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
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. % of detected alleles that did not come from a known contributor
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% Hand %
Hand % Hand %
2P PenD 8.9% Hand %
2P PenE 11.9% Hand %
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)
ID 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 %
Pen _ I % Pen _ F %
Staple remover 4.72% Scissor %
Bucket % Knife %
Stapler % Paperclip 0%
Plastic bowl 2.72% Tape_Dispenser 6.30%
Plastic % Ceramic_Bowl %
Divider % CD_Case %
Stapler 0% Bucket 0%
Tin_Can 8.33% Divider %
Lunch_Box 6.33% Pen_F %
Pen_A % Pen_G %
Pen_C % Ave. %: %
Pen_B %
Pen_D %
Pen_E %
Ave. %: 6.82%

23. Studies 4A and 4B (estimated % contaminant alleles)
ID31 ID28
Clean Item 2 – Glowstick 2 8.3% Clean Item 1 – Glowstick %
Clean Item 3 – Glowstick 3 7.3% Clean Item 5 – blueshoe 3.8%
Clean Item 4 – Glowstick % Clean Item 6 – Shotglass 1 1.2%
Clean Item 7 – Shotglass 2 7.5% Clean Item 8 – Plastic dome 18.1%
4P_Pen_A % 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