to detect methylation via unconverted CpGs is a well established method but the state of the art is based on sequencing subcloned PCR products or using special sequencing technologies. We developed a software that allows us to interpret common tracefiles from direct PCR sequencing using today's standard high throughput sequencers.

Our method normalizes trace file data from different dyes and allows the use of multi dye traces. Basecalling errors are corrected, incomplete bisulphite conversion is taken into account and measurements at CpG positions are corrected using this information. We combine information from both amplifiable strands that might be sequenced from both sides. Because of automatized analysis, we are able to carry out methylation studies on larger datasets with a minimum of effort. First applications to biological samples look very encouraging.