METHODS AND COMPOSITIONS FOR HIGH-THROUGHPUT BISULPHITE DNA-SEQUENCING AND UTILITIES

Abstract

The novel methods and compositions to produce DNA templates suitable for chemical modifications and high-throughput DNA-sequencing are disclosed. A method of a DNA adaptor design where constituent cytosines are substituted with 5-methylcytosines rendering the resulting adaptor resistant to bisulphite mediated deamination is also disclosed. When said adaptor is ligated onto double stranded DNA template, subsequent DNA denaturation and bisulphite treatment deaminates template DNA cytosine differentially to uracil whilst the 5-methylcytosines of the ligated adaptor resist chemical conversion resulting in the adaptor sequence remaining unaltered. Both strands of bisulphite treated DNA can thus be amplified with a single primer set that hybridizes to the unaltered adaptor sequence. Also the methods to produce control template of a defined methylation composition to optimize conditions for the bisulphite reaction are disclosed. In a preferred embodiment, the present invent can be used to produce templates suitable for genome-wide bisulphite-DNA sequencing using conventional, SolexaTM, SOLiDTM or 454TM_ type DNA sequencing platforms to study DNA methylation.