ORCID Profile
0000-0002-9676-5387
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Publisher: Springer Science and Business Media LLC
Date: 20-12-2117
Publisher: Elsevier BV
Date: 2021
DOI: 10.2139/SSRN.3830017
Publisher: Cold Spring Harbor Laboratory
Date: 06-04-2021
DOI: 10.1101/2021.04.06.438497
Abstract: The primary objective of the FDA-led Sequencing and Quality Control Phase 2 (SEQC2) project is to develop standard analysis protocols and quality control metrics for use in DNA testing to enhance scientific research and precision medicine. This study reports a targeted next generation sequencing (NGS) method that enables more accurate detection of actionable mutations in circulating tumor DNA (ctDNA) clinical specimens. This advancement was enabled by designing a synthetic internal standard spike-in for each actionable mutation target, suitable for use in NGS following hybrid-capture enrichment and unique molecular index (UMI) or non-UMI library preparation. When mixed with contrived ctDNA reference s les, internal standards enabled calculation of technical error rate, limit of blank, and limit of detection for each variant at each nucleotide position, in each s le. True positive mutations with variant allele fraction too low for detection by current practice were detected with this method, thereby increasing sensitivity.
Publisher: Springer Science and Business Media LLC
Date: 24-08-2014
DOI: 10.1038/NBT.2957
Publisher: Springer Science and Business Media LLC
Date: 16-04-2021
DOI: 10.1186/S13059-021-02315-0
Abstract: Targeted sequencing using oncopanels requires comprehensive assessments of accuracy and detection sensitivity to ensure analytical validity. By employing reference materials characterized by the U.S. Food and Drug Administration-led SEquence Quality Control project phase2 (SEQC2) effort, we perform a cross-platform multi-lab evaluation of eight Pan-Cancer panels to assess best practices for oncopanel sequencing. All panels demonstrate high sensitivity across targeted high-confidence coding regions and variant types for the variants previously verified to have variant allele frequency (VAF) in the 5–20% range. Sensitivity is reduced by utilizing VAF thresholds due to inherent variability in VAF measurements. Enforcing a VAF threshold for reporting has a positive impact on reducing false positive calls. Importantly, the false positive rate is found to be significantly higher outside the high-confidence coding regions, resulting in lower reproducibility. Thus, region restriction and VAF thresholds lead to low relative technical variability in estimating promising biomarkers and tumor mutational burden. This comprehensive study provides actionable guidelines for oncopanel sequencing and clear evidence that supports a simplified approach to assess the analytical performance of oncopanels. It will facilitate the rapid implementation, validation, and quality control of oncopanels in clinical use.
Publisher: Springer Science and Business Media LLC
Date: 11-2021
Publisher: Springer Science and Business Media LLC
Date: 09-2021
Publisher: Elsevier BV
Date: 11-2021
Publisher: Cold Spring Harbor Laboratory
Date: 03-05-2019
DOI: 10.1101/626440
Abstract: Clinical applications of precision oncology require accurate tests that can distinguish tumor-specific mutations from errors introduced at each step of next generation sequencing (NGS). For NGS to successfully improve patient lives, discriminating between true mutations and artifacts is crucial. We systematically interrogated somatic mutations in paired tumor-normal cell lines to identify factors affecting detection reproducibility and accuracy. Different types of s les with varying input amount and tumor purity were processed using multiple library construction protocols. Whole-genome and whole-exome sequencing were carried out at six sequencing centers followed by processing with nine bioinformatics pipelines to evaluate their reproducibility. We identified artifacts due to s le and library processing and evaluated the capabilities and limitations of bioinformatics tools for artifact detection and removal. By examining the interaction and effect of various wet lab and computational parameters concomitantly, here we recommend actionable best practices for mutation detection in clinical applications using NGS technologies.
Publisher: Springer Science and Business Media LLC
Date: 13-04-2022
DOI: 10.1038/S41597-022-01276-8
Abstract: Recently we reported the accuracy and reproducibility of circulating tumor DNA (ctDNA) assays using a unique set of reference materials, associated analytical framework, and suggested best practices. With the rapid adoption of ctDNA sequencing in precision oncology, it is critical to understand the analytical validity and technical limitations of this cutting-edge and medical-practice-changing technology. The SEQC2 Oncopanel Sequencing Working Group has developed a multi-site, cross-platform study design for evaluating the analytical performance of five industry-leading ctDNA assays. The study used tailor-made reference s les at various levels of input material to assess ctDNA sequencing across 12 participating clinical and research facilities. The generated dataset encompasses multiple key variables, including a broad range of mutation frequencies, sequencing coverage depth, DNA input quantity, etc. It is the most comprehensive public-facing dataset of its kind and provides valuable insights into ultra-deep ctDNA sequencing technology. Eventually the clinical utility of ctDNA assays is required and our proficiency study and corresponding dataset are needed steps towards this goal.
Publisher: Springer Science and Business Media LLC
Date: 16-04-2021
DOI: 10.1186/S13059-021-02316-Z
Abstract: 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 s les 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 erse cancer cell lines in idually and their pool, termed S le A, to develop a reference s le with suitably large numbers of coding positions with known (variant) positives and negatives for properly evaluating oncopanel analytical performance. In reference S le 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 s les. 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 S le B can be admixed with S le A to create new s les with a similar number of known variants at much lower allele frequency than what exists in S le A natively, including known variants having allele frequency of 0.02%, a range suitable for assessing liquid biopsy panels. These new reference s les and their admixtures provide superior capability for performing oncopanel quality control, analytical accuracy, and validation for small to large oncopanels and liquid biopsy assays.
No related grants have been discovered for Wendell Jones.