Fig. 3 a) ROC curve for detection of m6A and m5C in E. coli gDNA at different levels of coverage b) m5C detection on NA12878 chr20
We applied Tombo’s de novo modified base model to the E. coli genome for detection of m6A and m5C at different levels of coverage: 1x, 30x and 376x. As might be expected, greater coverage led to higher AUCs. At 376x, the AUC for m6A is 0.975, and for m5C is 0.992, whereas at 30x the AUCs are 0.947 and 0.983 respectively (Fig. 3a). We then used Tombo to detect m5C in human genomic data generated on a PromethION from NA12878, genomic DNA. The optimisation of Tombo for use with PromethION data is not yet complete, but we obtained strong correspondence between our Tombo analysis and publicly available bisulphite data from the same genome. Fig. 3b shows raw nanopore signals (red lines) which deviate from the expected canonical levels (grey background distributions) around sites of methylation which were identified using bisulphite sequencing. The top two panels show methylation at a CpG site, which is symmetric on positive and negative strands. The bottom two panels show methylation in examples of CHG and CHH contexts. Here the methylation was asymmetric, only being present on the positive strands.
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