NCJ Number
240790
Journal
Forensic Science International: Genetics Volume: 6 Issue: 6 Dated: December 2012 Pages: 708-715
Date Published
December 2012
Length
8 pages
Annotation
This study 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).
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, the authors 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 percent of the alleles had a peak height below this value. Using 2,085 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 percent of the -1 repeat unit stutters, thirteen stutter ratio filters could be lowered by up to 1.79 percent and for two loci the stutter ratio filters had to be elevated slightly with a maximum of 0.06 percent. 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 percent and for D22S1045 it was determined to be 7.27 percent. To find the optimal strategy to sensitize genotyping for low template DNA samples, a comparison was made between enhancing the capillary electrophoresis settings (9kV for 10s) and increasing the number of PCR cycles (29+5 cycles). (Published Abstract)