Changes of ±10% Everolimus nmr in the final concentration of master mix and primer pair mix were tolerated by both the PowerPlex® ESI Fast and ESX Fast Systems. An increase of either master mix or primer pair mix to a final concentration of 1.2× had minimal effect on these systems. However, decreasing the concentration of master mix or primer pair mix to 0.8× adversely

affected the signal and balance, particularly for the PowerPlex® ESI Fast Systems (Fig. 1 and Supplemental Fig. 2). Direct amplification is facilitated by the inclusion of AmpSolution™ Reagent in the reaction. Inclusion of AmpSolution™ Reagent in the amplification reaction has no effect on the signal or balance of the profile obtained whether 500 pg of DNA is amplified for 30 cycles or 10 ng for 26 cycles (Supplemental Figs. 3 and 4). No additional amplification artefacts were seen in the presence of AmpSolution™ Reagent over those documented in the technical manuals (data not shown) [14], [15], [16] and [17].

Increasing cycle number from 28 to 30 cycles resulted in the anticipated PCI32765 increase in signal across all loci for the PowerPlex® ESI 17 Fast and ESX Fast 17 Systems. At 32 cycles, the increase in signal was not uniform across all loci, resulting in a locus-to-locus imbalance (Supplemental Fig. 5). Similar results were obtained for the two 16 plexes (data not shown). Increasing cycle number did not result in the appearance of additional artefact peaks in the no-template amplifications reactions (data not shown). We looked at the effect of increasing cycle number from 25 to 27 cycles on blood FTA® cards (Fig. 2) and buccal FTA® cards

(Supplemental Fig. 6), blood on ProteinSaver™ 903® cards (Supplemental Fig. 7), Bode Buccal Collectors (Supplemental Fig. 8), and SwabSolution™ extracts (Supplemental Fig. 9). Signal tended to increase with cycle number for all direct amplification samples. C-X-C chemokine receptor type 7 (CXCR-7) When using two 1.2 mm buccal FTA® punches, full profiles were obtained with all samples at all cycle numbers. Dropout at one locus (in this case one allele at SE33 in one replicate of donor 2) was seen at 25 cycles when using one 1.2 mm buccal FTA® punch (Supplemental Table 3, data not shown for 16 plexes), but not with any of the other direct amplification sample types at any cycle number. The genotypes obtained for a given donor were concordant with each other between cycle numbers and across direct amplification sample types tested. Increasing the annealing temperature to 62 °C from the recommended 60 °C resulted in a significant reduction in signal at amelogenin, D8S1179 and FGA with the PowerPlex® ESI Fast Systems (occasional drop-out at amelogenin and D8S1179 at 62 °C) and dropout occurring at 64 °C along with D2S441 and in some replicates at D2S1338 and D19S433. Overall, 90–100% of alleles were obtainable at 62 °C and 61–76% at 64 °C with the PowerPlex® ESI Fast Systems (Supplemental Fig. 10).