Standard operating procedure for somatic variant refinement of tumor sequencing data

Purpose Manual review of aligned sequencing reads is required to develop a high-quality list of somatic variants from massively parallel sequencing data (MPS). Despite widespread use in analyzing MPS data, there has been little attempt to describe methods for manual review, resulting in high inter- and intra-lab variability in somatic variant detection and characterization of tumors. Methods Open source software was used to develop an optimal method for manual review setup. We also developed a systemic approach to visually inspect each variant during manual review. Results We present a standard operating procedures for somatic variant refinement for use by manual reviewers. The approach is enhanced through representative examples of 4 different manual review categories that indicate a reviewer’s confidence in the somatic variant call and 19 annotation tags that contextualize commonly observed sequencing patterns during manual review. Representative examples provide detailed instructions on how to classify variants during manual review to rectify lack of confidence in automated somatic variant detection. Conclusion Standardization of somatic variant refinement through systematization of manual review will improve the consistency and reproducibility of identifying true somatic variants after automated variant calling.