doug raiford lesson 15. every cell has identical dna if know the sequence of a suspect can compare...

Doug RaifordLesson 15

Every cell has identical DNA If know the sequence of a suspect

can compare to evidence

But wait…

Do we have to sequence the entire genome of the

individual?

Do we have to sequence the entire genome of the

individual?

04/21/23 2DNA forensic evidence

Only small portions Portions where there are a small

number of differing alleles

What was an allele, again?

04/21/23 3DNA forensic evidence

•One of a series of different forms of a gene•But not really a gene—more of a location

•Locus (plural: loci)

•One of a series of different forms of a gene•But not really a gene—more of a location

•Locus (plural: loci)

One that varies in the number of Short Tandem Repeats

Describes a type of DNA polymorphism that: Repeats And has a short (usually 4 base pair) repeat unit

Length polymorphism: alleles differ in their number of repeats

04/21/23 4DNA forensic evidence

5 repeats: AATG AATG AATG AATG AATG

6 repeats: AATG AATG AATG AATG AATG AATG

4 repeats: AATG AATG AATG AATG

3 repeats: AATG AATG AATG

If given a locus: say, Chromosome 3, sequence 1358 (D3S1358)

And given the following four alleles 3,4,5,6

And given that there are two chromatids

04/21/23 DNA forensic evidence 5

5 repeats: AATG AATG AATG AATG AATG

6 repeats: AATG AATG AATG AATG AATG AATG

4 repeats: AATG AATG AATG AATG

3 repeats: AATG AATG AATG

A person might exhibit 2 different alleles (for

instance, the 4 and 5 alleles of the D3S1358

Locus)

A person might exhibit 2 different alleles (for

instance, the 4 and 5 alleles of the D3S1358

Locus)

Alleles 4 and

5

What if a test indicated a person only had one allele?

04/21/23 DNA forensic evidence 6

5 repeats: AATG AATG AATG AATG AATG

6 repeats: AATG AATG AATG AATG AATG AATG

4 repeats: AATG AATG AATG AATG

3 repeats: AATG AATG AATG

Just Allele

4

DQ-alpha (specific gene)

TEST STRIPAllele = BLUE

DOT

DQ-alpha (specific gene)

TEST STRIPAllele = BLUE

DOT

Restriction fragment

length polymorphism

(RFLP)AUTORAD

Allele = BAND

Restriction fragment

length polymorphism

(RFLP)AUTORAD

Allele = BAND

Automated STRELECTROPHEROGRAM

Allele = PEAK

Automated STRELECTROPHEROGRAM

Allele = PEAK

Differential extraction in sex assault cases separates out DNA from sperm cells

• Extract and purify DNA

• If have a suspect get Reference Sample

04/21/23 8DNA forensic evidence

Know the regions upstream and downstream of the STRs

DNA regions flanked by primers are amplified

Groups of amplified STR products are labeled with different colored dyes (blue, green, yellow)

•Amplified STR DNA injected onto column

•Electric current applied

•DNA separated out by size:

– Large STRs travel slower

– Small STRs travel faster

•DNA pulled towards the positive electrode

•Color of STR detected and recorded as it passes the detector

DetectorWindow

04/21/23 12DNA forensic evidence

04/21/23 13DNA forensic evidence

0.222 x 0.222 x 2

= 0.1

04/21/23 14DNA forensic evidence

= 0.1

1 in 79,531,528,960,000,000

1 in 80 quadrillion

1 in 10 1 in 111 1 in 20

1 in 22,200

x x

1 in 100 1 in 14 1 in 81

1 in 113,400

x x

1 in 116 1 in 17 1 in 16

1 in 31,552

x x

04/21/23 15DNA forensic evidence

04/21/23 DNA forensic evidence 16

Random Match Probability

Combined DNA Index System (CODIS)

FBI Database of profiles

Very compact Each entry has

information about the individual and which alleles at each locus04/21/23 DNA forensic evidence 17

Usually, sample from crime scene and sample from suspect are sent to the crime lab at the same time

04/21/23 DNA forensic evidence 18

Many samples are in the form of mixtures E.g. multiple assailants in a rape case

Usually state that “can’t rule out the suspect”

Sometimes still publish the random match probability

Is this right?

04/21/23 DNA forensic evidence 19

04/21/23 20Expression Prediction with CUB

• Two samples really do have the same source

Samples match coincidentallyAn error has occurred

04/21/23 21DNA forensic evidence

04/21/23 22DNA forensic evidence

Can “Tom” be excluded?

SuspectD3 vWA FGATom 17, 17 15, 17 25, 25

04/21/23 23DNA forensic evidence

Can “Tom” be excluded?

SuspectD3 vWA FGATom 17, 17 15, 17 25, 25

No -- the additional alleles at D3 and FGA are “technical artifacts.”

04/21/23 24DNA forensic evidence

Can “Dick” be excluded?

SuspectD3 vWA FGATom 17, 17 15, 17 25, 25Dick 12, 17 15, 17 20, 25

04/21/23 25DNA forensic evidence

Can “Dick” be excluded?

SuspectD3 vWA FGATom 17, 17 15, 17 25, 25Dick 12, 17 15, 17 20, 25

No -- stochastic effects explain peak height disparity in D3; blob in FGA masks 20 allele.

04/21/23 26DNA forensic evidence

Can “Harry” be excluded?

SuspectD3 vWA FGATom 17, 17 15, 17 25, 25Dick 12, 17 15, 17 20, 25Harry 14, 17 15, 17 20, 25

No -- the 14 allele at D3 may be missing due to “allelic drop out”; FGA blob masks the 20 allele.

04/21/23 27DNA forensic evidence

Can “Sally” be excluded?

SuspectD3 vWA FGATom 17, 17 15, 17 25, 25Dick 12, 17 15, 17 20, 25Harry 14, 17 15, 17 20, 25Sally 12, 17 15, 15 20, 22

No -- there must be a second contributor; degradation explains the “missing” FGA allele.

04/21/23 28DNA forensic evidence

04/21/23 29DNA forensic evidence

• What is signal and what is noise?• Distinguish between “real peaks”

and technical artifacts Deducing the number of

contributors to mixtures Accounting for relatives• Determine measurement variability

04/21/23 30DNA forensic evidence

• “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

04/21/23 31DNA forensic evidence

μb

μb + 3σb

μb + 10σb

Mean backgroundSignal

Detection limit

Quantification limit

Measu

red

sig

nal (I

n V

olt

s/R

FUS

/etc

)

Saturation

0

04/21/23 32DNA forensic evidence

04/21/23 33DNA forensic evidence

0

50

100

150

200

250

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

RFU

Co

un

t

04/21/23 34DNA forensic evidence

Positive Control b b b + 3b b + 10b

Maximum 6.7 6.9 27.4 75.7 Average 5.0 3.7 16.1 42.0 Minimum 3.7 2.4 10.9 27.7

Negative Control b b b + 3b b + 10b

Maximum 13.4 13.2 53.0 145.4 Average 5.4 3.9 17.1 44.4 Minimum 4.0 2.6 11.8 30.0

Reagent Blank b b b + 3b b + 10b

Maximum 6.5 11.0 39.5 116.5 Average 5.3 4.0 17.3 45.3 Minimum 4.0 2.6 11.8 30.0

All three controls averaged b b b + 3b b + 10b

Maximum 7.1 7.3 29.0 80.1 Average 5.2 3.9 16.9 44.2 Minimum 3.9 2.5 11.4 28.9

Average (b) and standard deviation (b) values with corresponding

LODs and LOQs from positive, negative and reagent blank controls in 50 different runs. BatchExtract: ftp://ftp.ncbi.nlm.nih.gov/pub/forensics/04/21/23 35DNA forensic evidence

04/21/23 36DNA forensic evidence

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). From Gilder et al., J. For. Sci, 2007, 52:97-101.

04/21/23 37DNA forensic evidence

• What is signal and what is noise?• Distinguish between “real peaks”

and technical artifacts Deducing the number of

contributors to mixtures Accounting for relatives• Determine measurement variability

04/21/23 38DNA forensic evidence

• Stutter peaksPull-up (bleed through)Spikes and blobs

04/21/23 39DNA forensic evidence

04/21/23 40DNA forensic evidence

0

20

40

60

80

100

120

140

0 1000 2000 3000 4000 5000

Primary peak ht. (RFUs)

n+

4 s

tutt

er

pe

ak

ht.

(R

FU

s)

Primary peak height vs. n+4 stutter peak height. Evaluation of 37 data points, R2=0.293, p=0.0005. From 224 reference samples in 52 different cases. A filter of 5.9% would be conservative. Rowland and Krane, accepted with revision by JFS.04/21/23 41DNA forensic evidence

Advanced Classic04/21/23 42DNA forensic evidence

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

0

5000

10000

15000

20000

25000

30000

0 500 1000 1500 2000 2500 3000 3500 4000

Peak height (in RFUs)

Pe

ak

are

a

04/21/23 43DNA forensic evidence

• What is signal and what is noise?• Distinguish between “real peaks”

and technical artifacts• Deducing the number of

contributors to mixtures Accounting for relatives• Determine measurement variability

04/21/23 44DNA forensic evidence

04/21/23 45DNA forensic evidence

How many contributors to a mixture if analysts can discard a locus?

Maximum # of alleles observed in a 3-person mixture # of occurrences Percent of cases

2 0 0.00

3 78 0.00

4 4,967,034 3.39

5 93,037,010 63.49

6 48,532,037 33.12

There are 146,536,159 possible different 3-person mixtures of the 959 individuals in the FB I database (Paoletti et al., November 2005 JFS).

3,398

7,274,823

112,469,398

26,788,540

0.00

4.96

76.75

18.28

How many contributors to a mixture if analysts can discard a locus?

Maximum # of alleles observed in a 3-person mixture # of occurrences Percent of cases

2 0 0.00

3 310 0.00

4 2,498,139 5.53

5 29,938,777 66.32

6 12,702,670 28.14

There are 45,139,896 possible different 3-person mixtures of the 648 individuals in the MN BCI database (genotyped at only 12 loci).

8,151

1,526,550

32,078,976

11,526,219

0.02

3.38

71.07

25.53

Maximum # of alleles observed in a 4-person mixture # of occurrences Percent of cases

4 13,480 0.02

5 8,596,320 15.03

6 35,068,040 61.30

7 12,637,101 22.09

8 896,435 1.57

There are 57,211,376 possible different 4-way mixtures of the 194 individuals in the FB I Caucasian database (Paoletti et al., November 2005 JFS). (35,022,142,001 4-person mixtures with 959 individuals.)

0%

10%

20%

30%

40%

50%

60%

70%

80%

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170

Number of additional simulated loci

Mis

char

acte

riza

tio

n r

ate

(%)

Five simulations are shown with each data point representing 57,211,376 4-person mixtures (average shown in black). (Paoletti et al., November 2005 JFS). Mischaracterization rate of 76.34% for original 13 loci.

04/21/23 49DNA forensic evidence

• What is signal and what is noise?• Distinguish between “real peaks”

and technical artifacts Deducing the number of

contributors to mixtures• Accounting for relatives• Determine measurement variability

04/21/23 50DNA forensic evidence

0%

2%

4%

6%

8%

10%

12%

14%

16%

18%

20%

2 4 6 8 10 12 14 16 18 20 22 24

Number of pairwise shared alleles

Per

cen

t o

f to

tal (

%)

Randomized Individuals

Simulated Cousins

Simulated Siblings

04/21/23 51DNA forensic evidence

• Original FBI dataset’s mischaracterization rate for 3-person mixtures (3.39%) is more than two above the average observed in five sets of randomized individuals

• Original FBI dataset has more shared allele counts above 19 than five sets of randomized individuals (3 vs. an average of 1.4)

04/21/23 52DNA forensic evidence

• Maximum allele count by itself is not a reliable predictor of the number of contributors to mixed forensic DNA samples.

• Simply reporting that a sample “arises from two or more individuals” is reasonable and appropriate.

• Analysts should exercise great caution when invoking discretion.

• Excess allele sharing observed in the FBI allele frequency database is most easily explained by the presence of relatives in that database.

04/21/23 53DNA forensic evidence

Database search yields a close but imperfect DNA match

Can suggest a relative is the true perpetrator

Great Britain performs them routinely

Reluctance to perform them in US since 1992 NRC report

Current CODIS software cannot perform effective searches

04/21/23 54DNA forensic evidence

Search for rare alleles (inefficient)

Count matching alleles (arbitrary)

Likelihood ratios with kinship analyses

04/21/23 55DNA forensic evidence

0%

2%

4%

6%

8%

10%

12%

14%

16%

18%

20%

2 4 6 8 10 12 14 16 18 20 22 24

Number of pairwise shared alleles

Per

cen

t o

f to

tal (

%)

Randomized Individuals

Simulated Cousins

Simulated Siblings

04/21/23 56DNA forensic evidence

Given a closely matching profile, who is more likely to match, a relative or a randomly chosen, unrelated individual?

Use a likelihood ratio )|(

|

randomEP

relativeEPLR

04/21/23 57DNA forensic evidence

HF = 1 for homozygous loci and 2 for heterozygous loci; Pa is the frequency of the allele shared by the evidence sample and the individual in a database.

2,4

1

1,4

0,4

)|(

sharedifHFPPPP

sharedifHFPPP

sharedifHFPP

sibEP

baba

bab

ba

04/21/23 58DNA forensic evidence

HF = 1 for homozygous loci and 2 for heterozygous loci; Pa is the frequency of the allele shared by the evidence sample and the individual in a database.

2,2

1,2

0,0

)/|(

sharedifPP

sharedifP

sharedif

childparentEP

ba

b

04/21/23 59DNA forensic evidence

Cousins:

2,8

6

1,8

6

0,8

6

)|(

sharedifHFPPPP

sharedifHFPPP

sharedifHFPP

cousinsEP

baba

bab

ba

2,4

2

1,4

2

0,4

2

)//|(

sharedifHFPPPP

sharedifHFPPP

sharedifHFPP

HSAUNNGGEP

baba

bab

baGrandparent-grandchild; aunt/uncle-nephew-neice;half-sibings:

HF = 1 for homozygous loci and 2 for heterozygous loci; Pa is the frequency of the allele shared by the evidence sample and the individual in a database.

04/21/23 60DNA forensic evidence

• What is signal and what is noise?• Distinguish between “real peaks”

and technical artifacts Deducing the number of

contributors to mixtures Accounting for relatives• Determine measurement variability

04/21/23 61DNA forensic evidence

Can “Sally” be excluded?

SuspectD3 vWA FGATom 17, 17 15, 17 25, 25Dick 12, 17 15, 17 20, 25Harry 14, 17 15, 17 20, 25Sally 12, 17 15, 15 20, 22

Is the 12 allele at the D3 locus really 47 RFUs tall?

04/21/23 62DNA forensic evidence

Data from 18 samples, each amplified twice and with each amplification product injected two times (n = 1,316).

Amp 1: Injection 1 vs. Injection 2 y = 1.1991x - 26.84

R2 = 0.9582

0

500

1000

1500

2000

2500

0 500 1000 1500 2000 2500

First injection PH

Sec

on

d in

ject

ion

PH

04/21/23 63DNA forensic evidence

Amp 1: Injection 1 vs. Injection 2 y = 1.1991x - 26.84

R2 = 0.9582

0

500

1000

1500

2000

2500

0 500 1000 1500 2000 2500

First injection PH

Sec

on

d in

ject

ion

PH

04/21/23 64DNA forensic evidence

(1-1) vs. (2-1) and (2-2)(amplification + detection variability)

y = 1.1175x + 152.11

R2 = 0.5599y = 1.1416x + 137.42

R2 = 0.586

0

500

1000

1500

2000

2500

0 500 1000 1500 2000 2500

(1-1) PH (in RFUs)

(2-1

) an

d (

2-2)

PH

(in

RF

Us)

(1-1) vs. (2-1)

(1-1) vs. (2-2)

Linear ((1-1) vs. (2-1))

Linear ((1-1) vs. (2-2))

04/21/23 65DNA forensic evidence

--the tendency to interpret data in a manner consistent with expectations or prior theories (sometimes called “examiner bias”)

Most influential when: Data being evaluated are ambiguous or

subject to alternate interpretations Analyst is motivated to find a particular

result04/21/23 66DNA forensic evidence

04/21/23 67DNA forensic evidence

04/21/23 68DNA forensic evidence

04/21/23 69DNA forensic evidence

DNA Lab Notes (Commonwealth v. Davis) “I asked how they got their suspect. He is a

convicted rapist and the MO matches the former rape…The suspect was recently released from prison and works in the same building as the victim…She was afraid of him. Also his demeanor was suspicious when they brought him in for questioning…He also fits the general description of the man witnesses saw leaving the area on the night they think she died…So, I said, you basically have nothing to connect him directly with the murder (unless we find his DNA). He said yes.”

04/21/23 70DNA forensic evidence

DNA Lab Notes “Suspect-known crip gang member--keeps

‘skating’ on charges-never serves time. This robbery he gets hit in head with bar stool--left blood trail. Miller [deputy DA] wants to connect this guy to scene w/DNA …”

“Death penalty case! Need to eliminate Item #57 [name of individual] as a possible suspect”

04/21/23 71DNA forensic evidence

Resolve ambiguous data in a manner consistent with expectations

Miss or disregard evidence of problems Miss or disregard alternative interpretations of the

data Thereby undermining the scientific validity of

conclusions See, Risinger, Saks, Thompson, & Rosenthal, The

Daubert/Kumho Implications of Observer Effects in Forensic Science: Hidden Problems of Expectation and Suggestion. 93 California Law Review 1 (2002).

04/21/23 72DNA forensic evidence

• Simply interpret evidence with no knowledge of reference samples

Minimizes subjectivity of interpretations

Forces analysts to be truly conservative in their interpretations

See, Krane et al., Sequential unmasking: a solution for context effects in DNA profiling. June, 2008 issue of the Journal of Forensic Sciences.

04/21/23 73DNA forensic evidence

04/21/23 74DNA forensic evidence

QuickTime™ and a TIFF (Uncompressed) decompressor are needed to see this picture.

What we do:Review DNA testing results

Typically work with defendants

Rely heavily upon Genophiler™

Incorporated April 2, 2002

25 reviews in 2002, more than 250 reviews in 2007 to date

04/21/23 75DNA forensic evidence

Publications Forensic Bioinformatics Website: http://www.bioforensics.com/

Collaborators Larry Mueller and Bill Thompson (UC Irvine) Simon Ford (Lexigen Inc., San Francisco, CA) William Shields (SUNY, Syracuse, NY) Sandy Zabell (Northwestern University, Chicago, IL) Travis Doom (Wright State, Dayton, OH) Marc Taylor (Technical Associates, Ventura, CA) Keith Inman (Forensic Analytical, Hayword, CA) D. Michael Risinger (Seton Hall University, South Orange, NJ) Allan Jamieson (The Forensics Institute, Glasgow, UK)

Testing laboratories Technical Associates (Ventura, CA) Forensic Analytical (Hayword, CA) Indiana State Police Laboratory (Indianapolis, IN)

04/21/23 76DNA forensic evidence

Toddler disappears in bizarre circumstances: found dead six months later

Mother’s boy friend is tried and acquitted.

Unknown female profile on clothing.

Cold hit to a rape victim.

RMP: 1 in 227 million.

Lab claims “adventitious match.”

04/21/23 77DNA forensic evidence

Condom with rape victim’s DNA was processed in the same lab 1 or 2 days prior to Leskie samples.

Additional tests find matches at 5 to 7 more loci.

Review of electronic data reveals low level contributions at even more loci.

Degradation study further suggests contamination.

04/21/23 78DNA forensic evidence

When biological samples are exposed to adverse environmental conditions, they can become degraded Warm, moist, sunlight, time

Degradation breaks the DNA at random Larger amplified regions are affected first Classic ‘ski-slope’ electropherogram Degradation and inhibition are unusual and noteworthy.

LARGE

SMALL

04/21/23 79DNA forensic evidence

The Leskie Inquest, a practical application

Undegraded samples can have “ski-slopes” too.

How negative does a slope have to be to an indication of degradation?

Experience, training and expertise.

Positive controls should not be degraded.

04/21/23 80DNA forensic evidence

The Leskie Inquest

DNA profiles in a rape and a murder investigation match.

Everyone agrees that the murder samples are degraded.

If the rape sample is degraded, it could have contaminated the murder samples.

Is the rape sample degraded?

04/21/23 81DNA forensic evidence

The Leskie Inquest

04/21/23 82DNA forensic evidence

“8. During the conduct of the preliminary investigation (before it was decided to undertake an inquest) the female DNA allegedly taken from the bib that was discovered with the body was matched with a DNA profile in the Victorian Police Forensic Science database. This profile was from a rape victim who was subsequently found to be unrelated to the Leskie case.”

04/21/23 83DNA forensic evidence

“8. The match to the bib occurred as a result of contamination in the laboratory and was not an adventitious match. The samples from the two cases were examined by the same scientist within a close time frame.”

www.bioforensics.com/articles/Leskie_decision.pdf

04/21/23 84DNA forensic evidence

04/21/23 85DNA forensic evidence

04/21/23 86DNA forensic evidence

04/21/23 87DNA forensic evidence