It is essential to validate all candidate somatic mutations. False-positive calls can arise from DNA sequencing errors, contamination with germline variants, chimeric molecules generated during single-cell WGA, PCR-induced nucleotide substitutions, and the failure to amplify certain genomic regions. False-negative calls are dependent on the allele frequency of the somatic mutation within the sample, the type of mutation, and the method of detection. Orthologous experimental methods are required to eliminate false-positives and to calibrate the confidence of detection for different types of somatic mutations. Validation experiments can then be performed on either the tissue of origin or amplified material used to discover the variant. The first approach represents a biological validation, which establishes the presence of a variant call in unamplified DNA from the source sample. The second approach represents a technical validation, which establishes the presence/absence of variant calls in the DNA source material used for discovery.
