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Target enrichment using parallel nanoliter quantitative PCR amplification.
De Wilde, Bram; Lefever, Steve; Dong, Wes; Dunne, Jude; Husain, Syed; Derveaux, Stefaan; Hellemans, Jan; Vandesompele, Jo.
Afiliação
  • Vandesompele J; Center of Medical Genetics Ghent, Ghent University, Ghent, Belgium. Joke.Vandesompele@ugent.be.
BMC Genomics ; 15: 184, 2014 Mar 10.
Article em En | MEDLINE | ID: mdl-24612714
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.
Assuntos

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Reação em Cadeia da Polimerase Limite: Humans Idioma: En Ano de publicação: 2014 Tipo de documento: Article

Texto completo: 1 Base de dados: MEDLINE Assunto principal: Reação em Cadeia da Polimerase Limite: Humans Idioma: En Ano de publicação: 2014 Tipo de documento: Article