Validation and implementation of a modular targeted capture assay for the detection of clinically significant molecular oncology alterations
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rapid discovery of clinically significant genetic variants has translated to next-generation sequencing assays becoming out-of-date by the time they are designed, validated, and implemented.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 sig...
| Main Authors: | , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2020-03-01
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| Series: | Practical Laboratory Medicine |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2352551719301003 |
| _version_ | 1818611149680148480 |
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| author | Ayako J. Kuo Vera A. Paulson Jennifer A. Hempelmann Mallory Beightol Sheena Todhunter Brice G. Colbert Stephen J. Salipante Eric Q. Konnick Colin C. Pritchard Christina M. Lockwood |
| author_facet | Ayako J. Kuo Vera A. Paulson Jennifer A. Hempelmann Mallory Beightol Sheena Todhunter Brice G. Colbert Stephen J. Salipante Eric Q. Konnick Colin C. Pritchard Christina M. Lockwood |
| author_sort | Ayako J. Kuo |
| collection | DOAJ |
| description | 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rapid discovery of clinically significant genetic variants has translated to next-generation sequencing assays becoming out-of-date by the time they are designed, validated, and implemented.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. Keywords: Next generation sequencing, Precision medicine, Molecular diagnostics, Assay validation, Molecular oncology, OncoPlex |
| first_indexed | 2024-12-16T15:25:44Z |
| format | Article |
| id | doaj.art-c9d2733df5de49c1ab6b4390e50bce81 |
| institution | Directory Open Access Journal |
| issn | 2352-5517 |
| language | English |
| last_indexed | 2024-12-16T15:25:44Z |
| publishDate | 2020-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Practical Laboratory Medicine |
| spelling | doaj.art-c9d2733df5de49c1ab6b4390e50bce812022-12-21T22:26:31ZengElsevierPractical Laboratory Medicine2352-55172020-03-0119Validation and implementation of a modular targeted capture assay for the detection of clinically significant molecular oncology alterationsAyako J. Kuo0Vera A. Paulson1Jennifer A. Hempelmann2Mallory Beightol3Sheena Todhunter4Brice G. Colbert5Stephen J. Salipante6Eric Q. Konnick7Colin C. Pritchard8Christina M. Lockwood9Department of Laboratory Medicine, University of Washington Medical Center, 1959 NE Pacific Street, Seattle, WA, 98195, USACorresponding author.; Department of Laboratory Medicine, University of Washington Medical Center, 1959 NE Pacific Street, Seattle, WA, 98195, USADepartment of Laboratory Medicine, University of Washington Medical Center, 1959 NE Pacific Street, Seattle, WA, 98195, USADepartment of Laboratory Medicine, University of Washington Medical Center, 1959 NE Pacific Street, Seattle, WA, 98195, USADepartment of Laboratory Medicine, University of Washington Medical Center, 1959 NE Pacific Street, Seattle, WA, 98195, USADepartment of Laboratory Medicine, University of Washington Medical Center, 1959 NE Pacific Street, Seattle, WA, 98195, USADepartment of Laboratory Medicine, University of Washington Medical Center, 1959 NE Pacific Street, Seattle, WA, 98195, USADepartment of Laboratory Medicine, University of Washington Medical Center, 1959 NE Pacific Street, Seattle, WA, 98195, USADepartment of Laboratory Medicine, University of Washington Medical Center, 1959 NE Pacific Street, Seattle, WA, 98195, USADepartment of Laboratory Medicine, University of Washington Medical Center, 1959 NE Pacific Street, Seattle, WA, 98195, USAObjectives: 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. Keywords: Next generation sequencing, Precision medicine, Molecular diagnostics, Assay validation, Molecular oncology, OncoPlexhttp://www.sciencedirect.com/science/article/pii/S2352551719301003 |
| spellingShingle | Ayako J. Kuo Vera A. Paulson Jennifer A. Hempelmann Mallory Beightol Sheena Todhunter Brice G. Colbert Stephen J. Salipante Eric Q. Konnick Colin C. Pritchard Christina M. Lockwood Validation and implementation of a modular targeted capture assay for the detection of clinically significant molecular oncology alterations Practical Laboratory Medicine |
| title | Validation and implementation of a modular targeted capture assay for the detection of clinically significant molecular oncology alterations |
| title_full | Validation and implementation of a modular targeted capture assay for the detection of clinically significant molecular oncology alterations |
| title_fullStr | Validation and implementation of a modular targeted capture assay for the detection of clinically significant molecular oncology alterations |
| title_full_unstemmed | Validation and implementation of a modular targeted capture assay for the detection of clinically significant molecular oncology alterations |
| title_short | Validation and implementation of a modular targeted capture assay for the detection of clinically significant molecular oncology alterations |
| title_sort | validation and implementation of a modular targeted capture assay for the detection of clinically significant molecular oncology alterations |
| url | http://www.sciencedirect.com/science/article/pii/S2352551719301003 |
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