Abstract

Long-read whole-genome sequencing analysis of DNA methylation would provide useful information on the chromosomal context of gene expression regulation. Here we describe the development of a method that improves the read length generated by using the bisulfite-sequencing-based approach. In this method, we combined recently developed enzymatic base conversion, where an unmethylated cytosine (C) should be converted to thymine (T), with nanopore sequencing. After methylation-sensitive base conversion, the sequencing library was constructed using long-range polymerase chain reaction. This type of analysis is possible using a minimum of 1 ng genomic DNA, and an N50 read length of 3.4-7.6 kb is achieved. To analyze the produced data, which contained a substantial number of base mismatches due to sequence conversion and an inaccurate base read of the nanopore sequencing, a new analytical pipeline was constructed. To demonstrate the performance of long-read methylation sequencing, breast cancer cell lines and clinical specimens were subjected to analysis, which revealed the chromosomal methylation context of key cancer-related genes, allele-specific methylated genes, and repetitive or deletion regions. This method should convert the intractable specimens for which the amount of available genomic DNA is limited to the tractable targets.

Article

Publication : 「Nucleic Acids Research」（オンライン版：5月21日）

Title : Long-read whole-genome methylation patterning using enzymatic base conversion and nanopore sequencing

Author : Yoshitaka Sakamoto, Suzuko Zaha, Satoi Nagasawa, Shuhei Miyake, Yasuyuki Kojima, Ayako Suzuki, Yutaka Suzuki*, Masahide Seki*

DOI : 10.1093/nar/gkab397

URL : https://academic.oup.com/nar/advance-article/doi/10.1093/nar/gkab397/6279847?searchresult=1