The ability of nanopore sequencing to simultaneously detect modified nucleotides while producing long reads makes it ideal for detecting and phasing allele-specific methylation. However, there is currently no complete software for detecting SNPs, phasing haplotypes, and mapping methylation to these from nanopore sequence data. Here, we present NanoMethPhase, a software tool to phase 5-methylcytosine from nanopore sequencing. We also present SNVoter, which can post-process nanopore SNV calls to improve accuracy in low coverage regions. Together, these tools can accurately detect allele-specific methylation genome-wide using nanopore sequence data with low coverage of about ten-fold redundancy.

Imprinting is a critical part of normal embryonic development in mammals, controlled by defined parent-of-origin (PofO) differentially methylated regions (DMRs) known as imprinting control regions. Direct nanopore sequencing of DNA provides a means to detect allelic methylation and to overcome the drawbacks of methylation array and short-read technologies. Here, we used publicly available nanopore sequencing data for 12 standard B-lymphocyte cell lines to acquire the genome-wide mapping of imprinted intervals in humans. Using the sequencing data, we were able to phase 95% of the human methylome and detect 94% of the previously well-characterized, imprinted DMRs. In addition, we found 42 novel imprinted DMRs (16 germline and 26 somatic), which were confirmed using whole-genome bisulfite sequencing (WGBS) data. Analysis of WGBS data in mouse (Mus musculus), rhesus monkey (Macaca mulatta), and chimpanzee (Pan troglodytes) suggested that 17 of these imprinted DMRs are conserved. Some of the novel imprinted intervals are within or close to imprinted genes without a known DMR. We also detected subtle parental methylation bias, spanning several kilobases at seven known imprinted clusters. At these blocks, hypermethylation occurs at the gene body of expressed allele(s) with mutually exclusive H3K36me3 and H3K27me3 allelic histone marks. These results expand upon our current knowledge of imprinting and the potential of nanopore sequencing to identify imprinting regions using only parent-offspring trios, as opposed to the large multi-generational pedigrees that have previously been required.