Standard operating procedure for somatic variant refinement of sequencing data with paired tumor and normal samples

Following automated variant calling, manual review of aligned read sequences is required to identify a high-quality list of somatic variants. Despite widespread use in analyzing sequence data, methods to standardizemanual reviewhave not been described, resulting in high inter- and intralab variability. This manual review standard operating procedure(SOP) consists of methods to annotate variants with four different calls and 19 tags. The calls indicate a reviewer’s confidence in each variant and the tags indicate commonly observed sequencing patterns and artifacts that inform the manual review call. Four individuals were asked to classify variants prior to, and after, reading the SOP and accuracy was assessed by comparing reviewer calls with orthogonal validation sequencing. After reading the SOP, average accuracy in somatic variant identification increased by 16.7% (p value = 0.0298) and average interreviewer agreement increased by 12.7% (p value < 0.001). Manual review conducted after reading the SOP did not significantly increase reviewer time. This SOP supports and enhances manual somatic variant detection by improving reviewer accuracy while reducing the interreviewer variability for variant calling and annotation.