We have developed a rapid, cost-effective, high-throughput readout for single nucleotide polymorphism (SNP) genotyping using flow cytometric analysis performed on a Luminex 100 flow cytometer. This robust technique employs a PCR-derived target DNA containing the SNP, a synthetic SNP-complementary ZipCode-bearing capture probe, a fluorescent reporter molecule, and a thermophilic DNA polymerase. An array of fluorescent microspheres, covalently coupled with complementary ZipCode sequences (cZipCodes), was hybridized to the reaction products and sequestered them for flow cytometric analysis. The single base chain extension (SBCE) reaction was used to assay 20 multiplexed SNPs for 633 patients in 96-well format. Comparison of the microsphere-based SBCE assay results to gel-based oligonucleotide ligation assay (OLA) results showed 99.3% agreement in genotype assignments. Substitution of direct-labeled R6G dideoxynucleotide with indirect-labeled phycoerythrin dideoxynucleotide enhanced signal five- to tenfold while maintaining low noise levels. A new assay based on allele-specific primer extension (ASPE) was validated on a set of 15 multiplexed SNPs for 96 patients. ASPE offers both the advantage of streamlining the SNP analysis protocol and the ability to perform multiplex SNP analysis on any mixture of allelic variants.

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Microhaplotypes (MHs) are loci of two or more SNPs within a short distance from each other (< 300 nucleotides) with three or more allelic combinations. Massively parallel sequencing (MPS) is clonal sequencing of individual strands, thus it can distinguish the parental haplotypes at a locus. MH alleles within a locus all have the same size, do not generate stutter fragments, and have lower mutation rates than STRs. The goal of this project was to determine whether MHs could provide biogeographic ancestry prediction.A total of 278 samples including 76 African-Americans (AAs), 110 European-Americans (EAs) and 92 South West Hispanics (SWHISPs) were selected and genotyped using a 74-plex assay on the Ion Chef™ and Ion S5™ MPS platform. Results were used to generate allele frequencies for the three populations. A set of test samples ( 28) was genotyped and used to calculate the random match probability (RMP) in the three populations. Additionally, PHASE inferred allele frequencies from 24 populations obtained from ALFRED were used to calculate the RMP of the test samples in each population. A mixture study was also performed to evaluate the detection limit for the minor contributor of the MH 74-plex.Among the populations studied, the RMP averaged remarkably higher in each individual's self-identified population of "origin". For example, AAs had the highest RMP in African and African-American populations. In the mixture study results showed that alleles from the minor contributor were detectable at an 80:1 major/minor ratio when the input DNA was 10 ng and 40:1 when the input amount was 1 ng with a RMP equal to or greater than a typical 15 loci STR profile demonstrating that MHs can be an effective tool for ancestry prediction and mixture analysis.

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