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Type: Journal article
Title: Guidelines for whole genome bisulphite sequencing of intact and FFPET DNA on the Illumina HiSeq X Ten
Author: Nair, S.
Luu, P.-L.
Qu, W.
Maddugoda, M.
Huschtscha, L.
Reddel, R.
Chenevix-Trench, G.
Toso, M.
Kench, J.
Horvath, L.
Hayes, V.
Stricker, P.
Hughes, T.
White, D.
Rasko, J.
Wong, J.-L.
Clark, S.
Citation: Epigenetics and Chromatin, 2018; 11(1):24-24
Publisher: BioMed Central
Issue Date: 2018
ISSN: 1756-8935
Statement of
Shalima S. Nair, Phuc-Loi Luu, Wenjia Qu, Madhavi Maddugoda, Lily Huschtscha, Roger Reddel, Georgia Chenevix-Trench, Martina Toso, James G. Kench, Lisa G. Horvath, Vanessa M. Hayes, Phillip D. Stricker, Timothy P. Hughes, Deborah L. White, John E. J. Rasko, Justin J.-L. Wong and Susan J. Clark
Abstract: Background: Comprehensive genome-wide DNA methylation profiling is critical to gain insights into epigenetic reprogramming during development and disease processes. Among the different genome-wide DNA methylation technologies, whole genome bisulphite sequencing (WGBS) is considered the gold standard for assaying genome-wide DNA methylation at single base resolution. However, the high sequencing cost to achieve the optimal depth of coverage limits its application in both basic and clinical research. To achieve 15× coverage of the human methylome, using WGBS, requires approximately three lanes of 100-bp-paired-end Illumina HiSeq 2500 sequencing. It is important, therefore, for advances in sequencing technologies to be developed to enable cost-effective high-coverage sequencing. Results: In this study, we provide an optimised WGBS methodology, from library preparation to sequencing and data processing, to enable 16–20× genome-wide coverage per single lane of HiSeq X Ten, HCS 3.3.76. To process and analyse the data, we developed a WGBS pipeline (METH10X) that is fast and can call SNPs. We performed WGBS on both high-quality intact DNA and degraded DNA from formalin-fixed paraffin-embedded tissue. First, we compared different library preparation methods on the HiSeq 2500 platform to identify the best method for sequencing on the HiSeq X Ten. Second, we optimised the PhiX and genome spike-ins to achieve higher quality and coverage of WGBS data on the HiSeq X Ten. Third, we performed integrated whole genome sequencing (WGS) and WGBS of the same DNA sample in a single lane of HiSeq X Ten to improve data output. Finally, we compared methylation data from the HiSeq 2500 and HiSeq X Ten and found high concordance (Pearson r > 0.9×). Conclusions: Together we provide a systematic, efficient and complete approach to perform and analyse WGBS on the HiSeq X Ten. Our protocol allows for large-scale WGBS studies at reasonable processing time and cost on the HiSeq X Ten platform.
Keywords: DNA methylation
HiSeq X Ten
HiSeq 2500
Whole genome bisulphite sequencing
Rights: © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
DOI: 10.1186/s13072-018-0194-0
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