AmpliSeq Focus Panel

This test does not detect all possible variants in the genes tested. Only regions with recurrent (i.e. hotspot) somatic DNA and RNA alterations are covered by this assay. Furthermore, a negative result does not rule out the presence of an alteration that may be present but below the limits of detection of this assay. The minimum percentage of tumour cells required in the submitted sample is 10%; lower percentages may result in false negatives. All samples with less than 10% tumour nuclei will be reported as unsatisfactory. This test does not allow definitive differentiation between germline and somatic variants.

Test sensitivity can vary depending on DNA/RNA quality, tumour cell percentage, and tumour heterogeneity. Only clinically relevant variants (i.e. Tier I and II variants) are reported. For SNVs and indels, the threshold for variant calling is set at five or more reads, at a minimal sequencing depth of 250 reads, which are the recommended criteria to confidently call variants at an allele frequency of 5% (PMID: 28341590). For CNVs, only amplifications are reported, with a threshold of five copies for identifying a copy number amplification. For DNA sequences, at least 95% of targeted bases must be covered by 250 reads or more to qualify a sample as valid for analysis. For gene fusions and other aberrant splicing events, a minimum of 20,000 total mapped reads and 20,000 reads mapped to expression control genes are required to qualify a sample as valid. Only fusions supported by at least 20 reads are detected and reported.

This test is an amplicon-based assay. Therefore, variants located in primer binding sites can result in inaccurate diagnosis due to allelic dropout.

The presence or absence of an alteration may not be predictive of response to therapy in all patients.