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

Title: Methanol-based fixation is superior to buffered formalin for next-generation sequencing of DNA from clinical cancer samples
Authors: Piskorz, AM
Ennis, D
Macintyre, G
Goranova, TE
Eldridge, M
Segui-Gracia, N
Valganon, M
Hoyle, A
Orange, C
Moore, L
Jimenez-Linan, M
Millan, D
McNeish, IA
Brenton, JD
Item Type: Journal Article
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.
Issue Date: 17-Dec-2015
Date of Acceptance: 1-Dec-2015
URI: http://hdl.handle.net/10044/1/55479
DOI: https://dx.doi.org/10.1093/annonc/mdv613
ISSN: 0923-7534
Publisher: Oxford University Press (OUP)
Start Page: 532
End Page: 539
Journal / Book Title: Annals of Oncology
Volume: 27
Issue: 3
Copyright Statement: © The Author 2015. Published by Oxford University Press on behalf of the European Society for Medical Oncology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/ 4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Keywords: Science & Technology
Life Sciences & Biomedicine
Oncology
fixation
NBF
UMFIX
HGSOC
next-generation sequencing
copy-number abnormalities
SNVs
TISSUE FIXATION
MUTATIONS
QUALITY
RNA
FORMALDEHYDE
EXTRACTION
ARTIFACTS
INTEGRITY
TP53
Base Sequence
DNA
Formaldehyde
High-Throughput Nucleotide Sequencing
Humans
Methanol
Middle Aged
Neoplasms
Paraffin Embedding
Sequence Analysis, DNA
Tissue Fixation
1112 Oncology And Carcinogenesis
Oncology & Carcinogenesis
Publication Status: Published
Appears in Collections:Division of Surgery
Division of Cancer
Faculty of Medicine



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