title: Methanol-based fixation is superior to buffered formalin for next-generation sequencing of DNA from clinical cancer samples.
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.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge.
authors.forename: A M
authors.initials: AM
authors.lastname: Piskorz
authors.affiliation: Institute of Cancer Sciences, University of Glasgow, Glasgow.
authors.forename: D
authors.initials: D
authors.lastname: Ennis
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge.
authors.forename: G
authors.initials: G
authors.lastname: Macintyre
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge.
authors.forename: T E
authors.initials: TE
authors.lastname: Goranova
authors.affiliation: Cancer Research UK Cambridge Institute, University of Cambridge, Cambridge.
authors.forename: M
authors.initials: M
authors.lastname: Eldridge
authors.affiliation: Cancer Molecular Diagnostics Laboratory, Department of Oncology, University of Cambridge, Cambridge.
authors.forename: N
authors.initials: N
authors.lastname: Segui-Gracia
authors.affiliation: Cancer Molecular Diagnostics Laboratory, Department of Oncology, University of Cambridge, Cambridge.
authors.forename: M
authors.initials: M
authors.lastname: Valganon
authors.affiliation: Department of Pathology, Queen Elizabeth University Hospital, Glasgow.
authors.forename: A
authors.initials: A
authors.lastname: Hoyle
authors.affiliation: Institute of Cancer Sciences, University of Glasgow, Glasgow.
authors.forename: C
authors.initials: C
authors.lastname: Orange
authors.affiliation: 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.
authors.forename: L
authors.initials: L
authors.lastname: Moore
authors.affiliation: 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.
authors.forename: M
authors.initials: M
authors.lastname: Jimenez-Linan
authors.affiliation: Department of Pathology, Queen Elizabeth University Hospital, Glasgow.
authors.forename: D
authors.initials: D
authors.lastname: Millan
authors.affiliation: Institute of Cancer Sciences, University of Glasgow, Glasgow iain.mcneish@glasgow.ac.uk.
authors.forename: I A
authors.initials: IA
authors.lastname: McNeish
authors.affiliation: 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.
authors.forename: J D
authors.initials: JD
authors.lastname: Brenton
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000032:analysis / Q000187:drug effects*; D005557:Formaldehyde / Q000737:chemistry*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D000432:Methanol / Q000737:chemistry*; D008875:Middle Aged; D009369:Neoplasms / Q000175:diagnosis*; D016612:Paraffin Embedding; D017422:Sequence Analysis, DNA; D016707:Tissue Fixation / Q000379:methods*
chemical_list: D005557:Formaldehyde; D004247:DNA; D000432:Methanol
keywords: HGSOC; NBF; SNVs; UMFIX; copy-number abnormalities; fixation; next-generation sequencing