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Ann Oncol. 2016 Mar;27(3):532-9. doi: 10.1093/annonc/mdv613. Epub 2015 Dec 17.

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

Author information

1
Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge.
2
Institute of Cancer Sciences, University of Glasgow, Glasgow.
3
Cancer Molecular Diagnostics Laboratory, Department of Oncology, University of Cambridge, Cambridge.
4
Department of Pathology, Queen Elizabeth University Hospital, Glasgow.
5
Addenbrooke's Hospital, Cambridge University Hospital NHS Foundation Trust and National Institute for Health Research Cambridge Biomedical Research Centre, Cambridge Department of Histopathology, Addenbrooke's Hospital, Cambridge, UK.
6
Institute of Cancer Sciences, University of Glasgow, Glasgow iain.mcneish@glasgow.ac.uk.
7
Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge Addenbrooke's Hospital, Cambridge University Hospital NHS Foundation Trust and National Institute for Health Research Cambridge Biomedical Research Centre, Cambridge.

Abstract

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.

KEYWORDS:

HGSOC; NBF; SNVs; UMFIX; copy-number abnormalities; fixation; next-generation sequencing

PMID:
26681675
PMCID:
PMC4769995
DOI:
10.1093/annonc/mdv613
[Indexed for MEDLINE]
Free PMC Article

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