Magnitude-dependent variation in peak height balance at heterozygous STR loci.

When the smaller of two peaks at an STR locus is less than 70% the magnitude of the larger peak at that locus, the disparity is typically taken to be an indication that there is more than one contributor of template DNA to the sample being tested. An analysis of 1,763 heterozygous allele pairs suggests that a peak height imbalance threshold that varies with the magnitude of the peaks being evaluated at a locus is superior to a fixed threshold. Identifying samples that are likely to be mixtures and those that are likely to have arisen from a single source is accomplished more reliably when a statistically based, magnitude-dependent peak height imbalance threshold is used. The amelogenin locus was found to behave in a similar fashion and was also found to have no systematic bias that favored the amplification of Y or X alleles.

Run-specific limits of detection and quantitation for STR-based DNA testing.

STR-based DNA profiling is an exceptionally sensitive analytical technique that is often used to obtain results at the very limits of its sensitivity. The challenge of reliably distinguishing between signal and noise in such situations is one that has been rigorously addressed in numerous other analytical disciplines. However, an inability to determine accurately the height of electropherogram baselines has caused forensic DNA profiling laboratories to utilize alternative approaches. Minimum thresholds established during laboratory validation studies have become the de facto standard for distinguishing between reliable signal and noise/technical artifacts. These minimum peak height thresholds generally fail to consider variability in the sensitivity of instruments, reagents, and the skill of human analysts involved in the DNA profiling process over the course of time. Software (BatchExtract) made publicly available by the National Center for Biotechnology Information now provides an alternative means of establishing limits of detection and quantitation that is more consistent with those used in other analytical disciplines. We have used that software to determine the height of each data collection point for each dye along a control sample's electropherogram trace. These values were then used to determine a limit of detection (the average amount of background noise plus three standard deviations) and a limit of quantitation (the average amount of background noise plus 10 standard deviations) for each control sample. Analyses of the electropherogram data associated with the positive, negative, and reagent blank controls included in 50 different capillary electrophoresis runs validate that this approach could be used to determine run-specific thresholds objectively for use in forensic DNA casework.

Systematic differences in electropherogram peak heights reported by different versions of the GeneScan software.

DNA profiling using STRs on the 310 and 3100 Genetic Analyzers routinely generates electropherograms that are analyzed with the GeneScan software available from the instrument's manufacturer, Applied Biosystems. Users have been able to choose from three different smoothing options that have been known to result in significant differences in the peak heights that are reported. Improvements in the underlying algorithm of the most recent version of the software also result in significant and somewhat predictable differences in peak height values. Laboratories that have performed validation studies using older versions of GeneScan should either reanalyze the data generated in those validation studies with the newest version of the software or otherwise take into consideration the systematically higher peak height values obtained as they begin following the recommendation of the manufacturer and use the new algorithm.