A verified genomic reference sample for assessing performance of cancer panels detecting small variants of low allele frequency

Abstract

Background Oncopanel 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. Results 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. 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. Conclusion These 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.

Ultra-deep sequencing data from a liquid biopsy proficiency study demonstrating analytic validity

“…A set of contrived reference samples were created using the established Sample A, which was genotyped to have ~40,000 “known variants” and ~10.2 Mb of “known negatives” in defined coding regions (a.k.a., consensus target region (CTR)), and Sample B, which is a non-cancer background cell line 5 . Sample A and Sample B were mixed at different ratios and enzymatically fragmented to create Sample Df (a.k.a., LBx-high), Ef (a.k.a., LBx-low), and Ff, which harbor “known variants” at different variant allele frequency (VAF) levels.…”

The Sequencing Quality Control 2 study: establishing community standards for sequencing in precision medicine

“…To evaluate the detection of somatic mutations, the SEQC2 conduct two comprehensive analyses, one was focused on WGS which is emerging as a comprehensive technology in cancer genomics and the other was on oncopanel sequencing which is the default application practiced in many clinical settings. The former was relied on a paired tumor and normal tissues as a reference sample while the latter assembled mock tumor and normal reference samples by mixing cell-line DNA samples at different ratios to emulate different mutation frequencies [ 9 ].…”

FDA-led consortium studies advance quality control of targeted next generation sequencing assays for precision oncology

“…All other available reference samples either had variants with too high of an allele frequency [population genetics reference samples ( 20 )], too few variants total [based on a single cell line ( 21 – 23 )], or a limited source (from a tumor). To overcome these three limitations, the SEQC2 working group pooled genomic DNA extracted from 10 Universal Human Reference RNA (UHRR, Agilent Technologies, Inc., Santa Clara, California, USA) cell lines ( Figure 2 ) to make a reference sample ( 15 ). This mixture of cancer samples increased the mutation density per gene, which is ideal for being able to assess oncopanel performance across conditions.…”

“…The broad and deep collection of variants spanning key driver genes at various frequencies enabled assessments of tumor relevant oncopanels in the context of rare subclonal mutations. The group has made the set of known variants and negative positions openly available ( 15 ) to the community, enabling others to interrogate additional approaches to targeted oncopanel somatic mutation detection. Lastly, the study serves as a guide to others who may want to establish a reference sample to answer related but different questions.…”

“…In brief, we have developed a translational scientific infrastructure and applied it to common precision oncology situations. This includes the design and construction of a comprehensive and robust set of nucleic acid (DNA, RNA) reference standards ( 15 ) that were used in related SEQC2 studies ( Figure 1 ), which will become publicly available. These related studies include: (I) comprehensive solid tumor onco-panel testing ( 16 ); (II) liquid biopsy testing ( 17 ); (III) testing involving formalin fixed paraffin embedded (FFPE) materials ( 18 ) and; (IV) testing involving spike-in materials ( 19 ).…”