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High-throughput Single Nucleotide Polymorphism Genotyping Using Nanofluidic Dynamic Arrays

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High-throughput Single Nucleotide Polymorphism Genotyping Using Nanofluidic Dynamic Arrays

Jun Wang et al. BMC Genomics.

Abstract

Background: 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. 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.

Results: We 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.

Conclusion: Our 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.

Figures

Figure 1
Figure 1
A 48.48CS dynamic array showing the position of the sample inlets and the assay inlets.
Figure 2
Figure 2
SNP Genotyping data analysis. The Fluidigm SNP Genotyping Analysis software automatically analyzes the end-point image of a genotyping chip run and generates genotyping calls for each sample. (a). Raw image from a 48.48CS chip run in both FAM and VIC fluorescent channels. (b). The software generated call map view of the genotyping calls for each of the 2304 reaction chambers. (c) Software generated scatter plot for 48 samples in one SNP assay with genotype calls automatically. Four different color coded, 3 genotypes plus negative controls (NTC, black dots) are observed (d) Genotyping scatter plot of samples from 22 chip runs.
Figure 3
Figure 3
Comparison of genotyping results from 48.48CS dynamic array with microtiter plates run on Applied Biosystems 7900 HT. (a) Call rate comparison; (b) Concordance/Accuracy with HapMap results.
Figure 4
Figure 4
Comparison of genotyping calls from the same sets of 15 samples that were either genotyped directly, or after the STA step. The circled dots are data points generated from the STA samples.
Figure 5
Figure 5
Comparison of genotype call accuracy related to input DNA copy number. Three genomic DNA samples carrying different genotypes with varied input amount were genotyped on SNP rs513349. The scatter plots of different DNA copy number ends in each reaction chambers are shown, (a) 0.9 copies; (b) 4.5 copies; (c) 9 copies; (d) 45 copies; (e) 90 copies.

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