Challenges in Dealing With DNA Evidence (Noureddine)

7/27/2019 Challenges in Dealing With DNA Evidence (Noureddine)

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3/22/20

Challenges in Dealing wi th DNA Evidence:

Lab Reports and Data Interpretations

Maher “Max” Noureddi ne, PhD, MS

President: ForensiGen, LLC

Founder and President

The Institute for Advanced Career Development

03/22/2012

The “ Culture” of Forensic DNA Evidence

DNA: the ultimate ID card?

Forensic DNA Science is “Evolvi ng” as we speak!

Are we ap proaching DNA eviden ce w ith a

healthy dose of skepticism?

DNA evidence evaluation is primarily a science

experiment: T or F?

DNA Science vs Forensic DNA Evidence

Idea / Observation

Hypothesis Testing

Flexible Experimental Design

Crime / Incident

Evidence / Leads / Theories

Restricted Experimental Design

Results and Interpretations

Peer Review

Results

Interpretations / Internal Review?

Trial / JuryLiterature



-Laboratory Accreditation / Audits

-Laboratory Management / Case Management

-Staff Proficiency / Education

Quality Assurance Standards

- a a on o n s rumen s, es s, pro oco s

-Quality Control, sample prep, processing, integrity

-Results and Interpretations Standards

The Laboratory “Should” establish most standards

Chromosomes in Living Organisms

Human Chromosomesx3,000

Human DNA: Chromosomes

23 Pairs of chromosomes in every human cell (with few exceptions):

One copy from Mom, one from Dad

Human Chromosomes



DNA in the Cell

GAAACTTGCA…..

CTTT GAACGT…..

Every Chromosome

is a continuous

double strand of

DNA…Think of it as a

long string !

…AAGTTCATGTAAACGTAGTCCCAGTCCAGTCAGCC

ATTTGACAAGTTCATGTAAACGTAGTCCCAGTCCAGT

CAGCCATTTGACAAGTTCATGTAAACGTAGTCCCAGT

CCAGTCAGCCATTTGAC GATA GATA GATA GATA

GATA GATA CCCTCTTC…

Short Tandem Repeats (STRs) in DNA

6 Repeats

…AAGTTCATGTAAATGTAGTCCCAGTCCAGTCAGCC

ATTTGACAAGTTCATGTAAACGTAGTCCCAGTCCAGT

CAGCCATTTGACAAGTTCATGTAAACGTAGTCCCAGT

CCAGTCAGCCATTTGAC GATA GATA GATA GATA

GATA GATA GATA GATA CCCTCTTC…

Chromosom e 16 Marker D16S539

8 Repeats

Locus (Loci): Location on a Chromosome

Alleles 6 and 8

6 8

Chromosome 16

Marker D16S539



All ele In her itance fo r Locus D16S538

Frank Judy

a b a c

Anna Paul Tony Phil

caabcbaa

Short Tandem Repeats (STRs) in DNA

35413244# repeats

Chr 8(D8S1179)

Chr 21(D21S11)

Chr 7(D7S820)

Chr 13(D13S317)

Chromosome #

Marker Name

Genotype: 4 , 4 2 , 3 1 , 4 5 , 3

Or: 4

General Steps: STR Forensic DNA Analysis:

Sample Collection

DNA Extraction

(RFLP)

Restriction

Fragment Length

Polymorphism

Cut with Enzymes

Ampl ify w/ PCR Ampl if y w / PCR

Gel

Electrophoresis

Gel

Electrophoresis

Capillary

Electrophoresis

Mid 80s-90s Mid 90s-early 2000 Current



Which Suspect Matches the Evidence?

(14)

(12)

(11)

(13)

1

TH01alleles

Alleleladder

Sperm Female S1 S2 S3

(9)

(8)

(7)

(6)

(5)

(4)

(3)

D3S1358 VWA FGA

Matching DNA Profiles: e-grams or peaks

Electropherograms or “ e-grams”



STR Loci in Modern Forensics

There are more than 100,000 STRs in the hum an genome

Electropherogram

RFU

Or Peak Height (1336) All ele Call s f or

Marker TH01 (8 , 9.3)

RFU=Relative Fluorescence Units

Evidence Sample



Sample Types

Positive Controls

Ne at ive Controls

Evidence Samples

Positive Control: Ladder

Negative Control

Note any peaks, a sign of contamination



DNA Single Contributor

7

What are the possible genotypes?

DNA Single Contributor

7


8

DNA Mixtures

Type A: No Major Contributor

7 8 10




DNA Mixtures

Type A: No Major Contributor

7 8 10 11


Type A:Type A: No Major Contributor

DNA Mixtures

7 8 10 11


13

Type B: Clear Major and Minor contributors

DNA Mixtures

7 8 10 11




Type B: Clear Major and Minor contributors

DNA Mixtures

7 8 10 11


13

Type C: Low signals, possible allele drop outs.

No Interpretation possible

DNA Mixtures

250 RFUs Stochastic Threshold

7 8 10 11


13

75 RFUs Analytical Threshold

Stutter

Artifacts / Anomalies / Challenges

Biologically InducedTechnically Induced

Split Peaks

Dye Blobs

Pull-Ups

Sample Induced

Contamination

Mixtures

Increased Complexity

MicrovariantsSpikes

Tri-alleles

Allele Drop In / Out

Allele Peak Imbal ance

Inhibition

Low Copy Number DNA (LCN)

Wrong Conclusions

Complex Mixtures



Stutter

76 8

<15% <8%

DNA Single Contributor: Allele Imbalance

>60%

Peak Height Ratio

(PHE)

7 8


Smaller Peak rfu

Larger Peak rfu

X 100

*Remember this, it will

be important shortly!

• Match – Peaks between the compared STR profiles have the same genotypes and no unexplainable differences exist between the samples. Statistical evaluation of the significance of the match is usually reported with the match report.

• Exclusion (Non

‐match) – The genotype comparison shows profile

Outcomes of Forensic DNA Analysis

differences that can only be explained by the two samples originating from different sources.

• Inconclusive – The data does not support a conclusion as to whether the profiles match. This finding might be reported if two analysts remain in disagreement after review and discussion of the data and it is felt that insufficient information exists to support any conclusion.



Random Match Probabilit y (RMP)

Combined Probability of Inclusion (CPI) or

Statistical Methods Currently Used:

om ne ro a y o xc us on or

Random Man Not Exclu ded (RMNE)

Likelihood Ratio (LR)

Allele Frequency in the Population

Frank Judy

8 4 8 7

Liz

8 12

Michelle

8 12

Anna Paul Tony Phil

7813612131414


Allele

Name

Size of

“Population”

Surveyed

# Chrom.

Detected in

Population

Allele

Frequency

Approx

%

Allele 4 16 1 0.0625 6%

Allele 6 16 1 0.0625 6%

Allele 7 16 2 0.125 12%

Allele 8 16 5 0.3125 31%

Allele 12 16 3 0.1875 18%

Allele 13 16 2 0.125 12%

Allele 14 16 2 0.125 12%




Joe (s)

14 14

Evidence 14, 14 Allele

Name

Allele

Frequency

Allele 4 0.0625

Allele 6 0.0625

e e .

Allele 8 0.3125

Allele 12 0.1875

Allele 13 0.125

Allele 14 0.125=(0.0125)2 =0.125 X 0.125 = 0.0156

Or 1 in 64

Homozygote: p2

Heterozygote: 2p ApB

p2


Joe (s)

4 6

Evidence 4, 6 Allele

Name

Allele

Frequency

Allele 4 0.0625

Allele 6 0.0625

e e .

Allele 8 0.3125

Allele 12 0.1875

Allele 13 0.125

Allele 14 0.125

Homozygote: p2

Heterozygote: 2p ApB

=2 X (0.0625) (0.0625)= 0.0078125

Or 1 in 128

2p ApB

Locus allele value allele value 1 in Combined

D3S1358 16 0.2533 17 0.2152 9.17 9.17

VWA 17 0.2815 18 0.2003 8.87 81

FGA 21 0.1854 22 0.2185 12.35 1005

D8S1179 12 0.1854 14 0.1656 16.29 16,364

D21S11 28 0.1589 30 0.2782 11.31 185,073

Where do these Astronomical Numbers come from?

. . . , ,

D5S818 12 0.3841 13 0.1407 9.25 44,818,259

D13S317 11 0.3394 14 0.0480 3 0. 69 1 .3 8 x 1 09

D7S820 9 0.1772 31.85 4.38 x 1010

D16S539 9 0.1126 11 0.3212 1 3. 8 6 .0 5 x 1011

THO1 6 0.2318 18.62 1.13 x 1013

TPOX 8 0.5348 3 .50 3 .94 x 1 013

CSF1PO 10 0.2169 21.28 8.37 x 1014

The Random Match Probability for this profile in the U.S. Caucasian

population is 1 in 837 trillion (1012)



Where do these Astronomical Numbers come from?

Sample Uniqueness in 4 NC Populations

Homozygote: p i2 + p i (1 - p i)Ø

Heterozygote: 2PiP j

Formulas used by most laboratories to calculate profile

uniqueness or RMP:

Homozygote: p i2

Heterozygote: 2PiP j

or

NC Crime Lab

Case Review Recommendations:

DNA Profiles, Allelic Calls, Peak Heights, Alternative Hypotheses, etc

Evaluate the Results / Interpretations (Match, non-exclusion, exclusion, etc)

DNA Sample: Collection to Analysis to Results, Contamination

Quality Control, Quality Assurance, Audits

Chain of Custody

Other case documents, pictures, etc

Is there anything that does not make sense???

Case Analysi s (1)

13%



Case Analysi s (2)

Case Analysi s (3)

Is this a mixture?

Case Analysi s (4)

Given the above profile, if a defendant

has allele (22) at Marker FGA, should t hat

person be excluded?



Perpetrator’s sperm

Centrifuge

REMOVE

SDS, EDTA and

proteinase K(cell lysis buffer)

Incubate at37 oC

Differential Extraction: Sexual Assault Cases

mixed with victim’sepithelial cells

supernatant

SDS, EDTA and

proteinase K + DTT

‘Sperm Fraction’

‘Non-sperm Fraction’

sperm

pelletModified from: Butler, J; NIST

Case Analysi s (5)

Questions…

Documents

Challenges in Dealing With DNA Evidence (Noureddine)