SNPlexTM System—A Multiplex High-throughput Genotyping Platform Based on the Oligonucleotide Ligation Assay
Xiaoqing You1, Nicolas Peyret2, Ryan T. Koehler, Joseph P. Day, Jason Evans, Lily Xu, Heinz Hemken, Annie Titus, Charles R. Scafe, Eugene Spier, Joanna Curlee, and Francisco M. De La Vega
1youxn@appliedbiosystems.com, Applied Biosystems; 2youxn@appliedbiosystems.com, Applied Biosystems
At Applied Biosystems, we have developed the SNPlexTM Assay System, a multiplexed high-throughput genotyping platform based on the oligonucleotide ligation assay (OLA) with capillary electrophoresis as the readout. At a 48-plex level, the platform can resolve over 500,000 genotypes per day with a high level of accuracy. SNPlexTM System provides a high-throughput, robust and cost-effective tool especially for the studies that require the ability to genotype hundreds of thousand of SNPs in thousands of samples.
The SNPlexTM assays are designed using proprietary software algorithm. The software rejects SNPs located on genome repeats. Based on thermodynamic properties and manufacturability rules, the final assays are selected with a high confidence to afford highly efficient ligation and PCR reactions and minimize interactions among pairs of assays within one multiplex. In this study, a set of 1250 high confidence SNPs yielded 93.3% successful in silico assay designs. 91.9% of the manufactured assays resulted in a successful genotyping call. The genotyping results from SNPlexTM System were compared with those from two established technologies: TaqManâ probe-based 5’-nuclease genotyping assay and dideoxy sequencing. SNPlexTM System has concordance rates of 99.2% with TaqManâ genotyping assay and of 98.7% with dideoxy sequencing. It is interesting to note that 5’-nuclease/SNPlexTM System concordance is slightly higher than 5’-nuclease/sequencing concordance, suggesting that calling alleles by dideoxy sequencing from PCR amplicons is subject to error.
In conjunction with our R&D effort in developing leading-edge genotyping technologies, we also developed an integrated data management system to evaluate and improve assay design pipelines, perform data analysis and compare genotyping performances among different technology platforms.