evaluating forensic dna evidence forensic bioinformatics () [email protected] dan e. krane...
TRANSCRIPT
![Page 1: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/1.jpg)
Evaluating forensic DNA evidence
Forensic Bioinformatics (www.bioforensics.com)[email protected]
Dan E. KraneBiological Sciences, Wright State University,
Dayton OH 45435
![Page 2: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/2.jpg)
Three generations of DNA testing
DQ-alphaTEST STRIPAllele = BLUE DOT
RFLPAUTORADAllele = BAND
Automated STRELECTROPHEROGRAMAllele = PEAK
![Page 3: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/3.jpg)
DNA in the Cell
Target Region for PCRTarget Region for PCR
chromosome
nucleus
Double stranded DNA
molecule AAAA
TTTTTTTT
CCCC
GGGGAAAA
AAAATTTT
cell
![Page 4: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/4.jpg)
DNA content of biological samples:Type of sample Amount of DNA
Blood 30,000 ng/mLstain 1 cm in area 200 ngstain 1 mm in area 2 ng
Semen 250,000 ng/mLPostcoital vaginal swab 0 - 3,000 ng
Hairpluckedshed
1 - 750 ng/hair1 - 12 ng/hair
Saliva
Urine5,000 ng/mL
1 - 20 ng/mL
2
2
![Page 5: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/5.jpg)
Basic terminology: Genetics
• DNA Polymorphism (“many forms”)– Regions of DNA which differ from person to
person • Locus (plural = loci)
– Site or location on a chromosome• Allele
– Different variants which can exist at a locus• DNA Profile
– The combination of alleles for an individual
![Page 6: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/6.jpg)
Basic terminology: Technology
• Amplification or PCR (Polymerase Chain Reaction)– A technique for ‘replicating’ DNA in the
laboratory (‘molecular Xeroxing’)– Region to be amplified defined by PRIMERS– Can be ‘color coded’
• Electrophoresis– A technique for separating molecules according
to their size
![Page 7: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/7.jpg)
STR• Short tandem repeat• Describes a type of DNA polymorphism in
which:– a DNA sequence repeats– over and over again– and has a short (usually 4 base pair) repeat
unit• A length polymorphism -- alleles differ in their
length
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
![Page 8: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/8.jpg)
Reading an electropherogramPeaks correspond to alleles
Electropherogram
D3 vWA FGA
D8 D21 D18
D5 D13 D7
BLUE
GREEN
YELLOW
RED
AmelogeninAmelogeninXX = femaleXY = male
75 100 139
150
160
200 245 300 bpsRed = ROX size standard
![Page 9: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/9.jpg)
Automated STR Test
![Page 10: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/10.jpg)
Crime Scene Samples & Reference Samples
Differential extraction in sex assault cases separates out DNA from sperm cells
• Extract and purify DNA
![Page 11: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/11.jpg)
Extract and Purify DNA
• Add primers and other reagents
![Page 12: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/12.jpg)
PCR Amplification
Groups of amplified STR products are labeled with different colored dyes (blue, green, yellow)
• DNA regions flanked by primers are amplified
![Page 13: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/13.jpg)
The ABI 310 Genetic Analyzer:SIZE, COLOR & AMOUNT
![Page 14: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/14.jpg)
ABI 310 Genetic Analyzer: Capillary Electrophoresis
•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
![Page 15: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/15.jpg)
Profiler Plus: Raw data
![Page 16: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/16.jpg)
D3 vWA FGA
D8 D21 D18
D5 D13 D7
Am
RAW DATARAW DATA
PROCESSED DATAPROCESSED DATA
•GENESCAN divides the raw data into a separate electropherogram for each color:
•Blue•Green•Yellow•Red
•GENOTYPER identifies the different loci and makes the allele calls
•The type of this sample is:–D3: 16, 17–vWA: 15, 15–FGA: 21,23–Amelogenin: X, Y–D8: 16, 16–D21: 28, 29–D18: 14, 19–D5: 8, 12–D13: 11, 13–D7: 10 10
![Page 17: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/17.jpg)
Statistical estimates: the product rule
0.222 x 0.222 x 2
= 0.1
![Page 18: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/18.jpg)
Statistical estimates: the product rule
= 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
![Page 19: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/19.jpg)
D3S1358 FGAVWA
AMEL D8S1179 D21S11 D18S51
D5S818 D13S317 D7S820
Profiler Plus
![Page 20: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/20.jpg)
D3S1358
AMEL
D7S820
D16S539
TH01TPOX CSF1PO
Cofiler
![Page 21: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/21.jpg)
D3S1358 D16S539
VWA
AMEL
D8S1179D21S11
D18S51
D19S433
D5S818 FGA
D2S1338
TPOX
TH01 D13S317
CSF1POD7S820
Identifiler
![Page 22: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/22.jpg)
LOOKING AT A DNA REPORT
![Page 23: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/23.jpg)
Components of a DNA report
• The samples tested– Evidence samples (crime scene)– Reference samples (defendant, suspect)
• The lab doing the testing• The test used:
– Profiler Plus, Cofiler, Identifiler, mtDNA• The analyst who did the testing• Results and conclusions:
– Table of alleles– Narrative conclusions
![Page 24: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/24.jpg)
Table of alleles
• Some labs include more information than others• Usually includes information about mixed samples• May also include:
– Indication of low level results– Indication of results not reported– Relative amounts of different alleles (in mixed
samples)• No standard format
![Page 25: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/25.jpg)
Narrative conclusions
• Indicates which samples match• Includes a statistical estimate• Identifies samples as mixed• May include an ‘identity statement’ i.e., samples are from the same
source to a scientific degree of certainty (FBI)• May allude to problems (e.g. interpretative ambiguity,
contamination)
![Page 26: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/26.jpg)
Looking beneath the report
![Page 27: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/27.jpg)
Sources of ambiguity in STR interpretation
• Degradation• Allelic dropout• False peaks• Mixtures• Accounting for relatives• Threshold issues -- marginal
samples
![Page 28: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/28.jpg)
Degradation
• 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• Peaks on the right lower than peaks on the left
LARGE
SMALL
![Page 29: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/29.jpg)
Allelic Dropout
• Peaks in evidence samples all very low– Mostly below 150 rfu
• Peaks in reference sample much higher– All well above 800 rfu
• At D13S817:– Reference sample: 8, 14 – Evidence sample: 8, 8
• 14 allele has dropped out -- or has it?• Tend to see with ‘marginal samples’
1500
Evidence sampleEvidence sample
Reference sampleReference sample
150
?
![Page 30: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/30.jpg)
False peaks & machine problems
• False peaks:– Contamination– Dye blob– Electrical spikes– Pull-up
• Machine problems:– Noise– Baseline instability– Injection failures
![Page 31: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/31.jpg)
Mixed DNA samples
QuickTime™ and aPhoto - JPEG decompressor
are needed to see this picture.
![Page 32: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/32.jpg)
How many contributors to a mixture if analysts can discard a locus?
How many contributors to a mixture?
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-way mixtures of the 648 individuals in the MN BCI database.
8,151
1,526,550
32,078,976
11,526,219
0.02
3.38
71.07
25.53
![Page 33: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/33.jpg)
How many loci must have six or fewer alleles to be confident there
were only three contributors?
Nine loci are commonly used.No kit tests at more than 16 loci.Approximately 144 loci must be examined for
95% confidence that there were only 3 contributors.
4-Way Mixtures, MN Data, Average
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
70.00%
80.00%
12 24 36 48 60 72 84 96 108 120 132 144
Loci
% Misclassified
![Page 34: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/34.jpg)
Opportunities for subjective interpretation?
![Page 35: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/35.jpg)
Opportunities for subjective interpretation?
D3: 12, 17 vWA: 15, 17 FGA: 22, 26
![Page 36: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/36.jpg)
Opportunities for subjective interpretation?
![Page 37: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/37.jpg)
Opportunities for subjective interpretation?
![Page 38: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/38.jpg)
Opportunities for subjective interpretation?
![Page 39: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/39.jpg)
Opportunities for subjective interpretation?
![Page 40: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/40.jpg)
Accounting for relatives
0.00%
2.00%
4.00%
6.00%
8.00%
10.00%
12.00%
14.00%
16.00%
18.00%
20.00%
1 3 5 7 9 11 13 15 17 19 21 23
Number of Shared Alleles
Percent of Total
Synthetic
Cousins
Siblings
![Page 41: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/41.jpg)
Likelihood ratios for allele sharing:
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
70.00%
80.00%
90.00%
100.00%
1 3 5 7 9 11 13 15 17 19 21 23
Number of Shared Alleles
Likelihood
Synthetic
Cousins
Siblings
![Page 42: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/42.jpg)
• Automatically runs GeneScan and GenoTyper
• Presents all output in a web page
• Performs expert analysis to identify problems
• Generates a report detailing all testing issues
![Page 43: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/43.jpg)
![Page 44: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/44.jpg)
Genophiler output:
![Page 45: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/45.jpg)
![Page 46: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/46.jpg)
Genophiler also flags potential problems for further review:
![Page 47: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/47.jpg)
Forensic BioInformatics (bioforensics.com)
• Uses Genophiler to generate easily interpreted files
• Objectively applies analysis parameters to all samples
• Fast turn around times
• Efficiently draws attention to problems requiring further review
![Page 48: Evaluating forensic DNA evidence Forensic Bioinformatics () help@bioforensics.com Dan E. Krane Biological Sciences, Wright State University,](https://reader036.vdocuments.site/reader036/viewer/2022062618/5514cfd8550346b0338b5025/html5/thumbnails/48.jpg)
Resources
• Books– ‘Forensic DNA Typing’ by John M. Butler (Academic Press)
• Internet– Applied Biosystems Website: http://www.appliedbiosystems.com/ (see
human identity and forensics)– Promega Website: http://www.promega.com/ (see Genetic Identity)– STR base: http://www.cstl.nist.gov/biotech/strbase/ (very useful)
• Scientists– Larry Mueller (UC Irvine)– Simon Ford (Lexigen, Inc. San Francisco, CA)– William C. Thompson (UC Irvine)– William Shields (SUNY, Syracuse, NY)– Marc Taylor (Technical Associates, Ventura, CA)– Carll Ladd (Connecticut State Police)
• Testing laboratories– Technical Associates (Ventura, CA)– Forensic Analytical (Haywood, CA)
• Other resources– Forensic BioInformatics (Dayton, OH)