The mutational signatures of formalin fixation on the human genome

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.