Whole-genome Methylation Analysis of APOBEC Enzyme-converted DNA (~5 kb) by Nanopore Sequencing

Suzuko Zaha; Yoshitaka Sakamoto; Satoi Nagasawa; Sumio Sugano; Ayako Suzuki; Yutaka Suzuki; Masahide Seki

doi:10.21769/BioProtoc.4345

Whole-genome Methylation Analysis of APOBEC Enzyme-converted DNA (~5 kb) by Nanopore Sequencing

Bio Protoc. 2022 Mar 5;12(5):e4345. doi: 10.21769/BioProtoc.4345.

Authors

Suzuko Zaha, Yoshitaka Sakamoto, Satoi Nagasawa, Sumio Sugano^{1

2}, Ayako Suzuki, Yutaka Suzuki, Masahide Seki

Affiliations

¹ Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Chiba, Japan.
² Institute of Kashiwa-no-ha Omics Gate, Kashiwa, Chiba, Japan.

Abstract

In recent years, DNA methylation research has been accelerated by the advent of nanopore sequencers. However, read length has been limited by the constraints of base conversion using the bisulfite method, making analysis of chromatin content difficult. The read length of the previous method combining bisulfite conversion and long-read sequencing was ~1.5 kb, even using targeted PCR. In this study, we have improved read length (~5 kb), by converting unmethylated cytosines to uracils with APOBEC enzymes, to reduce DNA fragmentation. The converted DNA was then sequenced using a PromethION nanopore sequencer. We have also developed a new analysis pipeline that accounts for base conversions, which are not present in conventional nanopore sequencing, as well as errors produced by nanopore sequencing.

Keywords: DNA methylation; Epigenetics; Long-read sequencing; Nanopore sequencing; Next generation sequencing.