Abstract
BackgroundOncopanel genomic testing, which identifies important somatic variants, is increasingly common in medical practice and especially in clinical trials. Currently, there is a paucity of reliable genomic reference samples having a suitably large number of pre-identified variants for properly assessing oncopanel assay analytical quality and performance. The FDA-led Sequencing and Quality Control Phase 2 (SEQC2) consortium analyze ten diverse cancer cell lines individually and their pool, termed Sample A, to develop a reference sample with suitably large numbers of coding positions with known (variant) positives and negatives for properly evaluating oncopanel analytical performance.ResultsIn reference Sample A, we identify more than 40,000 variants down to 1% allele frequency with more than 25,000 variants having less than 20% allele frequency with 1653 variants in COSMIC-related genes. This is 5–100× more than existing commercially available samples. We also identify an unprecedented number of negative positions in coding regions, allowing statistical rigor in assessing limit-of-detection, sensitivity, and precision. Over 300 loci are randomly selected and independently verified via droplet digital PCR with 100% concordance. Agilent normal reference Sample B can be admixed with Sample A to create new samples with a similar number of known variants at much lower allele frequency than what exists in Sample A natively, including known variants having allele frequency of 0.02%, a range suitable for assessing liquid biopsy panels.ConclusionThese new reference samples and their admixtures provide superior capability for performing oncopanel quality control, analytical accuracy, and validation for small to large oncopanels and liquid biopsy assays.
Highlights
Recent Sequencing and Quality Control Phase 2 (SEQC2) [1] consortium efforts have engaged in determining samples and methods for DNA-based next-generation sequencing (NGS) testing for a variety of translational and precision medicine applications
In reference Sample A, we identify more than 40,000 variants down to 1% allele frequency with more than 25,000 variants having less than 20% allele frequency with 1653 variants in COSMIC-related genes
Agilent normal reference Sample B can be admixed with Sample A to create new samples with a similar number of known variants at much lower allele frequency than what exists in Sample A natively, including known variants having allele frequency of 0.02%, a range suitable for assessing liquid biopsy panels
Summary
Recent Sequencing and Quality Control Phase 2 (SEQC2) [1] consortium efforts have engaged in determining samples and methods for DNA-based NGS testing for a variety of translational and precision medicine applications. The National Institute for Standards and Technology (NIST) has developed several cell lines of reference material for testing population genetics, which translates into a very high percentage of variants inherently at 50% and 100% allele frequency in germline cells [2] These samples are not appropriate in their current form for comprehensively evaluating the analytical performance of cancer panels as somatic mutations often at lower than 20% variant allele frequency (VAF). Other samples including reference standards developed for somatic mutation typically involve at most one cancer cell line and a matching normal, greatly limiting the number of relevant variants at low VAF available for evaluation [3,4,5,6]. The FDA-led Sequencing and Quality Control Phase 2 (SEQC2) consortium analyze ten diverse cancer cell lines individually and their pool, termed Sample A, to develop a reference sample with suitably large numbers of coding positions with known (variant) positives and negatives for properly evaluating oncopanel analytical performance.
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