Bi-PROF

Abstract

The use of next generation sequencing has expanded our view on whole mammalian methylome patterns. In particular, it provides a genome-wide insight of local DNA methylation diversity at single nucleotide level and enables the examination of single chromosome sequence sections at a sufficient statistical power. We describe a bisulfite-based sequence profiling pipeline, Bi-PROF, which is based on the 454 GS-FLX Titanium technology that allows to obtain up to one million sequence stretches at single base pair resolution without laborious subcloning. To illustrate the performance of the experimental workflow connected to a bioinformatics program pipeline (BiQ Analyzer HT) we present a test analysis set of 68 different epigenetic marker regions (amplicons) in five individual patient-derived xenograft tissue samples of colorectal cancer and one healthy colon epithelium sample as a control. After the 454 GS-FLX Titanium run, sequence read processing and sample decoding, the obtained alignments are quality controlled and statistically evaluated. Comprehensive methylation pattern interpretation (profiling) assessed by analyzing 10²-10⁴ sequence reads per amplicon allows an unprecedented deep view on pattern formation and methylation marker heterogeneity in tissues concerned by complex diseases like cancer.

Keywords: :

• DNA methylation
• bisulfite profiling
• next-generation sequencing
• BiQ Analyzer HT
• colorectal cancer

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Roche Diagnostics and 454 Life Sciences for providing consumables and technical support. The project was supported by the Bundesministerium für Bildung und Forschung (ColoNet, grant no. 0315417A and D) and the EU (NOTOX, grant no. FP7 267038).