Journal
ANNALS OF ONCOLOGY
Volume 27, Issue 3, Pages 532-539Publisher
OXFORD UNIV PRESS
DOI: 10.1093/annonc/mdv613
Keywords
fixation; NBF; UMFIX; HGSOC; next-generation sequencing; copy-number abnormalities; SNVs
Categories
Funding
- Ovarian Cancer Action [BriTROC project]
- Cancer Research UK [A15973, A15601, A18072]
- NHS Greater Glasgow and Clyde Biorepository
- University of Cambridge
- University of Glasgow
- National Institute for Health Research Cambridge Biomedical Research Centre
- National Cancer Research Network
- Cambridge and Glasgow Experimental Cancer Medicine Centres
- Hutchison Whampoa Limited
- Cancer Research UK [15601, 15973] Funding Source: researchfish
- Ovarian Cancer Action [OCA6] Funding Source: researchfish
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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.
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