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Publication Details
AFRICAN RESEARCH NEXUS
SHINING A SPOTLIGHT ON AFRICAN RESEARCH
biochemistry, genetics and molecular biology
Guidelines for whole genome bisulphite sequencing of intact and FFPET DNA on the Illumina HiSeq X Ten
Epigenetics and Chromatin, Volume 11, No. 1, Article 24, Year 2018
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Description
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. © 2018 The Author(s).
Authors & Co-Authors
Huschtscha, Lily Irene
Australia, Parramatta
Children's Medical Research Institute
Chenevix-Trench, Georgia
Australia, Brisbane
Qimr Berghofer Medical Research Institute
Kench, J. G.
Australia, Sydney
Royal Prince Alfred Hospital
Australia, Sydney
The University of Sydney
Horvath, Lisa G.
Australia, Sydney
The University of Sydney
Australia, Sydney
The Kinghorn Cancer Centre
Hayes, Vanessa M.
Australia, Sydney
Garvan Institute of Medical Research
Australia, Sydney
Unsw Sydney
Australia, Sydney
The University of Sydney
Stricker, Phillip D.
Australia, Sydney
St. Vincent's Hospital Sydney
Hughes, Timothy P.
Australia, Adelaide
South Australian Health and Medical Research Institute
Australia, Adelaide
The University of Adelaide
Rasko, John E.J.
Australia, Sydney
Centenary Institute of Cancer Medicine and Cell Biology
Australia, Sydney
The University of Sydney
Australia, Sydney
Royal Prince Alfred Hospital
Statistics
Citations: 32
Authors: 8
Affiliations: 11
Identifiers
Doi:
10.1186/s13072-018-0194-0
ISSN:
17568935
Research Areas
Genetics And Genomics
Health System And Policy