Assessment of concordance between fresh-frozen and formalin-fixed paraffin embedded tumor DNA methylation using a targeted sequencing approach
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Bruce Moran1,10,*, Sudipto Das1,10,*, Dominiek Smeets2,3, Gillian Peutman2,3, Rut Klinger1,10, Bozena Fender4, Kate Connor1,5,10, Matthias Ebert6, Timo Gaiser6, Jochen HM Prehn5, Orna Bacon5,7, Elaine Kay7, Bryan Hennessy7, Verena Murphy8, Bauke Ylstra9, Diether Lambrechts2,3, Annette T. Byrne5, William M. Gallagher4,10,** and Darran P. O’Connor1,10,**
1Department of Molecular and Cellular Therapeutics, Royal College of Surgeons in Ireland, Dublin, Ireland
2Department of Oncology, Laboratory of Translational Genetics, VIB Center for Cancer Biology, Leuven, Belgium
3Department of Oncology, Laboratory of Translational Genetics, Department of Oncology, KU Leuven, Leuven, Belgium
4OncoMark Ltd., NovaUCD, Belfield Innovation Park, Dublin, Ireland
5Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland
6Department of Internal Medicine, University of Heidelberg, Mannheim, Germany
7Department of Pathology, Beaumont Hospital, Dublin, Ireland
8Cancer Trials Ireland, Dublin, Ireland
9Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands
10Cancer Biology and Therapeutics Laboratory, UCD School of Biomolecular and Biomedical Science, UCD Conway Institute, University College, Dublin, Ireland
*These authors have contributed equally to this work
Darran P. O’Connor, email: firstname.lastname@example.org
Keywords: targeted bisulfite sequencing, methylation, epigenetics, FFPE, tumor preservation
Received: July 20, 2016 Accepted: April 03, 2017 Published: May 30, 2017
DNA methylation is altered in many types of disease, including metastatic colorectal cancer. However, the methylome has not yet been fully described in archival formalin-fixed paraffin embedded (FFPE) samples in the context of matched fresh-frozen (FF) tumor material at base-pair resolution using a targeted approach. Using next-generation sequencing, we investigated three pairs of matched FFPE and FF samples to determine the extent of their similarity. We identified a ‘bowing’ pattern specific to FFPE samples categorized by a lower CG proportion at the start of sequence reads. We have found no evidence that this affected methylation calling, nor concordance of results. We also found no significant increase in deamination, measured by C>T transitions, previously considered a result of crosslinking DNA by formalin fixation and a barrier to the use of FFPE in methylation studies. The methods used in this study have shown sensitivity of between 60-70% based on positions also methylated in colorectal cancer cell lines. We demonstrate that FFPE material is a useful source of tumor material for methylation studies using targeted sequencing.
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