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

• Published in: Nucleic acids research Vol. 49; no. 14; p. e81
• Main Authors: Sakamoto, Yoshitaka, Zaha, Suzuko, Nagasawa, Satoi, Miyake, Shuhei, Kojima, Yasuyuki, Suzuki, Ayako, Suzuki, Yutaka, Seki, Masahide
• Format: Journal Article
• Language: English
• Published: Oxford University Press 20.08.2021
• Subjects: Methods Online
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkab397

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.