METHOD FOR GENERATING DOUBLE STRANDED DNA LIBRARIES AND SEQUENCING METHODS FOR THE IDENTIFICATION OF METHYLATED CYTOSINES

摘要

The present invention relates to methods for the identification of methylated cytosines in a population of double stranded DNA molecules. The invention also relates to adapters and kits for synthesizing said adapters as well as to double stranded DNA libraries obtained by the methods of the invention.

主权项

1. A method for the identification of methylated cytosines in a population of double stranded DNA molecules comprising the steps of
(i) Ligating double-stranded DNA adaptors to at least one end of the strands of a plurality of double-stranded DNA molecules and pairing the strands of a plurality of double-stranded DNA molecules to provide a plurality of paired adaptor-modified DNA molecules; (ii) Transforming any (non-methylated) cytosine in the paired adaptor-modified DNA molecules to uracil in the paired adaptor-modified DNA molecules; (iii) Providing complementary strands of the paired and transformed adaptor-modified DNA molecules using the nucleotides A, G, C and T and primers the sequences of which are complementary to at least a portion of the double-stranded adaptors to provide partially transformed paired double-stranded molecules; (iv) Optionally amplifying the partially transformed paired double stranded DNA molecules obtained in step (iii) to provide amplified paired double stranded DNA molecules; (v) sequencing the paired DNA molecules obtained in step (iii) or in step (iv),wherein the presence of methylated cytosine at a given position is determined if a cytosine appears in one of the strands of the paired double stranded DNA molecules obtained in step (iii) or in step (iv) and a guanine appears in the corresponding position in the other strand of the paired double stranded DNA molecules, and/or wherein the presence of a non-methylated cytosine at a given position is determined if a uracil or thymine appears in one of the strands of the paired double stranded DNA molecules obtained in step (iii) or in step (iv) and a guanine appears in the corresponding position in the other strand of the paired double stranded DNA molecules.