Multiplexing Algorithms for High-Throughput Genomic Based Assays

After the completion of the Human Genome Project has provided a blueprint of the DNA present in each human cell, genomics research is now focusing on the study of DNA variations that occur between individuals, seeking to understand how these variations confer susceptibility to common diseases such as diabetes or cancer. The most common form of genomic variation are the so called single nucleotide polymorphisms (SNPs), i.e., the presence of different DNA nucleotides, or alleles, at certain chromosomal locations. Determining the identity of the alleles present in a DNA sample at a given set of SNP loci is called SNP genotyping. Among emerging genotyping technologies, one of the most promising is the use of universal tag arrays, which provide unprecedented assay customization flexibility while maintaining a high degree of multiplexing and low unit cost. In the first part of this thesis we study methods for improving the multiplexing rate (defined as the average number of reactions assayed per array) in SNP genotyping assays involving multiple universal tag arrays. In general, it is not possible to use all tags in an array experiment due to, e.g., unwanted hybridizations. An assay specific optimization that determines the multiplexing rate (and hence the number of required arrays for a large assay) is the tag assignment problem, whereby individual tags are assigned to the primers used to genotype each SNP. We observe that significant improvements in multiplexing rate can be achieved by combining primer selection with tag assignment. For most tag array applications there are multiple primers with the desired functionality; for example in SNP genotyping one can choose the corresponding primer from either the forward or reverse strands. Since different primers hybridize to different sets of tags, a higher multiplexing rate is achieved by integrating primer selection with tag assignment. This integrated optimization is shown to lead to a reduction of up to 50% in the number of required arrays. In the second part of the thesis, we propose a new genotyping assay architecture combining multiplexed solution-phase single-base extension (SBE) reactions with sequencing