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Flexible, scalable, and efficient targeted resequencing on a benchtop sequencer for variant detection in clinical practice

Kim De Leeneer (UGent) , Jan Hellemans (UGent) , Wouter Steyaert (UGent) , Steve Lefever (UGent) , Inge Vereecke (UGent) , Eveline Debals (UGent) , Brecht Crombez (UGent) , Machteld Baetens (UGent) , Mattias Van Heetvelde (UGent) , Frauke Coppieters (UGent) , et al.
(2015) HUMAN MUTATION. 36(3). p.379-387
Author
Organization
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 approximate to 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.
Keywords
NGS, targeted resequencing, uniform target enrichment, clinical implementation, ISO15189 accreditation, HEREDITARY HEARING-LOSS, DILATED CARDIOMYOPATHY, MOLECULAR DIAGNOSIS, CAPTURE METHOD, MUTATIONS, ENRICHMENT, TECHNOLOGIES, DEAFNESS, DISEASE, CANCER

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MLA
De Leeneer, Kim, et al. “Flexible, Scalable, and Efficient Targeted Resequencing on a Benchtop Sequencer for Variant Detection in Clinical Practice.” HUMAN MUTATION, vol. 36, no. 3, 2015, pp. 379–87, doi:10.1002/humu.22739.
APA
De Leeneer, K., Hellemans, J., Steyaert, W., Lefever, S., Vereecke, I., Debals, E., … Claes, K. (2015). Flexible, scalable, and efficient targeted resequencing on a benchtop sequencer for variant detection in clinical practice. HUMAN MUTATION, 36(3), 379–387. https://doi.org/10.1002/humu.22739
Chicago author-date
De Leeneer, Kim, Jan Hellemans, Wouter Steyaert, Steve Lefever, Inge Vereecke, Eveline Debals, Brecht Crombez, et al. 2015. “Flexible, Scalable, and Efficient Targeted Resequencing on a Benchtop Sequencer for Variant Detection in Clinical Practice.” HUMAN MUTATION 36 (3): 379–87. https://doi.org/10.1002/humu.22739.
Chicago author-date (all authors)
De Leeneer, Kim, Jan Hellemans, Wouter Steyaert, Steve Lefever, Inge Vereecke, Eveline Debals, Brecht Crombez, Machteld Baetens, Mattias Van Heetvelde, Frauke Coppieters, Jo Vandesompele, Annelies De Jaegher, Elfride De Baere, Paul Coucke, and Kathleen Claes. 2015. “Flexible, Scalable, and Efficient Targeted Resequencing on a Benchtop Sequencer for Variant Detection in Clinical Practice.” HUMAN MUTATION 36 (3): 379–387. doi:10.1002/humu.22739.
Vancouver
1.
De Leeneer K, Hellemans J, Steyaert W, Lefever S, Vereecke I, Debals E, et al. Flexible, scalable, and efficient targeted resequencing on a benchtop sequencer for variant detection in clinical practice. HUMAN MUTATION. 2015;36(3):379–87.
IEEE
[1]
K. De Leeneer et al., “Flexible, scalable, and efficient targeted resequencing on a benchtop sequencer for variant detection in clinical practice,” HUMAN MUTATION, vol. 36, no. 3, pp. 379–387, 2015.
@article{6922430,
  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 approximate to 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.}},
  author       = {{De Leeneer, Kim and Hellemans, Jan and Steyaert, Wouter and Lefever, Steve and Vereecke, Inge and Debals, Eveline and Crombez, Brecht and Baetens, Machteld and Van Heetvelde, Mattias and Coppieters, Frauke and Vandesompele, Jo and De Jaegher, Annelies and De Baere, Elfride and Coucke, Paul and Claes, Kathleen}},
  issn         = {{1059-7794}},
  journal      = {{HUMAN MUTATION}},
  keywords     = {{NGS,targeted resequencing,uniform target enrichment,clinical implementation,ISO15189 accreditation,HEREDITARY HEARING-LOSS,DILATED CARDIOMYOPATHY,MOLECULAR DIAGNOSIS,CAPTURE METHOD,MUTATIONS,ENRICHMENT,TECHNOLOGIES,DEAFNESS,DISEASE,CANCER}},
  language     = {{eng}},
  number       = {{3}},
  pages        = {{379--387}},
  title        = {{Flexible, scalable, and efficient targeted resequencing on a benchtop sequencer for variant detection in clinical practice}},
  url          = {{http://doi.org/10.1002/humu.22739}},
  volume       = {{36}},
  year         = {{2015}},
}
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