Figure 2

Whole genome amplification (WGA) as a method to generate double stranded DNA following DNA immunoprecipitation. (a) Schematic of the steps during the WGA. Red = WGA adapter sequences (b). Quantitative PCR (qPCR) analysis of several loci prior to WGA (post hmeDIP: light grey) as well as following WGA (dark grey) reveals little to no bias is introduced by the WGA. Analysis of the signals from the hmeDIP-SC-seq over these same regions highlights the retention of the patterns following sequencing (red bars). Loci were selected from previous work: 5hmC –ve = little/no 5hmC, 5hmC +ve = moderate/high 5hmC. Light grey bars: pre-amplified IP DNA, dark grey bars: post WGA DNA, red bars: normalised signals over locus used for qPCR taken from hmeDIP-SC-seq liver A dataset. qPCR data plotted against the primary y-axis, hmeDIP SC-seq data plotted against secondary y-axis (c). Glu-RES-qPCR results of three loci reveal quantitative information on 5hmC levels at a given site, confirming that pre and post WGA samples in fig. 2b reflect the expected 5hmC patterns. Tan: unmodified CpG, pink: methylated CpG, blue: hydroxymethylated CpG. (d) Examples of good and poor read length distributions following SC-seq. The majority of successfully sequenced DNA fragments should be >100 bp with a mean around 125–150 bp. (e) Plot of mapping efficiency vs read length. Reads less than 50 bp should be excluded due to poor mapping accuracy.