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.
Gilder
Jason R
JR
Forensic Bioinformatics Inc., 2850 Presidential Drive, Suite 150, Fairborn, OH 45324, USA.
Doom
Travis E
TE
Inman
Keith
K
Krane
Dan E
DE
D016172:DNA Fingerprinting / Q000379:methods*; D019075:Electrophoresis, Capillary; D005260:Female; D005453:Fluorescence; D006801:Humans; D008297:Male; D017422:Sequence Analysis, DNA; D012984:Software; D020080:Tandem Repeat Sequences