Fig. 1 RRMS using AS a) read length distribution b) bed workflow c) features d) sequenced vs mapped e) coverage f) example region g) overlap with RRBS h) CpGs called i) correlation with RRBS
Nanopore sequencing enables direct detection of methylated cytosines (e.g. at CpG sites), without the need for bisulfite conversion. CpG sites frequently occur in high density clusters called CpG islands (CGI) and >60% of human genes have their promoters embedded within CGIs. Changes in methylation patterns within promoters is associated with changes in gene expression. Reduced representation bisulfite sequencing (RRBS) is a method used to obtain genome-wide methylation analysis without the need to sequence the whole genome, but is expensive and time consuming. Furthermore, the complex library preparation method is imprecise and does not specifically target any promoter region. Adaptive sampling (AS) offers a fast, flexible and precise method to enrich for regions of interest (e.g. CGIs) by depleting off-target regions during sequencing itself (Fig. 1a), with no requirement for upfront sample manipulation. Here we combine Oxford Nanopore’s methylation detection with AS and compare the performance with RRBS, using two tumour/normal cell line pairs. Our file of AS target regions is prepared using the workflow shown (Fig. 1b) and covers 310 Mb of the genome, including ~28,000 CpG islands and other key features (Fig. 1c). AS retains a higher proportion of data than RRBS and gives a higher proportion of on-target reads (Figs. 1d and e). AS shows more even coverage compared than RRBS (Fig. 1f), recovers more CpGs (Figs. 1g and 1h) and is highly reproducible (Fig. 1i).