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Target enrichment using parallel nanoliter quantitative PCR amplification.

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.

SUBMITTER: De Wilde B

PROVIDER: S-EPMC4234423 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Target enrichment using parallel nanoliter quantitative PCR amplification.

De Wilde Bram B Lefever Steve S Dong Wes W Dunne Jude J Husain Syed S Derveaux Stefaan S Hellemans Jan J Vandesompele Jo J

BMC genomics 20140310

<h4>Background</h4>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.<h4>Results</h4>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 P ...[more]

PMID: 24612714

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Project description:The SlipChip platform was tested to perform high-throughput nanoliter multiplex PCR. The advantages of using the SlipChip platform for multiplex PCR include the ability to preload arrays of dry primers, instrument-free sample manipulation, small sample volume, and high-throughput capacity. The SlipChip was designed to preload one primer pair per reaction compartment and to screen up to 384 different primer pairs with less than 30 nanoliters of sample per reaction compartment. Both a 40-well and a 384-well design of the SlipChip were tested for multiplex PCR. In the geometries used here, the sample fluid was spontaneously compartmentalized into discrete volumes even before slipping of the two plates of the SlipChip, but slipping introduced additional capabilities that made devices more robust and versatile. The wells of this SlipChip were designed to overcome potential problems associated with thermal expansion. By using circular wells filled with oil and overlapping them with square wells filled with the aqueous PCR mixture, a droplet of aqueous PCR mixture was always surrounded by the lubricating fluid. In this design, during heating and thermal expansion, only oil was expelled from the compartment and leaking of the aqueous solution was prevented. Both 40-well and 384-well devices were found to be free from cross-contamination, and end point fluorescence detection provided reliable readout. Multiple samples could also be screened on the same SlipChip simultaneously. Multiplex PCR was validated on the 384-well SlipChip with 20 different primer pairs to identify 16 bacterial and fungal species commonly presented in blood infections. The SlipChip correctly identified five different bacterial or fungal species in separate experiments. In addition, the presence of the resistance gene mecA in methicillin resistant Staphylococcus aureus (MRSA) was identified. The SlipChip will be useful for applications involving PCR arrays and lays the foundation for new strategies for diagnostics, point-of-care devices, and immobilization-based arrays.

Dataset Information

Target enrichment using parallel nanoliter quantitative PCR amplification.

Publications

Target enrichment using parallel nanoliter quantitative PCR amplification.

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