Flexible, Scalable, and Efficient Targeted Resequencing on a Benchtop Sequencer for Variant Detection in Clinical Practice
ABSTRACT The release of benchtop next‐generation sequencing (NGS) instruments has paved the way to implement the technology in clinical setting. The need for flexible, qualitative, and cost‐efficient workflows is high. We used singleplex‐PCR for highly efficient target enrichment, allowing us to rea...
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| Veröffentlicht in: | Human mutation 2015-03, Vol.36 (3), p.379-387 |
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| creator | De Leeneer, Kim Hellemans, Jan Steyaert, Wouter Lefever, Steve Vereecke, Inge Debals, Eveline Crombez, Brecht Baetens, Machteld Van Heetvelde, Mattias Coppieters, Frauke Vandesompele, Jo De Jaegher, Annelies De Baere, Elfride Coucke, Paul Claes, Kathleen |
| description | ABSTRACT
The release of benchtop next‐generation sequencing (NGS) instruments has paved the way to implement the technology in clinical setting. The need for flexible, qualitative, and cost‐efficient workflows is high. We used singleplex‐PCR for highly efficient target enrichment, allowing us to reach the quality standards set in Sanger sequencing‐based diagnostics. For the library preparation, a modified NexteraXT protocol was used, followed by sequencing on a MiSeq instrument. With an innovative pooling strategy, high flexibility, scalability, and cost‐efficiency were obtained, independent of the availability of commercial kits. The approach was validated for ∼250 genes associated with monogenic disorders. An overall sensitivity (>99%) similar to Sanger sequencing was observed in combination with a positive predictive value of >98%. The distribution of coverage was highly uniform, guaranteeing a minimal number of gaps to be filled with alternative methods. ISO15189‐accreditation was obtained for the workflow. A major asset of the singleplex PCR‐based enrichment is that new targets can be easily implemented. Diagnostic laboratories have validated assays available ensuring that the proposed workflow can easily be adopted. Although our platform was optimized for constitutional variant detection of monogenic disease genes, it is now also used as a model for somatic mutation detection in acquired diseases. |
| doi_str_mv | 10.1002/humu.22739 |
| format | Article |
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The release of benchtop next‐generation sequencing (NGS) instruments has paved the way to implement the technology in clinical setting. The need for flexible, qualitative, and cost‐efficient workflows is high. We used singleplex‐PCR for highly efficient target enrichment, allowing us to reach the quality standards set in Sanger sequencing‐based diagnostics. For the library preparation, a modified NexteraXT protocol was used, followed by sequencing on a MiSeq instrument. With an innovative pooling strategy, high flexibility, scalability, and cost‐efficiency were obtained, independent of the availability of commercial kits. The approach was validated for ∼250 genes associated with monogenic disorders. An overall sensitivity (>99%) similar to Sanger sequencing was observed in combination with a positive predictive value of >98%. The distribution of coverage was highly uniform, guaranteeing a minimal number of gaps to be filled with alternative methods. ISO15189‐accreditation was obtained for the workflow. A major asset of the singleplex PCR‐based enrichment is that new targets can be easily implemented. Diagnostic laboratories have validated assays available ensuring that the proposed workflow can easily be adopted. Although our platform was optimized for constitutional variant detection of monogenic disease genes, it is now also used as a model for somatic mutation detection in acquired diseases.</description><identifier>ISSN: 1059-7794</identifier><identifier>EISSN: 1098-1004</identifier><identifier>DOI: 10.1002/humu.22739</identifier><identifier>PMID: 25504618</identifier><language>eng</language><publisher>United States: Blackwell Publishing Ltd</publisher><subject>clinical implementation ; Clinical medicine ; Genetic Diseases, Inborn - diagnosis ; Genetic disorders ; Genomics ; High-Throughput Nucleotide Sequencing - methods ; Humans ; ISO15189 accreditation ; Mutation ; NGS ; Polymerase Chain Reaction - methods ; Prognosis ; Sensitivity and Specificity ; Sequence Analysis, DNA - methods ; targeted resequencing ; uniform target enrichment</subject><ispartof>Human mutation, 2015-03, Vol.36 (3), p.379-387</ispartof><rights>2014 WILEY PERIODICALS, INC.</rights><rights>Copyright © 2015 Wiley Periodicals, Inc.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c4649-2c26f729224e0b9dc880246795bd7d583270218d7b5770fb7d5dcbf4846e34673</citedby><cites>FETCH-LOGICAL-c4649-2c26f729224e0b9dc880246795bd7d583270218d7b5770fb7d5dcbf4846e34673</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://onlinelibrary.wiley.com/doi/pdf/10.1002%2Fhumu.22739$$EPDF$$P50$$Gwiley$$H</linktopdf><linktohtml>$$Uhttps://onlinelibrary.wiley.com/doi/full/10.1002%2Fhumu.22739$$EHTML$$P50$$Gwiley$$H</linktohtml><link.rule.ids>314,780,784,1417,27924,27925,45574,45575</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/25504618$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>De Leeneer, Kim</creatorcontrib><creatorcontrib>Hellemans, Jan</creatorcontrib><creatorcontrib>Steyaert, Wouter</creatorcontrib><creatorcontrib>Lefever, Steve</creatorcontrib><creatorcontrib>Vereecke, Inge</creatorcontrib><creatorcontrib>Debals, Eveline</creatorcontrib><creatorcontrib>Crombez, Brecht</creatorcontrib><creatorcontrib>Baetens, Machteld</creatorcontrib><creatorcontrib>Van Heetvelde, Mattias</creatorcontrib><creatorcontrib>Coppieters, Frauke</creatorcontrib><creatorcontrib>Vandesompele, Jo</creatorcontrib><creatorcontrib>De Jaegher, Annelies</creatorcontrib><creatorcontrib>De Baere, Elfride</creatorcontrib><creatorcontrib>Coucke, Paul</creatorcontrib><creatorcontrib>Claes, Kathleen</creatorcontrib><title>Flexible, Scalable, and Efficient Targeted Resequencing on a Benchtop Sequencer for Variant Detection in Clinical Practice</title><title>Human mutation</title><addtitle>Human Mutation</addtitle><description>ABSTRACT
The release of benchtop next‐generation sequencing (NGS) instruments has paved the way to implement the technology in clinical setting. The need for flexible, qualitative, and cost‐efficient workflows is high. We used singleplex‐PCR for highly efficient target enrichment, allowing us to reach the quality standards set in Sanger sequencing‐based diagnostics. For the library preparation, a modified NexteraXT protocol was used, followed by sequencing on a MiSeq instrument. With an innovative pooling strategy, high flexibility, scalability, and cost‐efficiency were obtained, independent of the availability of commercial kits. The approach was validated for ∼250 genes associated with monogenic disorders. An overall sensitivity (>99%) similar to Sanger sequencing was observed in combination with a positive predictive value of >98%. The distribution of coverage was highly uniform, guaranteeing a minimal number of gaps to be filled with alternative methods. ISO15189‐accreditation was obtained for the workflow. A major asset of the singleplex PCR‐based enrichment is that new targets can be easily implemented. Diagnostic laboratories have validated assays available ensuring that the proposed workflow can easily be adopted. Although our platform was optimized for constitutional variant detection of monogenic disease genes, it is now also used as a model for somatic mutation detection in acquired diseases.</description><subject>clinical implementation</subject><subject>Clinical medicine</subject><subject>Genetic Diseases, Inborn - diagnosis</subject><subject>Genetic disorders</subject><subject>Genomics</subject><subject>High-Throughput Nucleotide Sequencing - methods</subject><subject>Humans</subject><subject>ISO15189 accreditation</subject><subject>Mutation</subject><subject>NGS</subject><subject>Polymerase Chain Reaction - methods</subject><subject>Prognosis</subject><subject>Sensitivity and Specificity</subject><subject>Sequence Analysis, DNA - methods</subject><subject>targeted resequencing</subject><subject>uniform target enrichment</subject><issn>1059-7794</issn><issn>1098-1004</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2015</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqNkU9T1TAUxTOOjiC48QM4mXHjOBbTNP-61Cfv4QgPRni4zKTpLQT60mfSjuCnN6XAwoXDKjc3v3NukoPQm5zs5YTQT5fDetijVBblM7Sdk1Jlqc2ejzUvMylLtoVexXhFCFGcFy_RFuWcMJGrbfRn3sKNq1r4iE-tac1dZXyN95vGWQe-x2cmXEAPNf4BEX4N4K3zF7jz2OAvaXPZdxt8Oh1AwE0X8LkJziTl1ySzvUuo83jWOu_SCHwSTGpa2EUvGtNGeH2_7qDVfP9sdpAdHi--zT4fZpYJVmbUUtFIWlLKgFRlbZUilAlZ8qqWNVcFlYTmqpYVl5I0VerVtmqYYgKKxBU76P3kuwldumXs9dpFC21rPHRD1LkQigrKKH8KyihhVIzou3_Qq24IPj1kpFIYXIgiUR8myoYuxgCN3gS3NuFW50SP4ekxPH0XXoLf3lsO1RrqR_QhrQTkE_DbtXD7Hyt9sDpaPZhmk8bFHm4eNSZc6_Q3kuufy4X-frRcKrE41_PiL8hmsgw</recordid><startdate>201503</startdate><enddate>201503</enddate><creator>De Leeneer, Kim</creator><creator>Hellemans, Jan</creator><creator>Steyaert, Wouter</creator><creator>Lefever, Steve</creator><creator>Vereecke, Inge</creator><creator>Debals, Eveline</creator><creator>Crombez, Brecht</creator><creator>Baetens, Machteld</creator><creator>Van Heetvelde, Mattias</creator><creator>Coppieters, Frauke</creator><creator>Vandesompele, Jo</creator><creator>De Jaegher, Annelies</creator><creator>De Baere, Elfride</creator><creator>Coucke, Paul</creator><creator>Claes, Kathleen</creator><general>Blackwell Publishing Ltd</general><general>Hindawi Limited</general><scope>BSCLL</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QP</scope><scope>7TK</scope><scope>8FD</scope><scope>FR3</scope><scope>K9.</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope></search><sort><creationdate>201503</creationdate><title>Flexible, Scalable, and Efficient Targeted Resequencing on a Benchtop Sequencer for Variant Detection in Clinical Practice</title><author>De Leeneer, Kim ; Hellemans, Jan ; Steyaert, Wouter ; Lefever, Steve ; Vereecke, Inge ; Debals, Eveline ; Crombez, Brecht ; Baetens, Machteld ; Van Heetvelde, Mattias ; Coppieters, Frauke ; Vandesompele, Jo ; De Jaegher, Annelies ; De Baere, Elfride ; Coucke, Paul ; Claes, Kathleen</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4649-2c26f729224e0b9dc880246795bd7d583270218d7b5770fb7d5dcbf4846e34673</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2015</creationdate><topic>clinical implementation</topic><topic>Clinical medicine</topic><topic>Genetic Diseases, Inborn - diagnosis</topic><topic>Genetic disorders</topic><topic>Genomics</topic><topic>High-Throughput Nucleotide Sequencing - methods</topic><topic>Humans</topic><topic>ISO15189 accreditation</topic><topic>Mutation</topic><topic>NGS</topic><topic>Polymerase Chain Reaction - methods</topic><topic>Prognosis</topic><topic>Sensitivity and Specificity</topic><topic>Sequence Analysis, DNA - methods</topic><topic>targeted resequencing</topic><topic>uniform target enrichment</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>De Leeneer, Kim</creatorcontrib><creatorcontrib>Hellemans, Jan</creatorcontrib><creatorcontrib>Steyaert, Wouter</creatorcontrib><creatorcontrib>Lefever, Steve</creatorcontrib><creatorcontrib>Vereecke, Inge</creatorcontrib><creatorcontrib>Debals, Eveline</creatorcontrib><creatorcontrib>Crombez, Brecht</creatorcontrib><creatorcontrib>Baetens, Machteld</creatorcontrib><creatorcontrib>Van Heetvelde, Mattias</creatorcontrib><creatorcontrib>Coppieters, Frauke</creatorcontrib><creatorcontrib>Vandesompele, Jo</creatorcontrib><creatorcontrib>De Jaegher, Annelies</creatorcontrib><creatorcontrib>De Baere, Elfride</creatorcontrib><creatorcontrib>Coucke, Paul</creatorcontrib><creatorcontrib>Claes, Kathleen</creatorcontrib><collection>Istex</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Neurosciences Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Human mutation</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>De Leeneer, Kim</au><au>Hellemans, Jan</au><au>Steyaert, Wouter</au><au>Lefever, Steve</au><au>Vereecke, Inge</au><au>Debals, Eveline</au><au>Crombez, Brecht</au><au>Baetens, Machteld</au><au>Van Heetvelde, Mattias</au><au>Coppieters, Frauke</au><au>Vandesompele, Jo</au><au>De Jaegher, Annelies</au><au>De Baere, Elfride</au><au>Coucke, Paul</au><au>Claes, Kathleen</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Flexible, Scalable, and Efficient Targeted Resequencing on a Benchtop Sequencer for Variant Detection in Clinical Practice</atitle><jtitle>Human mutation</jtitle><addtitle>Human Mutation</addtitle><date>2015-03</date><risdate>2015</risdate><volume>36</volume><issue>3</issue><spage>379</spage><epage>387</epage><pages>379-387</pages><issn>1059-7794</issn><eissn>1098-1004</eissn><abstract>ABSTRACT
The release of benchtop next‐generation sequencing (NGS) instruments has paved the way to implement the technology in clinical setting. The need for flexible, qualitative, and cost‐efficient workflows is high. We used singleplex‐PCR for highly efficient target enrichment, allowing us to reach the quality standards set in Sanger sequencing‐based diagnostics. For the library preparation, a modified NexteraXT protocol was used, followed by sequencing on a MiSeq instrument. With an innovative pooling strategy, high flexibility, scalability, and cost‐efficiency were obtained, independent of the availability of commercial kits. The approach was validated for ∼250 genes associated with monogenic disorders. An overall sensitivity (>99%) similar to Sanger sequencing was observed in combination with a positive predictive value of >98%. The distribution of coverage was highly uniform, guaranteeing a minimal number of gaps to be filled with alternative methods. ISO15189‐accreditation was obtained for the workflow. A major asset of the singleplex PCR‐based enrichment is that new targets can be easily implemented. Diagnostic laboratories have validated assays available ensuring that the proposed workflow can easily be adopted. Although our platform was optimized for constitutional variant detection of monogenic disease genes, it is now also used as a model for somatic mutation detection in acquired diseases.</abstract><cop>United States</cop><pub>Blackwell Publishing Ltd</pub><pmid>25504618</pmid><doi>10.1002/humu.22739</doi><tpages>9</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Human mutation, 2015-03, Vol.36 (3), p.379-387 |
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| subjects | clinical implementation Clinical medicine Genetic Diseases, Inborn - diagnosis Genetic disorders Genomics High-Throughput Nucleotide Sequencing - methods Humans ISO15189 accreditation Mutation NGS Polymerase Chain Reaction - methods Prognosis Sensitivity and Specificity Sequence Analysis, DNA - methods targeted resequencing uniform target enrichment |
| title | Flexible, Scalable, and Efficient Targeted Resequencing on a Benchtop Sequencer for Variant Detection in Clinical Practice |
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