Validation and implementation of a modular targeted capture assay for the detection of clinically significant molecular oncology alterations.
Identifieur interne : 002876 ( Main/Corpus ); précédent : 002875; suivant : 002877Validation and implementation of a modular targeted capture assay for the detection of clinically significant molecular oncology alterations.
Auteurs : 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. LockwoodSource :
- Practical laboratory medicine [ 2352-5517 ] ; 2020.
Abstract
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rapid discovery of clinically significant genetic variants has translated to next-generation sequencing assays becoming out-of-date by the time they are designed, validated, and implemented.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.
DOI: 10.1016/j.plabm.2020.e00153
PubMed: 32123717
PubMed Central: PMC7038441
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<front><div type="abstract" xml:lang="en"><p><b>Objectives</b>
</p>
<p>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.</p>
</div>
<div type="abstract" xml:lang="en"><p><b>Design</b>
</p>
<p>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.</p>
</div>
<div type="abstract" xml:lang="en"><p><b>Results</b>
</p>
<p>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.</p>
</div>
<div type="abstract" xml:lang="en"><p><b>Conclusions</b>
</p>
<p>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.</p>
</div>
</front>
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<Abstract><AbstractText Label="Objectives" NlmCategory="UNASSIGNED">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.</AbstractText>
<AbstractText Label="Design" NlmCategory="UNASSIGNED">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.</AbstractText>
<AbstractText Label="Results" NlmCategory="UNASSIGNED">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.</AbstractText>
<AbstractText Label="Conclusions" NlmCategory="UNASSIGNED">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.</AbstractText>
<CopyrightInformation>© 2020 The Authors.</CopyrightInformation>
</Abstract>
<AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Kuo</LastName>
<ForeName>Ayako J</ForeName>
<Initials>AJ</Initials>
<AffiliationInfo><Affiliation>Department of Laboratory Medicine, University of Washington Medical Center, 1959 NE Pacific Street, Seattle, WA, 98195, USA.</Affiliation>
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<Author ValidYN="Y"><LastName>Paulson</LastName>
<ForeName>Vera A</ForeName>
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<AffiliationInfo><Affiliation>Department of Laboratory Medicine, University of Washington Medical Center, 1959 NE Pacific Street, Seattle, WA, 98195, USA.</Affiliation>
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<Author ValidYN="Y"><LastName>Hempelmann</LastName>
<ForeName>Jennifer A</ForeName>
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<Author ValidYN="Y"><LastName>Beightol</LastName>
<ForeName>Mallory</ForeName>
<Initials>M</Initials>
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</AffiliationInfo>
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<Author ValidYN="Y"><LastName>Todhunter</LastName>
<ForeName>Sheena</ForeName>
<Initials>S</Initials>
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<Author ValidYN="Y"><LastName>Colbert</LastName>
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<Initials>BG</Initials>
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<Author ValidYN="Y"><LastName>Konnick</LastName>
<ForeName>Eric Q</ForeName>
<Initials>EQ</Initials>
<AffiliationInfo><Affiliation>Department of Laboratory Medicine, University of Washington Medical Center, 1959 NE Pacific Street, Seattle, WA, 98195, USA.</Affiliation>
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<Author ValidYN="Y"><LastName>Pritchard</LastName>
<ForeName>Colin C</ForeName>
<Initials>CC</Initials>
<AffiliationInfo><Affiliation>Department of Laboratory Medicine, University of Washington Medical Center, 1959 NE Pacific Street, Seattle, WA, 98195, USA.</Affiliation>
</AffiliationInfo>
</Author>
<Author ValidYN="Y"><LastName>Lockwood</LastName>
<ForeName>Christina M</ForeName>
<Initials>CM</Initials>
<AffiliationInfo><Affiliation>Department of Laboratory Medicine, University of Washington Medical Center, 1959 NE Pacific Street, Seattle, WA, 98195, USA.</Affiliation>
</AffiliationInfo>
</Author>
</AuthorList>
<Language>eng</Language>
<PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType>
</PublicationTypeList>
<ArticleDate DateType="Electronic"><Year>2020</Year>
<Month>02</Month>
<Day>03</Day>
</ArticleDate>
</Article>
<MedlineJournalInfo><Country>Netherlands</Country>
<MedlineTA>Pract Lab Med</MedlineTA>
<NlmUniqueID>101690848</NlmUniqueID>
<ISSNLinking>2352-5517</ISSNLinking>
</MedlineJournalInfo>
<KeywordList Owner="NOTNLM"><Keyword MajorTopicYN="N">Assay validation</Keyword>
<Keyword MajorTopicYN="N">Molecular diagnostics</Keyword>
<Keyword MajorTopicYN="N">Molecular oncology</Keyword>
<Keyword MajorTopicYN="N">Next generation sequencing</Keyword>
<Keyword MajorTopicYN="N">OncoPlex</Keyword>
<Keyword MajorTopicYN="N">Precision medicine</Keyword>
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<CoiStatement>The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: CC Pritchard and SJ Salipante: consult for Promega EQ Konnick: Travel & honoraria – Ventana, Medscape, Roche, Genentech CM Lockwood: spouse is employed by Bayer.</CoiStatement>
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