Fig. 2

Reidentifying TP53 variants with double batched sequencing (DoBSeq). Upper plot: Illustrates all non-synonymous coding variants in the TP53 gene called without any filtering, i.e., includes all low-coverage and low-confidence calls, showing DoBSeq variants found in batches from the explorative cohort (EC) in red and whole genome sequencing (WGS) variants found in the EC in teal. The x-axis shows the canonical TP53 protein product, with numbers indicating codon number and dotted lines indicating exonic borders. The y-axis shows variant allele frequency (VAF) for DoBSeq on the left and WGS on the right. The dotted line indicates the theoretical VAF for true heterozygous variants. On the x-axis, the variants found on WGS and reidentified with DoBSeq are indicated by lollipop markers with colors corresponding to ClinVar classifications of likely pathogenic (orange), variant of unknown significance (yellow), likely benign (blue), and benign (green). A marker (¨) was added to the protein change when the variant did not have a ClinVar classification; here color indicates in-house classification. A common polymorphism (p.Pro72Arg) found in 86 alleles was filtered out for the sake of clarity. Interpretation: A steady level of sequencing noise runs along the low end of the y-axis with individual sets of batches rising up towards or slightly above the expected VAF. This signal allows for pinpointing to individuals. Lower plot: As above; for the validation cohort. A common polymorphism (p.Pro72Arg) found in 92 alleles was filtered out for the sake of clarity