Dr. Heather Miller Coyle

People v. Jaquan Collins

People v. Andrew Peaks

Nov. 8, 2013

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

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

Scope of Peer Review1. 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)

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

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)

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)

Increased Peak Height Imbalance

Balanced at 1ng

Not balanced at<100 pg

Cannot accuratelyestablish majorcontributor to amixture

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

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

Timothy Callahan – University of New Haven, Honors Thesis, 2013(known buccal swab DNA samples; two replicates)

Scientific Accuracy and Reliability Decreases with Decreasing Template Concentrations (28 vs. 34 cycles)

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

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

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

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

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.

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

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%

Contamination Percentage for Total Allele Count Calculated the percentage of contaminant

alleles to the total allele count for each mixture.

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% Hand1830.0% 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%

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%

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%

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)

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