Validation and implementation of a modular targeted capture assay for the detection of clinically significant molecular oncology alterations

The rapid discovery of clinically significant genetic variants has translated to next-generation sequencing assays becoming out-of-date by the time they are designed, validated, and implemented. UW-OncoPlex addresses this through the adoption of a modular panel capable of redesign as significant alt...

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Bibliographic Details
Published in:Practical laboratory medicine Vol. 19; p. e00153
Main Authors: Kuo, Ayako J., Paulson, Vera A., Hempelmann, Jennifer A., Beightol, Mallory, Todhunter, Sheena, Colbert, Brice G., Salipante, Stephen J., Konnick, Eric Q., Pritchard, Colin C., Lockwood, Christina M.
Format: Journal Article
Language:English
Published: Netherlands Elsevier B.V 01.03.2020
Elsevier
Subjects:
Assay validation
Molecular diagnostics
Molecular oncology
Next generation sequencing
OncoPlex
Precision medicine
Molecular diagnostics
Precision medicine
Molecular oncology
Assay validation
Next generation sequencing
OncoPlex
ISSN:2352-5517, 2352-5517
Online Access:Get full text
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Summary:The rapid discovery of clinically significant genetic variants has translated to next-generation sequencing assays becoming out-of-date by the time they are designed, validated, and implemented. UW-OncoPlex addresses this through the adoption of a modular panel capable of redesign as significant alterations are identified. We describe the validation of OncoPlex version 6 (OPXv6) for the detection of single nucleotide variants (SNVs), insertions and deletions (indels), copy number variants (CNVs), structural variants (SVs), microsatellite instability (MSI), and tumor mutational burden (TMB) in a panel of 340 genes. One hundred twelve samples with diverse diagnoses were comprised of formalin-fixed-paraffin-embedded tissue, fresh-frozen tissue, plasma, peripheral blood, bone marrow, saliva, and cell-line DNA. Libraries were prepared from genomic and cell-free DNA, hybridized to a custom panel of xGen Lockdown probes, and sequenced on Illumina platforms. Sequences were processed through a custom bioinformatics pipeline, and variant calls were compared to prior orthogonal clinical results. Accuracy was 99% for SNVs ≥5% allele frequency, 98% for indels, 97% for SVs, 99% for CNVs, 100% for MSI, and 100% for TMB (compared to previous OncoPlex versions). Library preparation turnaround time decreased by 40%, and sequencing quality improved with a 2.5-fold increase in average sequencing coverage and 4-fold increase in percent on-target. OPXv6 demonstrates improvements over prior UW-OncoPlex versions including reduced capture cost, improved sequencing quality, and decreased time to results. The modular capture probe design also provides a nimble laboratory response in addressing the expansions necessary to meet the needs of the continuously evolving field of molecular oncology. •OncoPlex version 6 is a targeted, comprehensive next generation sequencing panel.•The panel of 340 genes includes full coverage of BRCA1/2 and mismatch repair genes.•While sequencing quality increased, library preparation time and cost decreased.•The panel is modular and capable of rapid redesign and expansion of targeted genes.
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Authors contributed equally.
ISSN:2352-5517
2352-5517
DOI:10.1016/j.plabm.2020.e00153