Assessment of the stochastic threshold, back- and forward stutter filters and low template techniques for NGM

Abstract The AmpFlSTR ® NGM™ kit shows an increased sensitivity compared to previous AmpFlSTR ® kits, and the addition of a 29th PCR cycle was found to be the major cause for this. During in-house validation, we evaluated whether the increased sensitivity requires elevation of the stochastic threshold (below which alleles are prone to drop out due to low template amplification effects). To determine the stochastic threshold, over 500 false homozygotes were examined and the threshold was set at the rfu value where 99% of the alleles had a peak height below this value. Using 2085 Dutch reference samples, locus-specific stutter ratios were empirically determined and compared with the ones provided by Applied Biosystems. Application of sharp stutter filters is especially important for the analysis of unequal mixtures. To prevent allele calling of 99% of the −1 repeat unit stutters, thirteen stutter ratio filters could be lowered by up to 1.79% and for two loci the stutter ratio filters had to be elevated slightly with a maximum of 0.06%. At all loci +1 repeat stutters were visible for the higher DNA inputs and for lower inputs at the tri-nucleotide repeat locus D22S1045 as well. The overall +1 stutter ratio filter was set to 2.50% and for D22S1045 it was determined to be 7.27%. To find the optimal strategy to sensitise genotyping for low template DNA samples, a comparison was made between enhancing the capillary electrophoresis settings (9 kV for 10 s) and increasing the number of PCR cycles (29 + 5 cycles).