Abstract
Modification of DNA resulting in 5-methylcytosine (5 mC) or 5-hydroxymethylcytosine (5hmC) has been shown to influence the local chromatin environment and affect transcription. Although recent advances in next generation sequencing technology allow researchers to map epigenetic modifications across the genome, such experiments are often time-consuming and cost prohibitive. Here we present a rapid and cost effective method of generating genome wide DNA modification maps utilising commercially available semiconductor based technology (DNA immunoprecipitation semiconductor sequencing; “DIP-SC-seq”) on the Ion Proton sequencer. Focussing on the 5hmC mark we demonstrate, by directly comparing with alternative sequencing strategies, that this platform can successfully generate genome wide 5hmC patterns from as little as 500 ng of genomic DNA in less than 4 days. Such a method can therefore facilitate the rapid generation of multiple genome wide epigenetic datasets.
Highlights
Modification of DNA resulting in 5-methylcytosine (5 mC) or 5-hydroxymethylcytosine (5hmC) has been shown to influence the local chromatin environment and affect transcription
The first phase of the hydroxymethyl DNA immunoprecipitation and semiconductor sequencing (“hmeDIP-SC-seq”) protocol relies on the fragmentation of high quality genomic DNA to generate a range of fragments 100–500 bp long, the majority of which are around 300 bp in length (Fig. 1)
We find that the 5hmC patterns generated though hmeDIP-SC-seq are in agreement with these findings and to patterns generated by hmeDIP-Hiseq and affinity Hiseq (Fig. 4a)
Summary
Modification of DNA resulting in 5-methylcytosine (5 mC) or 5-hydroxymethylcytosine (5hmC) has been shown to influence the local chromatin environment and affect transcription. We present a rapid and cost effective method of generating genome wide DNA modification maps utilising commercially available semiconductor based technology (DNA immunoprecipitation semiconductor sequencing; “DIP-SC-seq”) on the Ion Proton sequencer. Focussing on the 5hmC mark we demonstrate, by directly comparing with alternative sequencing strategies, that this platform can successfully generate genome wide 5hmC patterns from as little as 500 ng of genomic DNA in less than 4 days Such a method can facilitate the rapid generation of multiple genome wide epigenetic datasets. Today’s sequencing based experiments typically generate more data than alternative methods such as microarray based assays; allowing the researcher to carry out high resolution analyses such as quantification of both transcriptional activity (RNA sequencing: RNAseq) and epigenetic modification patterns
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