Using Nanoscale PCR and NGS Technologies to Detect Rare Sequence Variants in a Core Setting

Abstract
The University of Iowa DNA Facility has been providing sequence variant detection services using nanoscale PCR and NGS technologies for nearly three years. Detection of sequence variants that occur in a small percentage of the population has met with both technical and analytical challenges in avoiding or minimizing false calls. False positive calls can be very expensive to verify. False negative calls simply cannot happen as they result in a missed diagnosis. Using the Applied Biosystems OpenArray and Fluidigm Dynamic arrays, the ability of each platform to give the correct genotype for 32 assays over 575 individuals was evaluated. Both platforms were similar in their false call rate and the correct call rates improved with by conducting a specific target enhancement of the samples prior to the genotyping assay. Variant detection was also evaluated using NGS technologies. Amplicons from 300 individuals interrogating over 210 target regions from each individual were mixed together and run over the Roche GS FLX and Applied Biosystems SOLiD genome sequencers. False positive rates were initially high, but decreased as the specificity of the primers was improved. Workflows and/or experimental design enhancements to reduce false genotyping call rates will be discussed.