Abstract
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.
Highlights
Epigenetic modification including DNA methylation is regarded as one of the factors that regulate gene expression across a variety of diseases including cancer [1,2,3,4]
We 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 formalin-fixed paraffin embedded (FFPE) in methylation studies
A known issue with FFPE material is increased DNA fragmentation which can result in template DNA which is shorter than the number of bases being sequenced [18]
Summary
Epigenetic modification including DNA methylation is regarded as one of the factors that regulate gene expression across a variety of diseases including cancer [1,2,3,4]. Advantages of archival FFPE-derived cohorts include availability of extensive clinical, histological information and potentially longitudinal sampling, not necessarily available otherwise. This sample type has not been extensively used to generate high-resolution single base DNA methylation profiles with NGS, and this may have resulted in some trepidation in considering this option. We believe this to be partly due to the fact that FFPE-derived DNA presents several challenges in terms of overall quality as well as artifacts associated with preservation. Research of the inherent effects of formalin fixation on dsDNA has illustrated that denaturation occurs at AT-rich regions, which results in further chemical interactions such as hydrolysis of the phosphodiester bonds, causing fragmentation [18, 23,24,25]
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