Figure 5

Optimization and performance of primer sets. (a) Classification of reads based on known typing results using sequence and Q-values. PCR artifacts resulting in artificial hybrids of allele1 and allele2 are reported as “crossover“. (b) Optimization of primer sets - Allele balancing: Example of an optimized primer set (A Exon 2) demonstrating balanced amplification and sufficient read counts. (c) Optimization of primer sets - Allele amplification bias: Example of an unoptimized primer set (B Exon 3) demonstrating negative amplification bias for allele groups B*14 and B*27. (d) Crossover artifact quantification: 48 samples were amplified using 30 to 36 PCR cycles and the rate of crossover formation was quantified for each locus and exon. Sample-loci with homozygous results were not considered for analysis. Lowering the number of PRC cycles reduces the crossover-rate.