latest tools for human identification...latest tools for human identification lisa calandro, senior...

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Latest Tools for Human Identification

Lisa Calandro, Senior Forensic Scientist Applied Markets Division

To hear the audio portion of this seminar, please call the phone number for your country:Australia-1800002092 Austria-0 800281475 Belgium-0 80074215 Canada-1-800-603-9259 China-1 0 8001400431 Finland-0 800115347 France-0 800900053 Germany-0 8001815287 Hong Kong-800901700 Italy-800783240 Japan-0 0531160205 Malaysia-1800805439 Netherlands-0 8000224103 New Zealand-0 800445971 Norway 80 016162 Singapore-8001011350 South Korea-0 0308140327 Spain-900971520 Sweden-0 20791231 Switzerland-0 800835819 Taiwan- 02-2192-2576 Thailand-0 0 18001562037877 United Kingdom-0 8009174860

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And ask for conference ID # 2865329

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Latest Tools for Y Chromosome STR Analysis

Lisa Calandro, Senior Forensic ScientistApplied Markets Division

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©2004 Applied Biosystems All Rights Reserved

Important Aspects of Y STR Analysis

• Why Y STR Analysis?

• Screening samples prior to Y STR analysis

• Application of Y STR Analysis– Sexual assault crimes

– Male:female mixtures

– Paternity/lineage testing

• Interpretation of Y STR Results

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Why Y STR Analysis?

• Paternity applications– Identical by descent from father to son– Useful for male children only

• Lineage applications– Can establish paternal relationships along

family tree• Evolutionary studies

– Defining markers for population differentiation– Y STRs are more sensitive to genetic drift

resulting in statistically significant differences between populations

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Application of Y STR Analysis Paternity test: fatherless cases

• Y STR analysis can provide evidence of paternity through linkage to paternal relatives

Father ?

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Application of Y STR Analysis Paternity test: motherless cases

• Can supplement weaker paternity indices when obligate paternal allele is in doubt by providing additional evidence of paternity

• No need to identify obligate paternal allele with Y STR analysis

Father ?

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Why Y STR Analysis?

• Male specific – reduced competition for amplification from female DNA

• Solution for incomplete separation of male and female components in sexual assault cases

• Verification of gender – amelogenin mutation

• Solution for mixtures containing– Semen from vasectomized or azoospermic

males– Multiple male semen donors– Non-semen body fluid mixtures

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Application of Y STR Analysis Sexual assault crimes• Autosomal DNA testing of sexual assault

evidence often involves complex mixture analysis due to low contribution of male DNA

Differential extraction may result in carryover of female DNA into the sperm fraction

Competition between male and female DNA results in limited ability to detect minor male component in mixtures – typically restricted to ratios of <1:10-1:20

Partial profiles and/or masking of alleles are not uncommon

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Limitations of Y STR Analysis

• Lack of independence due to absence of recombination on Y chromosome

• Statistical power of inclusion is much lower than autosomal STR analysis– Cannot apply the product rule across Y STRs

due to linkage– Much greater capability of resolving two

individuals using autosomal STR analysis• Male relatives not excluded as potential

contributors

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Screen for semen

Few/No Spermatozoa

Quantifiler ™ kit /Quantifiler ™ Y kit

Analysis

Quantifiler/Quantifiler Y

Analysis

No Male

DNALow Male DNA

>1:10 Male DNA

Y STR Analysis

Autosomal STR Analysis

No Differential

Many Spermatozoa

Non-Sperm Fraction

Sperm Fraction

Differential

Quantifiler™ kit/Quantifiler ™ Y kit

Analysis

Male DNADetected

No Male DNA Detected

Y STR Analysis

Autosomal STR AnalysisSexual Assault Case

Workflow

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Integration of Quantifiler ™ AssaysProvide information regarding the nature of forensic DNA samples before amplification:

•Are inhibitors present?

•Interpretation of Internal PCR Control

•Is male DNA present?

•Specific amplification of a target found on the Y chromosome allows determination of amplifiable male DNA

•What is the ratio of male:female DNA?

•Dual interpretation of Quantifiler Human vs. Quantifiler Y quantification results

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Quantifiler™ Primer Mix Pre-Formulated as A Duplex Assay

Simultaneous amplification and detection in the same well

FAMTM dyeTarget Probe toAutosomal or Y-ChromosomeSpecific Region

VIC® dye

Internal PCR Control

MGB/NFQ

MGB/NFQR

Q

RQ

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PCR Amplification Plot

CT value = 27.8

Threshold

FAM™ Dye signalfrom sampleSmaller the CT number

the more DNA detected

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Quantifiler™Assay Internal PCR Control-Hematin inhibition study

0 µM10µM

12µM14µM

16, 18, 20, & 40µM

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FAM™ dye signal from sample below threshold

VIC® dye signal from internal PCR control

below threshold

Threshold

Quantifiler™ Assay Example of a sample that may be inhibited

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Threshold

FAM™ dye signalfrom sample

VIC® dye signalfrom PCR control

Quantifiler™ Assay Example of a Sample with no Detectable DNA

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Threshold

FAM™ dye signalfrom sample

VIC® dye signalfrom PCR control

Quantifiler™ Assay Example of a Positive Sample

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The combined use of Quantifiler™ Human and Y kits allows the analyst to

determine the Female/Male ratio

Low ratio of Female/Male:Autosomal STR analysis

High ratio of Female/Male:Y-STR analysis

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Quantifiler™Assay Male:Female Mixture Samples

Quantifiler Assays Results with Male:Female DNA Mixtures

0.010

0.100

1.000

10.000

100.000

1:1 1:5 1:16 1:64 1:256 1:1024

Male:Female Mixture Ratio

DN

A Q

ty (n

g/ul

)

Quantifiler YQuantifiler Human

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Integration of Quantifiler Assays

Incorporation of Quantifiler assays into casework workflow:

•Select the route of extraction processing which will yield the best results – Differential or not?

•Determine appropriate amplification method which will yield the best results

•Autosomal analysis if enough material is available and major:minor ratio is <1:20

•Y-STR analysis if the minor contributor is not present at levels sufficient for exclusionary purposes

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Quantifiler™ reactions measure the amount of amplifiable DNA in the reaction

Provide a more comparative indication of subsequent (STR) analysis

than other quantification methods

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Male DNA Quantity vs. Yfiler™ Peak HeightMale DNA Quantity

1.07 ng

478 pg

265 pg

133 pg

Relative Average Pk. Ht.

1x

0.57x

0.24x

0.14x

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Selection of Y STRs for Forensic Applications• Single copy Y STRs

– Repeat length ranges from 2-6 bp> Decreased stutter with longer repeats> 2 bp repeats – stutter relatively high

– DYS389I/DYS389II – same reverse primer but different forward primers

• Multi-copy Y STRs– e.g. DYS385 – two PCR products

amplified with single primer set

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Y STR Typing of duplicated region DYS385 a/b and DYS389 I/II

DYS389I/IIDYS385a/b

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AmpFSTR® Yfiler™ Kit Design Goals• Employs 5-dye chemistry for detection.

– Enables analysis of more loci within a smaller size range

– Improves ability to amplify challenging samples• Utilize existing ABI PRISM® systems for

analysis• Amplification of 17 Y-STR loci in a single

PCR reaction• No detectable cross reactivity with female

DNA

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YDYS19

DYS389I/II

DYS390

DYS391

DYS392

DYS393

DYS385a/b

DYS437

DYS438

DYS439

DYS448

DYS456

DYS458

DYS635

Y GATA H4

European

Minimal

Haplotype

SWGDAM

ABI


AmpFSTR® Yfiler™ Kit 17 Y-STR Loci

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DYS456 DYS389 I DYS390 DYS389 II

DYS458 DYS19 DYS385 a/b

DYS393 DYS391 DYS439 DYS635 DYS392

Y GATA H4 DYS437 DYS438 DYS448

ABI PRISM® 3100 Genetic Analyzer Data Collection Software v.1.1

AmpFSTR® Yfiler™ Kit 1 ng Male Control DNA 007

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Y-STR gene diversity in Three U.S. Populations

Y-STR AA G.D. Cauc G.D. Hisp G.D.n=786 n=778 n=381

DYS456 0.603 0.722 0.663DYS389I 0.527 0.520 0.559DYS390 0.644 0.708 0.680DYS389II 0.747 0.676 0.730DYS458 0.753 0.777 0.782DYS19 0.747 0.500 0.666DYS385a/b 0.950 0.842 0.935DYS393 0.607 0.381 0.504DYS391 0.404 0.546 0.541DYS439 0.629 0.648 0.665DYS635 0.715 0.643 0.724DYS392 0.411 0.604 0.664Y GATA H4 0.610 0.599 0.572DYS437 0.498 0.576 0.573DYS438 0.544 0.590 0.708DYS448 0.693 0.596 0.716

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Stutter Percentages for Yfiler Kit Loci

Pentanucleotide repeatHexanucleotide repeat

Tetranucleotide repeats

Trinucleotide repeat

Locus Stutter filter %

% 2 bp or + stutter

DYS438 4.28DYS448 4.96DYS437 8.59DYS390 10.4DYS635 10.75DYS19 11.04 10.21 (2 bp)Y GATA H4 11.08DYS439 11.18DYS391 11.62DYS389I 11.79DYS458 12.2DYS393 12.58DYS456 13.21DYS389II 13.85DYS385 13.9DYS392 16.22 7.9 (+ stutter)

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AmpFSTR® Yfiler™ Kit Mixtures of male and female DNA

Male DNA input decreased, Female DNA input fixed at 500 ng

500 pg male DNA

1:1000

1:2000

1:4000

1:8000

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Interpretation of Y STR Analysis:

Exclusion• Paternity test:

– Profiles from alleged father and male child do not match

> Consider mutation

• Forensic analysis: – Profile from evidence sample and known

sample do not match

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• Average mutation rate estimated at 2.8 x 10-3 (Kayser & Sajantila, 2001)

• Additional mutational type seen in Y STRs – duplication of STR region – DYS19 – duplication frequency of 0.12%

(Kayser & Sajantila, 2001)

• Must consider possibility of mutation in cases of exclusion

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CEPH family 1340 Profile of father of 5 sons (1340-7029)

DYS458-16

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CEPH family 1340 Profile of son (1340-7342) with 1 mismatch at DYS458

DYS458-17

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Typing Results – CEPH 1340Locus Father SonDYS456 14 14DYS3891 12 12DYS390 22 22DYS389II 28 28DYS458 16 17DYS19 15 15DYS385 13, 14 13, 14DYS393 13 13DYS391 10 10DYS439 11 11DYS635 22 22DYS392 11 11Y GATA H4 11 11DYS437 16 16

DYS438 10 10DYS448 20 20

Conclusion:

Cannot exclude based on a single locus mismatch

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Inclusion• Paternity test:

– Match between alleged father and male child> Establish # of occurences of matching haplotype in

population database

• Forensic analysis:– Match between evidence sample and known

sample> Establish # of occurences of matching haplotype in

population database

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Interpretation of Y STR Analysis:Strength of Inclusions

• Sharing of Y STR haplotypes– Identical by descent – male relatives

> Relationship may not be known

– Identical by state – identical haplotypes arise by chance or through mutation

• Increasing Y STR loci increases discrimination capacity

• Increasing the number of samples in the database increases the statistical power of a match

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Yfiler™ Kit Population Study>3,500 samples analyzed and typed at our test sites

Populations include: •Caucasian – European and North American•African American •Hispanic •Filipino •Vietnamese•Asian•Native American•Sub-Saharan African (South Africa)

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Population Data by Ethnicity

Caucasian35%

Hispanic17%

Filipino3%

Asian9%

African American28%

Vietnamese3%

Black African2%

Native American3%

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Statistical Analysis of Y STRs for Forensic Applications

• Arlequin Software – Freeware developed by Stefan Schneider, David Roessli and Laurent Excoffier for genetic analysis of population data

URL: http://anthro.unige.ch/arlequin

http://anthro.unige.ch/arlequin

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• Haplotype Diversity (HD) – the probability that two haplotypes chosen at random are different

1-HD x 100 = the probability (in %) of obtaining an identical haplotype in a pair of random unrelated males – Random Match Probability

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Haplotype Diversity for Three U.S. Populations

Y-STR marker combination

African American (N=786)

Caucasians (N=778)

Hispanics (N=381)

European minimal haplotype (9) .9985 .993 .9967

Eur. Minimal + SWGDAM (11) .9994 .9972 .9985

Minimal + SWGDAM + DYS437 (12)

.9995 .9978 .999

AmpFSTR®

Yfiler™ kit (17)

.9999 .9999 .9998

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• Discrimination capacity – the frequency with which two unrelated individuals can be discriminated– Calculated by dividing the number of different

haplotypes by the total number of haplotypes

• Unique haplotype – defined by a single occurrence in the population

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Discriminatory capacity* for three U.S. populations

Y-STR marker combination

African American (N=786)

Caucasians (N=778)

Hispanics (N=381)

European minimal haplotype (9) 75.8% 61.7% 79.8%

Eur. Minimal + SWGDAM (11) 86.8% 74.3% 85.6%

Minimal + SWGDAM + DYS437 (12)

87.6% 76.7% 88.2%

AmpFSTR®

Yfiler kit (17)

97.6% 95.5% 95.8%

*DC= (# of different haplotypes / pop. size) x 100

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Most common Haplotype identified by the European Minimal Haplotype markers

(20 individuals)

European Minimal Haplotype

0# of differenthaplotypes

20YfilerTM Kit

Sample Info DYS19 1DYS385 DYS389 DYS389 DYS390 DYS391 DYS392 DYS393 DYS438 DYS439 DYS437 DYS448 DYS456 DYS458 Y GATA DYS635 C37 14 11,14 13 29 24 11 13 13 13 12 14 19 15 17 12 23C330 14 11,14 13 29 24 11 13 13 13 11 15 19 15 19 11 23ATCC 14 11,14 13 29 24 11 13 13 12 13 15 19 15 17 13 23C304 14 11,14 13 29 24 11 13 13 12 13 15 19 17 18 12 23C327 14 11,14 13 29 24 11 13 13 12 13 15 19 16 16 12 24C63 14 11,14 13 29 24 11 13 13 12 13 15 17 16 17 11 23C177 14 11,14 13 29 24 11 13 13 12 12 15 21 17 19 12 24C198 14 11,14 13 29 24 11 13 13 12 12 15 18 16 17 12 23C276RL 14 11,14 13 29 24 11 13 13 12 12 15 19 15 19 12 23C294 14 11,14 13 29 24 11 13 13 12 12 15 19 15 17 11 24C236 14 11,14 13 29 24 11 13 13 12 12 14 19 15 18 12 23C85RL 14 11,14 13 29 24 11 13 13 12 12 14 19 16 16 11 24C12 14 11,14 13 29 24 11 13 13 12 11 15 19 17 18 12 23C194 14 11,14 13 29 24 11 13 13 12 11 15 19 17 17 12 23C197 14 11,14 13 29 24 11 13 13 12 11 15 19 15 16 >13 24C318 14 11,14 13 29 24 11 13 13 12 11 15 19 16 17 12 23C345 14 11,14 13 29 24 11 13 13 12 11 15 19 15 18 12 23C66RL 14 11,14 13 29 24 11 13 13 12 11 15 19 16 19 12 23C205 14 11,14 13 29 24 11 13 13 12 11 14 19 15 17 12 23C158 14 11,14 13 29 24 11 13 13 12 10 15 19 16 20 13 23

6SWGDAM



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AmpFSTR® Yfiler™ Kit Haplotype Database

• Internet-based haplotype search tool• Search results for entire database or

broken down by ethnic group• Accepts GeneMapper® ID and Genotyper®

Software export formats or manual input of data

• Allows search of haplotypes containing microvariant alleles

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User Interface Options1. Drop-down allele selections

2. Custom entry (recommended for microvariants)

3. Automatic upload of allele tables

http://www.appliedbiosystems.com/yfilerdatabase/

AmpFSTR® Yfiler™ KitHaplotype Database

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AmpFSTR® Yfiler™ Kit Haplotype Database

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Concordant with NIST 20plex and 11plex data

Microvariant Example

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DYS439

DYS389IIDYS456 DYS389I DYS390

DYS458 DYS19DYS385 a/b

DYS393 DYS391 DYS635 DYS392

Y GATA H4 DYS437 DYS438 DYS448

Duplication of DYS439 region

Duplication of DYS389II region

Duplication of DYS389I region

Duplication of DYS437 region

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Example of Microvariant Not Amenable to Database Search

3 alleles

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Creating a GeneMapper® ID Software v3.2 table for batch upload of samples to the

Yfiler™ Haplotype Database

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Yfiler™ Kit Haplotype DatabaseFile Upload

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Input Batch Haplotypes from GeneMapper® ID Software v3.2

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Haplotype Search Result

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Yfiler™ Kit Haplotype Database Results Print Screen

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Summary• Y STR analysis represents a valuable new tool for

the resolution of difficult paternity questions and forensic cases involving mixtures of male and female DNA

• The Quantifiler™ Human and Quantifiler™ Y Quantification kits provide information about forensic samples which can be used to make informed decisions about subsequent analyses

• The AmpFℓSTR Yfiler PCR Amplification kit represents a highly discriminating means of specifically targeting male DNA

• The online Yfiler Kit Haplotype Database provides a means of estimating the frequency of occurrence of a haplotype detected using the Yfiler kit

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AmpFlSTR Yfiler kits -

For Research, Forensic or Paternity Use Only. Not for use in diagnostic procedures.

The PCR process is covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd.

Quantifiler Kits -


The PCR process and 5' nuclease process are covered by patents owned by Roche Molecular Systems, Inc. and F. Hoffmann-La Roche Ltd.

GeneMapper ID software-


Notice to Purchaser:

Purchase of this software product alone does not imply any license under any process, instrument or other apparatus, system, composition, reagent or kit rights under patent claims owned or otherwise controlled by Applera Corporation, either expressly, or by estoppel.

GeneMapper ID Software version 3.2 has undergone a verification process defined by Applied Biosystems. However, human identification laboratories analyzing forensic, paternity, databasing, and single-source samples that choose to use GeneMapper ID Software for data analysis should perform their own appropriate validation studies.

Genotyper Software -

For Research Use Only. Not for use in diagnostic procedures.

Notice to Purchaser: License Disclaimer

Purchase of this software product alone does not imply any license under any process, instrument or other apparatus, system, composition, reagent or kit rights under patent claims owned or otherwise controlled by Applera Corporation, either expressly, or by estoppel.

Applied Biosystems, ABI PRISM, AMPFLSTR, VIC, GeneMapper, and Genotyper are registered trademarks and AB (Design), Applera, FAM, iScience (Design), Quantifiler, and Yfiler are trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries. All other trademarks are the sole property of their respective owners.

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