Genome Biology (Apr 2021)

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

  • Wendell Jones,
  • Binsheng Gong,
  • Natalia Novoradovskaya,
  • Dan Li,
  • Rebecca Kusko,
  • Todd A. Richmond,
  • Donald J. Johann,
  • Halil Bisgin,
  • Sayed Mohammad Ebrahim Sahraeian,
  • Pierre R. Bushel,
  • Mehdi Pirooznia,
  • Katherine Wilkins,
  • Marco Chierici,
  • Wenjun Bao,
  • Lee Scott Basehore,
  • Anne Bergstrom Lucas,
  • Daniel Burgess,
  • Daniel J. Butler,
  • Simon Cawley,
  • Chia-Jung Chang,
  • Guangchun Chen,
  • Tao Chen,
  • Yun-Ching Chen,
  • Daniel J. Craig,
  • Angela del Pozo,
  • Jonathan Foox,
  • Margherita Francescatto,
  • Yutao Fu,
  • Cesare Furlanello,
  • Kristina Giorda,
  • Kira P. Grist,
  • Meijian Guan,
  • Yingyi Hao,
  • Scott Happe,
  • Gunjan Hariani,
  • Nathan Haseley,
  • Jeff Jasper,
  • Giuseppe Jurman,
  • David Philip Kreil,
  • Paweł Łabaj,
  • Kevin Lai,
  • Jianying Li,
  • Quan-Zhen Li,
  • Yulong Li,
  • Zhiguang Li,
  • Zhichao Liu,
  • Mario Solís López,
  • Kelci Miclaus,
  • Raymond Miller,
  • Vinay K. Mittal,
  • Marghoob Mohiyuddin,
  • Carlos Pabón-Peña,
  • Barbara L. Parsons,
  • Fujun Qiu,
  • Andreas Scherer,
  • Tieliu Shi,
  • Suzy Stiegelmeyer,
  • Chen Suo,
  • Nikola Tom,
  • Dong Wang,
  • Zhining Wen,
  • Leihong Wu,
  • Wenzhong Xiao,
  • Chang Xu,
  • Ying Yu,
  • Jiyang Zhang,
  • Yifan Zhang,
  • Zhihong Zhang,
  • Yuanting Zheng,
  • Christopher E. Mason,
  • James C. Willey,
  • Weida Tong,
  • Leming Shi,
  • Joshua Xu

DOI
https://doi.org/10.1186/s13059-021-02316-z
Journal volume & issue
Vol. 22, no. 1
pp. 1 – 38

Abstract

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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.