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

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...

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Published inNucleic 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
LanguageEnglish
Published Oxford University Press 20.08.2021
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Summary: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.
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The authors wish it to be known that, in their opinion, the first two authors should be regarded as Joint First Authors.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkab397