Abstract
BackgroundSingle nucleotide polymorphisms (SNPs) have emerged as the genetic marker of choice for mapping disease loci and candidate gene association studies, because of their high density and relatively even distribution in the human genomes. There is a need for systems allowing medium multiplexing (ten to hundreds of SNPs) with high throughput, which can efficiently and cost-effectively generate genotypes for a very large sample set (thousands of individuals). Methods that are flexible, fast, accurate and cost-effective are urgently needed. This is also important for those who work on high throughput genotyping in non-model systems where off-the-shelf assays are not available and a flexible platform is needed.ResultsWe demonstrate the use of a nanofluidic Integrated Fluidic Circuit (IFC) - based genotyping system for medium-throughput multiplexing known as the Dynamic Array, by genotyping 994 individual human DNA samples on 47 different SNP assays, using nanoliter volumes of reagents. Call rates of greater than 99.5% and call accuracies of greater than 99.8% were achieved from our study, which demonstrates that this is a formidable genotyping platform. The experimental set up is very simple, with a time-to-result for each sample of about 3 hours.ConclusionOur results demonstrate that the Dynamic Array is an excellent genotyping system for medium-throughput multiplexing (30-300 SNPs), which is simple to use and combines rapid throughput with excellent call rates, high concordance and low cost. The exceptional call rates and call accuracy obtained may be of particular interest to those working on validation and replication of genome- wide- association (GWA) studies.
Highlights
Single nucleotide polymorphisms (SNPs) have emerged as the genetic marker of choice for mapping disease loci and candidate gene association studies, because of their high density and relatively even distribution in the human genomes
Chip Architecture The chip used in this study, the 48.48CS dynamic array, is mounted on a plastic carrier with interface and containment accumulators and 48 sample inlets and 48 assay inlets, with the dimensions of the inlets and the size of the plate conforming to the standards set by the Society for Biomolecular Sciences (SBS format)
The NanoFlexTM valves are made of an elastomeric material which deflects under pressure to create a tight seal and are used to regulate the flow of liquids in the Integrated Fluidic Circuit (IFC)
Summary
Single nucleotide polymorphisms (SNPs) have emerged as the genetic marker of choice for mapping disease loci and candidate gene association studies, because of their high density and relatively even distribution in the human genomes. Recent technological advances in ultra high-throughput genotyping platforms potentially permit the parallel analysis of millions of SNPs with a significant reduction in genotyping price, making GWA studies a reality [9,10,11,12,13] The impact of this approach is readily visible, since over 296 publications have been collected in the last three years in the National Cancer Institute (NCI)-National Human Genome Research Institute (NHGRI)'s catalog of published genome-wide association studies [14]. Homogenous detection methods such as TaqMan® [20] and molecular beacon -based [21] approaches provide uniplex reactions, and are readily applied to a large number of samples but multiplexing may be difficult Between these two (ultrahigh multiplexing and low/no multiplexing) methods there is a need for systems allowing medium multiplexing (ten to hundreds of SNPs) with high throughput, which can efficiently and cost-effectively generate genotypes for a very large sample set (thousands of individuals). This is important for those who work on high throughput genotyping in non-model systems where off-the-shelf assays are not available and a flexible platform is needed
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