Methanol-based fixation is superior to buffered formalin for next-generation sequencing of DNA from clinical cancer samples.

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Piskorz, AM 
Ennis, D 
Goranova, TE 

BACKGROUND: 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. PATIENTS AND METHODS: 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. RESULTS: 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. CONCLUSION: 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.

HGSOC, NBF, SNVs, UMFIX, copy-number abnormalities, fixation, next-generation sequencing, Base Sequence, DNA, Formaldehyde, High-Throughput Nucleotide Sequencing, Humans, Methanol, Middle Aged, Neoplasms, Paraffin Embedding, Sequence Analysis, DNA, Tissue Fixation
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Ann Oncol
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Elsevier BV
Cancer Research UK (A15973)
Cancer Research UK (A15601)
This work was supported by Ovarian Cancer Action [BriTROC project grant: IMcN, JDB], Cancer Research UK [grant numbers A15973, A15601, A18072]; NHS Greater Glasgow and Clyde Biorepository; the Universities of Cambridge and Glasgow; National Institute for Health Research Cambridge Biomedical Research Centre; National Cancer Research Network; Cambridge and Glasgow Experimental Cancer Medicine Centres and Hutchison Whampoa Limited. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We acknowledge the NIHR Cambridge Biomedical Research Centre for their support of this work. The authors would also like to thank Colin Nixon (CRUK Beatson Institute, Glasgow) for assistance with IHC optimisation.