5 pg with all four multiplexes on both the 3130 and 3500 series CE instruments (Fig. 4 and Supplemental Fig. 13). On the 3130 series CE instrument 61–87% and 28–56% of alleles were called at 31 pg and 15.5 pg, respectively whereas on the 3500 series these numbers were 86–94% and 51–71%. As the mass of DNA amplified decreased, the peak height ratio (PHR) at heterozygous loci became more variable with some alleles dropping out at 62.5 pg and below, resulting in PHR values of zero (Supplemental

Fig. 14). Full profiles were obtained at 400 μM hematin, 100 ng/μL humic acid, 200 ng/μL tannic acid and 0.5 mM calcium chloride. Above these concentrations, see more dropout of alleles was observed, the most significant inhibition occurring with calcium chloride and the least with humic acid (Supplemental Fig. 15). The performance in the presence of PCR inhibitors is comparable to that seen with the standard cycling systems [4] and [5]. All of the unique minor contributor alleles were detected at the 1:1 and 2:1 ratios with both mixture sets with the PowerPlex® ESI Fast and

ESX Fast Systems (Supplemental Fig. 16). At the 4:1 ratio, 94–100% of all unique minor contributor alleles were detected with all four multiplexes with the values dropping below 100% due to a minor contributor LY294002 allele that fell in a stutter position being filtered out by the stutter filter for that locus. As the mixture ratio increased to 9:1 and 19:1, there was a gradual decrease in the percentage of unique minor contributor alleles detected (Supplemental Fig. 16). Exposure to increasing Galeterone UV-C energy results in a classic degradation profile with both PowerPlex® ESI 17 Fast and ESX 17 Fast Systems (Supplemental Fig. 17). At 100 mJ of UV-C exposure drop-out

was seen at D10S1248 and D2S441 in PowerPlex® ESI 17 Fast and D18S51, D16S539, D2S1338, and FGA in PowerPlex® ESX 17 Fast (Supplemental Table 5). These loci correspond to some of the largest loci in each multiplex. For both multiplex configurations, the largest standard deviation of the mean size obtained for each ladder allele on the Applied Biosystems 3130 and 3500 series Genetic Analyzers did not exceed 0.11 bases and 0.10 bases, respectively whereas on the ABI PRISM® 310 Genetic Analyzer this value never exceeded 0.14 bases. The sizes of all alleles obtained with components A, B, and C of the Standard Reference Materials 2391c, PCR Based DNA Profiling Standard and 2800M Control DNA with both multiplex configurations were within ±0.5 bases of the size of the corresponding allele in the allelic ladder. Expected genotypes were obtained for components A–C of the Standard Reference Materials 2391c, PCR Based DNA Profiling Standard, and 2800M control DNA in amplification reactions performed at Promega (all four systems), Key Forensics (PowerPlex® ESI Fast Systems) and NBI (PowerPlex® ESX Fast Systems).