Target enrichment using parallel nanoliter quantitative PCR amplification View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2014-12

AUTHORS

Bram De Wilde, Steve Lefever, Wes Dong, Jude Dunne, Syed Husain, Stefaan Derveaux, Jan Hellemans, Jo Vandesompele

ABSTRACT

BACKGROUND: Next generation targeted resequencing is replacing Sanger sequencing at high pace in routine genetic diagnosis. The need for well validated, high quality enrichment platforms to complement the bench-top next generation sequencing devices is high. RESULTS: We used the WaferGen Smartchip platform to perform highly parallelized PCR based target enrichment for a set of known cancer genes in a well characterized set of cancer cell lines from the NCI60 panel. Optimization of PCR assay design and cycling conditions resulted in a high enrichment efficiency. We provide proof of a high mutation rediscovery rate and have included technical replicates to enable SNP calling validation demonstrating the high reproducibility of our enrichment platform. CONCLUSIONS: Here we present our custom developed quantitative PCR based target enrichment platform. Using highly parallel nanoliter singleplex PCR reactions makes this a flexible and efficient platform. The high mutation validation rate shows this platform's promise as a targeted resequencing method for multi-gene routine sequencing diagnostics. More... »

PAGES

184

Identifiers

URI

http://scigraph.springernature.com/pub.10.1186/1471-2164-15-184

DOI

http://dx.doi.org/10.1186/1471-2164-15-184

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1034841634

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/24612714


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45 schema:description BACKGROUND: Next generation targeted resequencing is replacing Sanger sequencing at high pace in routine genetic diagnosis. The need for well validated, high quality enrichment platforms to complement the bench-top next generation sequencing devices is high. RESULTS: We used the WaferGen Smartchip platform to perform highly parallelized PCR based target enrichment for a set of known cancer genes in a well characterized set of cancer cell lines from the NCI60 panel. Optimization of PCR assay design and cycling conditions resulted in a high enrichment efficiency. We provide proof of a high mutation rediscovery rate and have included technical replicates to enable SNP calling validation demonstrating the high reproducibility of our enrichment platform. CONCLUSIONS: Here we present our custom developed quantitative PCR based target enrichment platform. Using highly parallel nanoliter singleplex PCR reactions makes this a flexible and efficient platform. The high mutation validation rate shows this platform's promise as a targeted resequencing method for multi-gene routine sequencing diagnostics.
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