ERASE-Seq: An Ultra-Sensitive Variant Caller Applied to the TruSight® Tumor 15 Panel

Here we introduce ERASE-Seq, a method that relies on improved bioinformatics and a better utilization of the sequencing depth to work well down to mutations present at allelic frequencies (AF) down to 0.1%, presenting an improvement in resolution with respect to commercialized barcoding kits that cite sensitivities down to 0.5% (HaloplexHS Data Sheet, Agilent) without measuring specificity below 1%. The low error rate is dependent on the utilization of a high fidelity amplicon panel for which the background error rate has been well characterized.

To that end we used the TruSight Tumor 15 (TST 15), a newly introduced 250-amplicon cancer mutation detection panel developed by Illumina and aimed at somatic mutation detection. It covers portions of 15 genes commonly associated with actionable mutations present in common cancer types. 

Analytical validation using DNA mixtures demonstrates variant calling down to 0.3% allele frequency (AF) with 100% sensitivity and 100% specificity at a depth of 20,000x per sample. Between 0.05% and 0.3% AF, 80% sensitivity is achieved at a specificity of 99.996%.