1. Artifacts and noise in DNA profiling Forensic Bioinformatics (www.bioforensics.com) Dan E. Krane, Wright State University, Dayton, OH Forensic DNA Profiling Video Series

2. Factors that can complicate the interpretation of DNA profiles Technical artifacts –Stutter –Spikes and blobs –Peak height imbalance –Degradation/Inhibition Background noise

3. Stutter (little extra peaks)

4. Stutter (an artifact) 1418

5. Factors that can complicate the interpretation of DNA profiles Technical artifacts –Stutter –Spikes and blobs –Peak height imbalance –Degradation/Inhibition Background noise

6. Spikes (and blobs) 89 samples (references, pos controls, neg controls) 1010 “good” peaks 55 peaks associated with 24 spike events 95% boundaries shown

7. Factors that can complicate the interpretation of DNA profiles Technical artifacts –Stutter –Spikes and blobs –Peak height imbalance –Degradation/Inhibition Background noise

8. Peak height imbalances

9. Peak Height Ratios 44% PHR 51% PHR 98% PHR 96% PHR

10. Factors that can complicate the interpretation of DNA profiles Technical artifacts –Stutter –Spikes and blobs –Peak height imbalance –Degradation/Inhibition Background noise

11. Degradation (deterioration of DNA) Inhibition (poor PCR amplification)

12. Minimal degradation/inhibition

13. Slightly degraded/inhibited “Ski slope”

14. More degraded/inhibited Drop out on right DROP-OUT

15. Minimal degradation/inhibition

16. Factors that can complicate the interpretation of DNA profiles Technical artifacts –Stutter –Spikes and blobs –Peak height imbalance –Degradation/Inhibition Background noise

17. Sometimes signal is easy to see

18. Sometimes signal and noise are hard to tell apart 150 RFU minimum peak height threshold

19. Where do minimum peak height thresholds come from (originally)? Applied Biosystems validation study of 1998 Wallin et al., 1998, “TWGDAM validation of the AmpFISTR blue PCR Amplification kit for forensic casework analysis.” JFS 43:854-870.

20. Where do minimum peak height thresholds come from (originally)?

21. Where do minimum peak height thresholds come from? “Conservative” thresholds established during validation studies Eliminate noise (even at the cost of eliminating signal) Can arbitrarily remove legitimate signal Contributions to noise vary over time (e.g. polymer and capillary age/condition) Analytical chemists use LOD and LOQ

22. Signal Measure μbμb μ b + 3σ b μ b + 10σ b Mean background Signal Detection limit Quantification limit Measured signal (In Volts/RFUS/etc) Saturation 0

23. Many opportunities to measure baseline

24. Doesn’t someone either match or not?

25. Lines in the sand: a two-person mix? Two reference samples in a 1:10 ratio (male:female). Three different thresholds are shown: 150 RFU (red); LOQ at 77 RFU (blue); and LOD at 29 RFU (green).

26. Factors that can complicate the interpretation of DNA profiles Technical artifacts –Stutter –Spikes and blobs –Peak height imbalance –Degradation/Inhibition Background noise

27. Artifacts and noise in DNA profiling Forensic Bioinformatics (www.bioforensics.com) Dan E. Krane, Wright State University, Dayton, OH Forensic DNA Profiling Video Series