Development of a microfluidic-multiplex PCR-based targeted next generation sequencing method for genetic variant screening.

FASS M.; PUEBLA A.; ZUBRZYCKI J.; FILIPPI C.; NISHINAKAMSU V.; ALVAREZ D.; CORDES D.; HOPP H.E.; HEINZ R.A.; LIA V.V.; PANIEGO N.

Montevideo

Congreso; XVI Congreso Latinoamericano de Genética; 2016

Sociedad Argentina de Genética

Advances in high-throughput sequencing resulted in the development and optimization of a variety of methods allowing the generation of large amounts of genomic information. Targeted sequencing focuses on specific regions of interest reducing datasets to more manageable sizes, increasing the coverage levels, improving resolution and allowing the discovery and/or validation of rare genetic variants. The goal of this work was to develop and apply a technique which can generate multilocus datasets in any population of interest based on the microfluidic-multiplex PCR sequencing assay (mmPCR-seq) using the Fluidigm Access Array System®. This approach simultaneously undertakes the enrichment of targeted sequences and library preparation. To test the performance of the method, we carried out a mmPCR-seq assay for 48 stress resistance candidate genes and a set of 48 individuals from a mutagenized sunflower population. Therefore, we designed 48 tagged target-specific primer pairs and used them for amplification in combination with Illumina-specific primer pairs containing a barcode and an adaptor sequence. Amplicons were subjected to paired end sequencing with the Illuminas MiSeq platform. Our targeted approach generated a large dataset for the selected loci across the amplified samples, allowing the detection and description of mutants. This strategy proved to be cost- and time-effective for the collection of large numbers of target enriched sequences. Moreover, the system can be used for any sample of interest, expanding the potential of detecting genomic variants to many species.