If a somatic variant is only present in a single cell, it will be impossible to validate in bulk tissue. Likewise, a variant present in very few cells may be difficult to validate in the tissue of origin. Thus, technical validation in the source DNA used to discover a putative variant can be used to determine whether a call is true or false. Technical validation typically employs PCR, qPCR, and Sanger sequencing of the locus in the DNA source material (e.g., WGA DNA or DNA from a clonal cell population). Multiple true/false verdicts form the basis for estimating false-discovery and false-negative rates in the resultant call sets.
