Nucleic Acids Research Advance Access published online on May 21, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm330
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Methods Online |
Sequence analysis and editing for bisulphite genomic sequencing projects
Leeds Institute for Molecular Medicine, University of Leeds, Leeds, UK
*To whom correspondence should be addressed. Tel: +44 113 343 8649; Fax: +44 113 343 8702; Email: d.t.bonthron{at}leeds.ac.uk
Received March 22, 2007. Revised April 12, 2007. Accepted April 17, 2007.
Bisulphite genomic sequencing is a widely used technique for detailed analysis of the methylation status of a region of DNA. It relies upon the selective deamination of unmethylated cytosine to uracil after treatment with sodium bisulphite, usually followed by PCR amplification of the chosen target region. Since this two-step procedure replaces all unmethylated cytosine bases with thymine, PCR products derived from unmethylated templates contain only three types of nucleotide, in unequal proportions. This can create a number of technical difficulties (e.g. for some base-calling methods) and impedes manual analysis of sequencing results (since the long runs of T or A residues are difficult to align visually with the parent sequence). To facilitate the detailed analysis of bisulphite PCR products (particularly using multiple cloned templates), we have developed a visually intuitive program that identifies the methylation status of CpG dinucleotides by analysis of raw sequence data files produced by MegaBace or ABI sequencers as well as Staden SCF trace files and plain text files. The program then also collates and presents data derived from independent templates (e.g. separate clones). This results in a considerable reduction in the time required for completion of a detailed genomic methylation project.
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