Guidelines for whole genome bisulphite sequencing of intact and FFPET DNA on the Illumina HiSeq X Ten
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 15x 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 16-20x 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 > 0.9x). 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.
|ISBN||1756-8935 (Electronic) 1756-8935 (Linking)|
|Authors||Nair, S. S.; Luu, P. L.; Qu, W.; Maddugoda, M.; Huschtscha, L.; Reddel, R.; Chenevix-Trench, G.; Toso, M.; Kench, J. G.; Horvath, L. G.; Hayes, V. M.; Stricker, P. D.; Hughes, T. P.; White, D. L.; Rasko, J. E. J.; Wong, J. J.; Clark, S. J.|
|Responsible Garvan Author|
|Publisher Name||Epigenetics & Chromatin|
|URL link to publisher's version||https://www.ncbi.nlm.nih.gov/pubmed/29807544|