Rigorous software pipeline for clinical somatic mutation analyses of solid tumors

Mutational analyses of tumor DNA guide the use of targeted therapies and checkpoint inhibitors in management of solid tumors. Reducing false positive mutation calls without compromising sensitivity as gene panels increase in size, and whole exome and genome sequencing enters clinical use, remains a major challenge. Aiming for robust somatic mutation analyses in the clinical setting, we have developed VARify, an integrated, accurate and computationally efficient software for cancer genome analyses encompassing all steps from pre-processing of sequencing reads to mutation identification. Benchmarking to two state-of-the-art open-source somatic mutation analysis pipelines demonstrated accurate detection of clinically actionable point mutations, all while strongly reducing the number of false positive mutations reported, at comparable or faster speed. Further, the VARify output classified microsatellite unstable colorectal cancers by tumor mutation burden better than the other pipelines. In comparisons where the same tumors were subjected to different panel enrichment and sequencing technologies, VARify had the most consistent intersection of consensus mutations. False positive calls were produced when the same data was used as tumor and reference by the other pipelines, while VARify did not produce such calls. The calling uniformity across sequencing technologies of VARify and its tumor-only analysis derivative pipeline ALTOmate was also demonstrated. Taken together, these two novel pipelines can improve clinical mutation analysis to the benefit of cancer patients. ### Competing Interest Statement The use of PBR in this and other applications is governed by patents WO2016043659A1 and US10325183B2. IS, TA, IC, VL and TS are co-founders of Oncodia AB which holds all rights to VARify and ALTOmate. MR, TA, LN are employees of Oncodia AB. ### Funding Statement This work was funded by grants from the Swedish Agency for Innovation Systems (Vinnova) and EU FP7 (MERIT). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The mutational analyses of patient samples were approved by the Ethical Review Board in Uppsala (EPN Uppsala, C116/2007). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors