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Preparation of genome-wide DNA fragment-libraries using bisulfite in polyacrylamide gel electrophoresis slices (Bis-PAGE), with formamide denaturation, and QC for massively parallel sequencing by oligonucleotide ligation and detection (SOLiD).
Anal Biochem. 2009 Apr 17;
Authors: Ranade SS, Chung CB, Zon G, Boyd VL
Bisulfite sequencing is widely used for analysis of DNA methylation status, i.e. 5-methylcytosine (5mC) vs. cytosine (C), in CpG-rich or other loci in genomic DNA (gDNA). Such methods typically involve reaction of gDNA with bisulfite followed by PCR amplification of specific regions of interest that, overall, converts C-->T and 5mC-->C, and then capillary sequencing to measure C vs. T composition at CpG sites. Massively parallel sequencing by oligonucleotide ligation and detection (SOLiD) has recently enabled relatively low-cost whole genome sequencing, and it would be highly desirable to apply such massively parallel sequencing to bisulfite-converted whole genomes to determine DNA methylation status of an entire genome, which has heretofore not been reported. As an initial step toward achieving this goal, we have extended our ongoing interest in improving bisulfite-conversion sample preparation to include a human genome-wide fragment-library for SOliD. The present report features novel use of formamide denaturant during bisulfite conversion of a suitably constructed library directly in a band-slice from polyacryamide gel electrophoresis (PAGE). To validate this new protocol for 5mC-protected fragment-library conversion, which we refer to as Bis-PAGE, capillary-based size-analysis and Sanger sequencing were carried out for individual amplicons derived from single-molecule PCR (smPCR) of randomly selected library-fragments. smPCR/capillary Sanger-sequencing of approximately 200 amplicons unambiguously demonstrated >99% C-->T conversion. All of these approximately 200 Sanger sequences were analyzed with a previously published web-accessible bioinformatics tool (methBLAST) for mapping to human chromosomes, the results of which indicated random distribution of analyzed fragments across all chromosomes. While these particular Bis-PAGE conversion and QC methods were exemplified in the context of a fragment-library for SOLiD, the concepts can be generalized to include other genome-wide library constructions intended for DNA methylation analysis by alternative high-throughput or massively parallelized methods that are currently available.
PMID: 19379703 [PubMed - as supplied by publisher]
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Preparation of genome-wide DNA fragment-libraries using bisulfite in polyacrylamide gel electrophoresis slices (Bis-PAGE), with formamide denaturation, and QC for massively parallel sequencing by oligonucleotide ligation and detection (SOLiD).
Anal Biochem. 2009 Apr 17;
Authors: Ranade SS, Chung CB, Zon G, Boyd VL
Bisulfite sequencing is widely used for analysis of DNA methylation status, i.e. 5-methylcytosine (5mC) vs. cytosine (C), in CpG-rich or other loci in genomic DNA (gDNA). Such methods typically involve reaction of gDNA with bisulfite followed by PCR amplification of specific regions of interest that, overall, converts C-->T and 5mC-->C, and then capillary sequencing to measure C vs. T composition at CpG sites. Massively parallel sequencing by oligonucleotide ligation and detection (SOLiD) has recently enabled relatively low-cost whole genome sequencing, and it would be highly desirable to apply such massively parallel sequencing to bisulfite-converted whole genomes to determine DNA methylation status of an entire genome, which has heretofore not been reported. As an initial step toward achieving this goal, we have extended our ongoing interest in improving bisulfite-conversion sample preparation to include a human genome-wide fragment-library for SOliD. The present report features novel use of formamide denaturant during bisulfite conversion of a suitably constructed library directly in a band-slice from polyacryamide gel electrophoresis (PAGE). To validate this new protocol for 5mC-protected fragment-library conversion, which we refer to as Bis-PAGE, capillary-based size-analysis and Sanger sequencing were carried out for individual amplicons derived from single-molecule PCR (smPCR) of randomly selected library-fragments. smPCR/capillary Sanger-sequencing of approximately 200 amplicons unambiguously demonstrated >99% C-->T conversion. All of these approximately 200 Sanger sequences were analyzed with a previously published web-accessible bioinformatics tool (methBLAST) for mapping to human chromosomes, the results of which indicated random distribution of analyzed fragments across all chromosomes. While these particular Bis-PAGE conversion and QC methods were exemplified in the context of a fragment-library for SOLiD, the concepts can be generalized to include other genome-wide library constructions intended for DNA methylation analysis by alternative high-throughput or massively parallelized methods that are currently available.
PMID: 19379703 [PubMed - as supplied by publisher]
More...