The identification of small sequence variants remains a challenging but critical step in the analysis of next?generation sequencing data. Our variant?calling tool, VarScan 2, employs heuristic and statistic thresholds based on user?defined criteria to call variants using SAMtools mpileup data as input. Here, we provide guidelines for generating that input, and describe protocols for using VarScan 2 to (1) identify germline variants in individual samples; (2) call somatic mutations, copy?number alterations, and LOH events in tumor?normal pairs; and (3) identify germline variants, de novo mutations, and Mendelian inheritance errors in family trios. Further, we describe a strategy for variant filtering that removes likely false positives associated with common sequencing? and alignment?related artifacts.