Whole-genome long-read TAPS deciphers DNA methylation patterns at base resolution using PacBio SMRT sequencing technology

Long-read sequencing provides valuable information on difficult-to-map genomic regions, which can complement short-read sequencing to improve genome assembly, yet limited methods are available to accurately detect DNA methylation over long distances at a whole-genome scale. By combining our recently developed TET-assisted pyridine borane sequencing (TAPS) method, which enables direct detection of 5-methylcytosine and 5-hydroxymethylcytosine, with PacBio single-molecule real-time sequencing, we present here whole-genome long-read TAPS (wglrTAPS). To evaluate the performance of wglrTAPS, we applied it to mouse embryonic stem cells as a proof of concept, and an N50 read length of 3.5 kb is achieved. By sequencing wglrTAPS to 8.2x depth, we discovered a significant proportion of CpG sites that were not covered in previous 27.5x short-read TAPS. Our results demonstrate that wglrTAPS facilitates methylation profiling on problematic genomic regions with repetitive elements or structural variations, and also in an allelic manner, all of which are extremely difficult for short-read sequencing methods to resolve. This method therefore enhances applications of third-generation sequencing technologies for DNA epigenetics.

Automated summary

Long-read sequencing provides valuable information on difficult-to-map genomic regions and can improve genome assembly when combined with short-read sequencing. This study presents a method called whole-genome long-read TAPS (wglrTAPS), which combines TET-assisted pyridine borane sequencing with PacBio single-molecule real-time sequencing. Application of wglrTAPS to mouse embryonic stem cells showed the ability to detect previously uncovered CpG sites in problematic genomic regions. This method enhances the use of third-generation sequencing technologies for DNA epigenetics.