Experimental validation of subclonal TP53 mutations identified by ultra-deep-NGS. (A) Representation of the variant frequency of 2 exemplificative subclonal TP53 mutations (c.743G>A p.R248Q and c.673-2A>T) of very low allelic abundance (<0.5%). The first bar of the graphs shows the variant allele frequency in the discovery ultra-deep-NGS experiment. The second and third bars show the variant allele frequency in independent ultra-deep-NGS validation experiments. The number of mutated read outs of the total number of reads covering the variant position is reported. (B) Conventional agarose-gel electrophoresis of the AS-PCR products. Patient 10642, harboring the subclonal TP53 c.743G>A p.R248Q missense substitution (left), and patient 7561, harboring the subclonal TP53 c.673-2A>T splice site mutation (right), are represented. After AS-PCR for the mutant allele, a mutation-specific band is amplified from the patient sample and from the mutated plasmid DNA (positive control). No bands are amplified from the wild-type plasmid DNA and the wild-type genomic DNA from a healthy donor (negative controls), thus confirming the specificity of the assay. (C) Due to their low clonal abundance (<0.5%), the subclonal TP53 c.743G>A p.R248Q missense substitution (left) and the subclonal TP53 c.673-2A>T splice site mutation (right) are not detectable by conventional Sanger sequencing in patient 10 642 and patient 7561, respectively. Asterisks point to the positions of the subclonal variants.