Abstract
Next-generation sequencing (NGS) of tumour samples is a critical component of personalised cancer treatment, but it requires high-quality DNA samples. Routine neutral-buffered formalin (NBF) fixation has detrimental effects on nucleic acids, causing low yields, as well as fragmentation and DNA base changes, leading to significant artefacts. We have carried out a detailed comparison of DNA quality from matched samples isolated from high-grade serous ovarian cancers from 16 patients fixed in methanol and NBF. These experiments use tumour fragments and mock biopsies to simulate routine practice, ensuring that results are applicable to standard clinical biopsies. Using matched snap-frozen tissue as gold standard comparator, we show that methanol-based fixation has significant benefits over NBF, with greater DNA yield, longer fragment size and more accurate copy-number calling using shallow whole-genome sequencing (WGS). These data also provide a new approach to understand and quantify artefactual effects of fixation using non-negative matrix factorisation to analyse mutational spectra from targeted and WGS data. We strongly recommend the adoption of methanol fixation for sample collection strategies in new clinical trials. This approach is immediately available, is logistically simple and can offer cheaper and more reliable mutation calling than traditional NBF fixation.
Highlights
Microscopic examination of formalin-fixed paraffin embedded (FFPE) material remains crucial in cancer diagnosis, next-generation sequencing (NGS) of tumour DNA has emerged as a powerful diagnostic tool [1] and is a central component of personalised medicine initiatives
Methanol fixation yields higher yield and size of DNA fragments than buffered formalin There was no significant difference in tumour cellularity and TP53 allele fraction between Universal molecular fixative (UMFIX) and neutral-buffered formalin (NBF) samples, allowing direct comparison of DNA metrics (Supplementary Figure 1)
Copy-number calling in methanol-fixed material is superior to formalin Copy number profiles from shallow whole genome sequencing were compared for correlation and variance of Copy Number Abnormality (CNA) estimation, using SF as gold standard
Summary
Microscopic examination of formalin-fixed paraffin embedded (FFPE) material remains crucial in cancer diagnosis, next-generation sequencing (NGS) of tumour DNA has emerged as a powerful diagnostic tool [1] and is a central component of personalised medicine initiatives. Universal molecular fixative (UMFIX) has been shown to be superior for IHC to neutral-buffered formalin (NBF), and gives higher yield and molecular weight of extracted DNA and RNA [5, 6, 8]. We have tested the suitability of DNA extracted after methanol-based fixation for NGS assays compared to DNA from matched NBF and fresh-frozen tissues.
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