Pyrosequencing is a sequencing-by-synthesis method for DNA analysis that has emerged as a platform not only for de novo sequencing applications, but also for quantitative analysis of genomic methylation, single-nucleotide polymorphisms, and allele quantification. In this unit, we describe a complete workflow from sample to result that is suitable for each of these applications. As cytosine conversion is a key element of successful methylation analysis using pyrosequencing, a support protocol for bisulfite treatment is also included.
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