Dr. Heather Miller Coyle

1. Dr. Heather Miller Coyle People v. Jaquan Collins People v. Andrew Peaks Nov. 8, 2013

2. Validation • Purpose of validation • To ensure a method is reliable and robust and reproducible for use in casework • Requisite for new methodologies • Reliable method defined as one where results are accurate and reflect the sample being tested • Reproducible method defined as the same or very similar results obtained each time tested

3. Validation • Purpose of validation • Developmental validation – method development • Internal validation – a method validated in-house in a lab means it is generally accepted in the lab but not necessarily accepted by the scientific community as reliable • If accepted by scientific community, general use by all laboratories would follow • Low copy number (LCN) is problematic and not generally used in criminal casework across the United States

4. Scope of Peer Review • Peer review is open scientific evaluation by appropriate scientific community as a step toward general acceptance or rejection of method • Dr. Caragine’s testimony: • Question: And then when an article is published in the peer review journal, what does that mean? • Answer: It means that your results have been accepted by the sci-by the your peers, by the scientific community. • Publication in a peer reviewed journal does not equal general acceptance • Publication in peer reviewed journal does not equal exhaustive peer review since all of the data was not submitted to journal (summary)

5. Auditing Is Not Peer Review • Auditing is a quality assurance/quality control process • It is aimed at ensuring that a laboratory follows protocols and the DNA Advisory Board standards and is not primarily aimed at ensuring that the underlying scientific methodology is reliable

6. What is Low Copy Number Testing? • Methodology employing increased sensitivity techniques on low template samples (<100 pg) • Generally accepted forensic DNA tests employ 28 cycles of a PCR method • LCN uses 31 cycles of PCR (cycle enhancement) and different interpretation criteria for samples and controls (not the same method) • Samples and controls should be reproducible from PCR amplification to PCR amplification but are not due to low template concentrations of DNA (<100pg) (stochastic effects)

7. Characteristics of Low Copy Number DNA Testing: Stochastic Effects Stochastic effects: • Increased heterozygote peak imbalance • “Drop-out” (extreme form of PHI) • Increased baseline • Increased stutter (artifactual PCR peaks) • Increased “drop-in” or contamination • “Drop-in” and “drop-out” confounds the ability to determine with accuracy the true number of contributors (source attribution error in LCN)

8. Increased Peak Height Imbalance Balanced at 1ng Not balanced at <100 pg Cannot accurately establish major contributor to a mixture

9. Increased Peak Height Imbalance • “Drop out” is an extreme example • Drop out rate is especially variable in the stochastic range of under 100 pg, particularly below 50 pg • Affects interpretation of results how • False homozygote • Difficulty in deconvoluting mixtures with accuracy • May incorrectly conclude two peaks are “sisters”, masking • Error rates or false inclusion rate is high

11. Characteristics of Low Copy Number DNA Testing: “Drop-In” • “Drop-In” is contamination • OCME defines as “drop-in” as observation of a single DNA peak once in triplicate amplifications; “contamination” is if DNA peak is observed more than once • Problematic to distinguish between authentic DNA representative of sample and contamination from an outside source • Clean facilities and UV treatment of tubes still yields 8-11% extraneous DNA in negative controls • Extremely confounding when interpreting DNA results

12. Scientific Accuracy and Reliability Decreases with Decreasing Template Concentrations (28 vs. 34 cycles) Timothy Callahan – University of New Haven, Honors Thesis, 2013 (known buccal swab DNA samples; two replicates)

13. Complex Mixtures Add Another Layer of Uncertainty • Complex mixture interpretation is difficult enough at 28 cycle, high template DNA testing • Controversy in scientific community about how to interpret and report complex DNA mixtures • Low level LCN testing is that much worse • Stochastic effects make it difficult to establish number of true contributors and major donors • Coincidental matching makes it difficult to identify which DNA fragments go to which individual

14. Consequence of Underestimating # of Contributors and/or Deconvoluting Mixtures • Underestimating number of contributors increases the source attribution error (force fit of data) • Source attribution error = deducing profiles (major donor) from a mixture is not valid in stochastic range • Both result in scientific unreliability or scientific inaccuracy of a result

15. Negative Control Contamination • Purpose of negative controls is to establish whether extraneous DNA is present that was not originally in the sample • OCME-NYC allows up to 9 spurious alleles in the negative controls before contamination is at an unacceptable level • LCN validation data shows different injections, resulting in different alleles detected in negative controls • Not generally accepted to have contamination in negative controls

16. OCME Performed Ten Touch Studies • Six clean studies (used various items: hands, pens, CD case, lunch box, etc.) • These six studies were performed using two-, three-, and four-person touched items. • Two studies were mixed (clean and dirty) • One study was performed using three-person touched items; the second study used four-person touched items. • These studies had nine clean items and nine dirty items. • One study used dirty items • These were two person touched items. • One study was performed on degraded items

17. Clean Touch Studies • Examined 85 clean touch samples when calculating alleles that were unaccounted for by the known contributors. • ID 28 = 41 samples • ID 31 = 44 samples • Summaries 19, 20, and 21: designed to replicate case work protocols followed in cleaning items before items were touched. • OCME cleaning protocols unable to eliminate contamination.

18. Analysis • Determined the number of unaccounted alleles in • 28 cycles • 31 cycles Total # of unaccounted alleles/(# samples x # of replicates x # loci) = % of detected alleles that did not come from a known contributor

19. Standard PCR v. LCN Testing • Conventional 28 cycle testing= 12.0% of alleles detected that did not come from a known contributor • LCN 31 cycle testing= 23.2% of alleles detected that did not come from a known contributor • Drop-in reported by OCME for LCN 31 cycle testing= 8-11%

20. Contamination Percentage for Total Allele Count • Calculated the percentage of contaminant alleles to the total allele count for each mixture.

21. Studies 2A and 2B (estimated % contaminant alleles) ID31 ID28 Hand2 7.6% Hand3 0% Hand4 35.0% Hand9 2.4% Hand5 26.0% Hand10 2.7% Hand18 30.0% Hand11 4.4% 2P PenD 8.9% Hand14 16.8% 2P PenE 11.9% Hand15 3.7% 2P PenG 0.0% Hand16 0% 2P PenH 7.6% 2P Stapler 1.7% 2P PenJ 16.5% 2P CD case 0% 2P Mouse 21.4% 2P plastic cover 0% Ave. %: 16.0% Ave. %: 2.3%

22. Studies 3A, 3B, and 3E (estimated % contaminant alleles) • ID31 ID28 • Clean Item 3 – Sharpie 6.8% Clean Item 1 – CD Case 1.30% • Clean Item 5 – Glowstick 0% Clean Item 2 – Plastic Dome 0% • Clean Item 7 – Shot glass 12.5% Clean Item 4 – Ceramic Shoe 3.07% • Clean Item 9 – Glowstick 11.6% Clean Item 6 – Glowstick 1.62% • Pen _ A 12.8% Clean Item 8 – Glowstick 0% • Pen _ B 13.2% Pen _ C 1.42% • Pen _ I 7.14% Pen _ F 5.61% • Staple remover 4.72% Scissor 9.34% • Bucket 1.25% Knife 8.21% • Stapler 4.22% Paperclip 0% • Plastic bowl 2.72% Tape_Dispenser 6.30% • Plastic 13.6% Ceramic_Bowl 21.33% • Divider 4.86% CD_Case 4.70% • Stapler 0% Bucket 0% • Tin_Can 8.33% Divider 3.77% • Lunch_Box 6.33% Pen_F 1.31% • Pen_A 10.0% Pen_G 4.41% • Pen_C 5.67% Ave. %: 4.25% • Pen_B 6.74% • Pen_D 2.80% • Pen_E 7.82% • Ave. %: 6.82%

23. Studies 4A and 4B (estimated % contaminant alleles) • ID31 ID28 • Clean Item 2 – Glowstick 2 8.3% Clean Item 1 – Glowstick 1 11.8% • Clean Item 3 – Glowstick 3 7.3% Clean Item 5 – blueshoe 3.8% • Clean Item 4 – Glowstick 4 10.6% Clean Item 6 – Shotglass 1 1.2% • Clean Item 7 – Shotglass 2 7.5% Clean Item 8 – Plastic dome 18.1% • 4P_Pen_A 11.7% Clean Item 9 – CD Case 19.8% • 4P_Pen_C 7.3% 4P_Pen_B 2.2% • 4P_Sharpie_C 0% 4P_Pen_D 0% • 4P_Sharpie_D 3.7% 4P_Pen_E 7.5% • 4P_Marker 2.9% 4P_Sharpie_A 1.1% • 4P_Glowstick_A 7.7% 4P_Sharpie_B 0.9% • 4P_Glowstick_C 2.2% 4P_Glowstick_B 0% • 4P_Glowstick_D 7.2% 4P_Paperclip 0% • 4P_Sharpie_E 8.2% 4P_ceramic 0% • Avg. %: 6.5% 4P_Plastic 12% Avg.% 5.6%

24. Conclusion: LCN DNA Testing is Unreliable and Not Reproducible for Criminal Casework • Study: Timothy Callahan, University of New Haven • Increased cycle numbers also increased artifacts and confounded interpretation (low % correct alleles) • Study: Mesha Smithen, University of New Haven • Cellular basis of DNA from thumbprints on a smooth surface, 30 seconds of pressure, variable results, could generate artificial composite DNA profiles confounding interpretation of a true contributor (source attribution error)

25. Lack of Scientific Accuracy and General Acceptance of LCN Use in Forensic Community • Other forensic science laboratories do not utilize this LCN process in United States • “Drop-out” rates make it difficult to establish number of contributors with accuracy • Contamination rates are high (more than 8-11%) so DNA sample does not accurately reflect contributor • Kits are not optimized to <100 pg so stochastic effects and PCR artifacts are high and confounds interpretation • Statistics are challenging with results being inconclusive or with FST software, a high false inclusion rates due to coincidental matching of DNA