DNA methylome profiling at single-base resolution through bisulfite sequencing of 5mC-immunoprecipitated DNA
Authors: Rongfa Bu
Yueyi Shi
Zhen Jia
Baorong Zhang
Guolan Gao
Yipeng Ren
Lei Zhang
Lejun Xing
Tong Wang
Journal: BMC biotechnology (2018)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: DNA methylome; MB-seq; MRB-seq; Novel technology; Single-base resolution.
Affiliations: Department of Obstetrics and Gynecology, Beijing Aviation General Hospital, Beijing, 100012, China., Department of Stomatology, Chinese PLA General Hospital, Beijing, 100853, People's Republic of China., Medical School, Nankai University, No.94 Weijin Road, Tianjin, 300070, China., Department of Stomatology, Beijing Aviation General Hospital, Beijing, 100012, People's Republic of China., Department of Obstetrics and Gynecology, Beijing Aviation General Hospital, Beijing, 100012, China. ., Department of Stomatology, Chinese PLA General Hospital, Beijing, 100853, People's Republic of China. .

Abstract:  Background: Detection of DNA methylome at single-base resolution is a significant challenge but promises to shed considerable light on human disease etiology. Current technologies could not detect DNA methylation genome-wide at single-base resolution with small amount of sequencing data and could not avoid detecting the methylation of repetitive elements which are considered as "junk DNA". Methods: In this study, we have developed a novel DNA methylome profiling technology named MB-seq with its ability to identify genome-wide 5mC and quantify DNA methylation levels by introduced an assistant adapter AluI-linker This linker can be ligated to sonicated DNA and then be digested after the bisulfite treatment and amplification, which has no effect of MeDIP enrichment. Because many researchers are interested in investigating the methylation of functional regions such as promoters and gene bodies, we have also developed a novel alternative method named MRB-seq, which can be used to investigate the DNA methylation of functional regions by removing the repeats with Cot-1 DNA. Results: In this study, we have developed MB-seq, a novel DNA methylome profiling technology combining MeDIP-seq with bisulfite conversion, which can precisely detect the 5mC sites and determine their DNA methylation level at single-base resolution in a cost-effective way. In addition, we have developed a new alternative method, MRB-seq (MeDIP-repetitive elements removal-bisulfite sequencing), which interrogates 5mCs in functional regions by depleting nearly half of repeat fragments enriched by MeDIP. Comparing MB-seq and MRB-seq to whole-genome BS-seq using the same batch of DNA from YH peripheral blood mononuclear cells. We found that the sequencing data of MB-seq and MRB-seq almost reaches saturation after generating 7-8 Gbp data, whereas BS-seq requires about 100 Gbp data to achieve the same effect. In comparison to MeDIP-seq and BS-seq, MB-seq offers several key advantages, including single-base resolution, discriminating the methylated sites within a CpG and non-CpG pattern and overcoming the false positive of MeDIP-seq due to the non-specific binding of 5-methylcytidine antibody to genomic fragments. Conclusion: Our novel developed method MB-seq can accelerate the decoding process of DNA methylation mechanism in human diseases because it requires 7-8 Gbp data to measure human methylome with enough coverage and sequencing depth, affording it a direct and practical application in the study of multiple samples. In addition, we have also provided a novel alternative MRB-seq method, which removes most repetitive sequences and allows researchers to genome-wide characterize DNA methylation of functional regions. 

Retracted: Yes

PubPeer comments:
 2020 retraction. https: bmcbiotechnol.biomedcentral.com articles 10.1186 s12896-020-00619-w The Editor has retracted this article [1] due to substantial similarities with an unpublished manuscript by Yan Wang, Guanyu Ji, Jinyu Wu, Mingzhi Ye, Lee M. Butcher, Ning Li, Wanshi Cai, Xu Han, Wei Dai, Qi Liu, Zhimei Yuan, Xiuqing Zhang, and Zhong Sheng Sun. The authors have not responded to correspondence about this retraction. Reference 1. Jia, et al. BMC Biotechnology. 2018;18:7. https: doi.org 10.1186 s12896-017-0409-7. ,