Guidelines for whole genome bisulphite sequencing of intact and FFPET DNA on the Illumina HiSeq X Ten
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...
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doaj-4b0c244e8fac47f18cdf2eed2689bdf42020-11-25T00:28:17ZengBMCEpigenetics & Chromatin1756-89352018-05-0111112010.1186/s13072-018-0194-0Guidelines for whole genome bisulphite sequencing of intact and FFPET DNA on the Illumina HiSeq X TenShalima S. Nair0Phuc-Loi Luu1Wenjia Qu2Madhavi Maddugoda3Lily Huschtscha4Roger Reddel5Georgia Chenevix-Trench6Martina Toso7James G. Kench8Lisa G. Horvath9Vanessa M. Hayes10Phillip D. Stricker11Timothy P. Hughes12Deborah L. White13John E. J. Rasko14Justin J.-L. Wong15Susan J. Clark16Genomics and Epigenetics Division, Garvan Institute of Medical ResearchGenomics and Epigenetics Division, Garvan Institute of Medical ResearchGenomics and Epigenetics Division, Garvan Institute of Medical ResearchGenomics and Epigenetics Division, Garvan Institute of Medical ResearchCancer Research Unit, Children’s Medical Research Institute, University of SydneyCancer Research Unit, Children’s Medical Research Institute, University of SydneyQIMR BerghoferQIMR BerghoferDepartment of Tissue Pathology and Diagnostic Oncology, Royal Prince Alfred HospitalCentral Clinical School, Sydney Medical School, University of SydneyGenomics and Epigenetics Division, Garvan Institute of Medical ResearchDepartment of Urology, St. Vincent’s HospitalCancer Theme, South Australian Health and Medical Research InstituteCancer Theme, South Australian Health and Medical Research InstituteGene and Stem Cell Therapy Program, Centenary Institute, University of SydneyGene and Stem Cell Therapy Program, Centenary Institute, University of SydneyGenomics and Epigenetics Division, Garvan Institute of Medical ResearchAbstract 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.http://link.springer.com/article/10.1186/s13072-018-0194-0DNA methylationWhole genome bisulphite sequencingHiSeq X TenHiSeq 2500EpigeneticsSNP |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
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 Susan J. Clark |
spellingShingle |
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 Susan J. Clark Guidelines for whole genome bisulphite sequencing of intact and FFPET DNA on the Illumina HiSeq X Ten Epigenetics & Chromatin DNA methylation Whole genome bisulphite sequencing HiSeq X Ten HiSeq 2500 Epigenetics SNP |
author_facet |
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 Susan J. Clark |
author_sort |
Shalima S. Nair |
title |
Guidelines for whole genome bisulphite sequencing of intact and FFPET DNA on the Illumina HiSeq X Ten |
title_short |
Guidelines for whole genome bisulphite sequencing of intact and FFPET DNA on the Illumina HiSeq X Ten |
title_full |
Guidelines for whole genome bisulphite sequencing of intact and FFPET DNA on the Illumina HiSeq X Ten |
title_fullStr |
Guidelines for whole genome bisulphite sequencing of intact and FFPET DNA on the Illumina HiSeq X Ten |
title_full_unstemmed |
Guidelines for whole genome bisulphite sequencing of intact and FFPET DNA on the Illumina HiSeq X Ten |
title_sort |
guidelines for whole genome bisulphite sequencing of intact and ffpet dna on the illumina hiseq x ten |
publisher |
BMC |
series |
Epigenetics & Chromatin |
issn |
1756-8935 |
publishDate |
2018-05-01 |
description |
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. |
topic |
DNA methylation Whole genome bisulphite sequencing HiSeq X Ten HiSeq 2500 Epigenetics SNP |
url |
http://link.springer.com/article/10.1186/s13072-018-0194-0 |
work_keys_str_mv |
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