The mutational signatures of formalin fixation on the human genome

Abstract

Background: Formalin fixation and paraffin embedding (FFPE) of patient material remains standard practice in clinical pathology labs around the world. Clinical archives of patient material near-exclusively consist of FFPE blocks. The ability to perform high quality genome sequencing on FFPE-derived DNA would accelerate a broad spectrum of medical research. However, formalin is a recognised mutagen and sequencing of DNA derived from FFPE material is known to be riddled with artefactual mutations. Results: Here we derive genome-wide mutational signatures caused by formalin fixation, and provide a computational method to correct mutational profiles for these formalin-induced artefacts. We show that the FFPE-signature is dominated by C>T transitions caused by cytosine deamination, and has very high similarity to COSMIC signature SBS30 (base excision repair deficiency due to inactivation mutations in NTHL1). Further, we demonstrate that chemical repair of formalin-induced DNA lesions, a process that is routinely performed as part of sequencing library preparation, leads to a signature highly similar to COSMIC signature SBS1 (spontaneous deamination of methylated cytosine). Next, we design FFPEsig, a computational method to remove the formalin-induced artefacts from mutational counts. We prove the efficacy of this method by generating synthetic FFPE samples using 2,780 cancer genomes from the Pan-Cancer Analysis of Whole Genome (PCAWG) project, and via analysis of FFPE-derived genome sequencing data from colorectal cancers. Conclusions: Formalin fixation leaves a predictable mutational footprint across the genome. The application of our FFPEsig software corrects the mutational profile for the influence of formalin, enabling robust mutational signature analysis in FFPE-derived patient material.

Comprehensive Molecular Analysis of Inflammatory Myofibroblastic Tumors Reveals Diverse Genomic Landscape and Potential Predictive Markers for Response to Crizotinib

“…The mutational signature of IMFT was characterized by predominant C>T transitions, as seen in most solid tumors (40). Additionally, cytosine deamination in formalin-fixed, paraffinembedded (FFPE) material is known to induce an artefact of C>T transitions with a signature highly similar to SBS30 (41). However, the clock-like signatures of SBS5 and SBS1 observed in our IMFT cohort suggest the etiology linked with ageing and allow us to disregard the artefactual contribution from FFPE material in this study.…”

Single-strand mismatch and damage patterns revealed by single-molecule DNA sequencing

“…4e ). The COSMIC dsDNA signature SBS30 has been previously associated with NTHL1 and UNG biallelic loss of function mutations 31,59,60 and with formalin fixation 68 . NTHL1 and UNG encode DNA glycosylases that initiate base excision repair of oxidized pyrimidines, including uracil-species that result from cytosine oxidation 61,62 .…”

Somatic Mutation Profiling in Head and Neck Paragangliomas

“…The SBS5 and SBS1 signatures were highly enriched by mutations, and, notably, are both associated with aging. However, the SBS1-like signature may be caused by repair of FFPE artifacts in the process of library preparation for sequencing ( 66 ). The SBS29 signature was enriched by a quarter of all mutations and was characterized with the highest mutation burden.…”