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Updated: Dec 27, 2025
Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing
Published on: October 18, 2013
Validation and implementation of a modular targeted capture assay for the detection of clinically significant
Ayako J Kuo1, Vera A Paulson1, Jennifer A Hempelmann1
1Department of Laboratory Medicine, University of Washington Medical Center, 1959 NE Pacific Street, Seattle, WA, 98195, USA.
Objectives:
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rapid discovery of clinically significant genetic variants has translated to next-generation sequencing assays becoming out-of-date by the time they are designed, validated, and implemented.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.
Design:
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.
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.
Conclusions:
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.
Insights
UW-OncoPlex version 6 (OPXv6) offers a modular design for rapid updates in cancer gene panels. This next-generation sequencing assay improves accuracy and efficiency for detecting genetic variants, including SNVs, indels, CNVs, SVs, MSI, and TMB.
Area of Science:
- Molecular oncology
- Genomic variant detection
- Next-generation sequencing (NGS) assays
Background:
- Rapid genetic variant discovery necessitates adaptable NGS assays.
- Existing assays risk obsolescence due to the pace of genomic research.
- UW-OncoPlex utilizes a modular panel for continuous redesign.
Purpose of the Study:
- Validate OncoPlex version 6 (OPXv6) for comprehensive genetic variant detection.
- Assess OPXv6's performance across various variant types and sample matrices.
- Evaluate improvements in assay efficiency and quality compared to previous versions.
Main Methods:
- Validated OPXv6 on 112 diverse samples (tissue, plasma, blood, etc.).
- Employed genomic and cell-free DNA libraries with custom xGen Lockdown probes.
- Sequenced on Illumina platforms and processed via a custom bioinformatics pipeline.
Main Results:
- Achieved high accuracy: 99% for SNVs, 98% for indels, 97% for SVs, 99% for CNVs, 100% for MSI and TMB.
- Reduced library preparation time by 40%.
- Enhanced sequencing quality with 2.5x coverage and 4x on-target rate.
Conclusions:
- OPXv6 surpasses previous versions in reduced cost, improved sequencing quality, and faster turnaround.
- The modular probe design enables agile laboratory adaptation to evolving molecular oncology needs.
- OPXv6 provides a robust and efficient solution for clinical genomic variant detection.
