Latest Tools for Human Identification

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 United States 1 800 603 9259 Standard International Dial-in 706 645 9105 And ask for conference ID # 2865329

Latest Tools for Y Chromosome STR Analysis Lisa Calandro, Senior Forensic Scientist Applied Markets Division

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

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

Application of Y STR Analysis Paternity test: fatherless cases Y STR analysis can provide evidence of paternity through linkage to paternal relatives Father?

Application of Y STR Analysis Paternity test: motherless cases Father? 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

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

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

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

Screen for semen Few/No Spermatozoa Many Spermatozoa Quantifiler/ Quantifiler Y kit/ Quantifiler Analysis Y kit Analysis No Differential Differential Non-Sperm Fraction Sperm Fraction No Male DNA Low Male DNA >1:10 Male DNA Quantifiler kit / Quantifiler Y kit Analysis Y STR Analysis Autosomal STR Analysis Male DNA Detected No Male DNA Detected Sexual Assault Case Workflow Y STR Analysis Autosomal STR Analysis

Integration of Quantifiler Assays Provide 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

Quantifiler Primer Mix Pre-Formulated as A Duplex Assay FAM TM dye R MGB/NFQ Q Target Probe to Autosomal or Y-Chromosome Specific Region VIC dye R MGB/NFQ Q Internal PCR Control Simultaneous amplification and detection in the same well

PCR Amplification Plot Smaller the C T number the more DNA detected FAM Dye signal from sample C T value = 27.8 Threshold

QuantifilerAssay Internal PCR Control-Hematin inhibition study 0 µm 10µM 12µM 14µM 16, 18, 20, & 40µM

Quantifiler Assay Example of a sample that may be inhibited FAM dyesignal from sample below threshold Threshold VIC dye signal from internal PCR control below threshold

Quantifiler Assay Example of a Sample with no Detectable DNA VIC dye signal from PCR control Threshold FAM dye signal from sample

Quantifiler Assay Example of a Positive Sample FAM dye signal from sample Threshold VIC dye signal from PCR control

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

QuantifilerAssay Male:Female Mixture Samples Quantifiler Assays Results with Male:Female DNA Mixtures 100.000 10.000 DNA Qty (ng/ul) 1.000 0.100 Quantifiler Y Quantifiler Human 0.010 1:1 1:5 1:16 1:64 1:256 1:1024 Male:Female Mixture Ratio

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

Quantifiler reactions measure the amount of amplifiable DNA in the reaction Provide a more comparative indication of subsequent (STR) analysis than other quantification methods

Male DNA Quantity vs. Yfiler Peak Height Male DNA Quantity 1.07 ng Relative Average Pk. Ht. 1x 478 pg 0.57x 265 pg 0.24x 133 pg 0.14x

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

Y STR Typing of duplicated region DYS385 a/b and DYS389 I/II DYS385a/b DYS389I/II

AmpF STR 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

AmpF STR Yfiler Kit 17 Y-STR Loci Y DYS19 DYS389I/II DYS390 European DYS391 Minimal DYS392 Haplotype DYS393 DYS385a/b DYS437 DYS438 SWGDAM DYS439 DYS448 DYS456 ABI DYS458 DYS635 Y GATA H4

AmpF STR Yfiler Kit 1 ng Male Control DNA 007 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

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

Stutter Percentages for Yfiler Kit Loci Pentanucleotide repeat Hexanucleotide repeat Tetranucleotide repeats Trinucleotide repeat Locus Stutter % 2 bp or + stutter filter % DYS438 4.28 DYS448 4.96 DYS437 8.59 DYS390 10.4 DYS635 10.75 DYS19 11.04 10.21 (2 bp) Y GATA H4 11.08 DYS439 11.18 DYS391 11.62 DYS389I 11.79 DYS458 12.2 DYS393 12.58 DYS456 13.21 DYS389II 13.85 DYS385 13.9 DYS392 16.22 7.9 (+ stutter)

AmpF STR 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

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

Interpretation of Y STR Analysis: 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

CEPH family 1340 Profile of father of 5 sons (1340-7029) DYS458-16

CEPH family 1340 Profile of son (1340-7342) with 1 mismatch at DYS458 DYS458-17

Typing Results CEPH 1340 Locus Father Son DYS456 14 14 DYS3891 12 12 DYS390 22 22 DYS389II 28 28 DYS458 16 17 DYS19 15 15 DYS385 13, 14 13, 14 DYS393 13 13 DYS391 10 10 DYS439 11 11 DYS635 22 22 DYS392 11 11 Y GATA H4 11 11 DYS437 16 16 DYS438 10 10 DYS448 20 20 Conclusion: Cannot exclude based on a single locus mismatch

Interpretation of Y STR Analysis: 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

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

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)

Population Data by Ethnicity Filipino 3% Hispanic 17% Asian 9% African American 28% Native American 3% Vietnamese 3% Black African 2% Caucasian 35%

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

Statistical Analysis of Y STRs for Forensic Applications 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

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) AmpF STR Yfiler kit (17).9995.9978.999.9999.9999.9998

Statistical Analysis of Y STRs for Forensic Applications 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

Discriminatory capacity* for three Y-STR marker combination U.S. populations 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) AmpF STR Yfiler kit (17) 87.6% 76.7% 88.2% 97.6% 95.5% 95.8% *DC= (# of different haplotypes / pop. size) x 100

Most common Haplotype identified by the European Minimal Haplotype markers (20 individuals) 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 23 C330 14 11,14 13 29 24 11 13 13 13 11 15 19 15 19 11 23 ATCC 14 11,14 13 29 24 11 13 13 12 13 15 19 15 17 13 23 C304 14 11,14 13 29 24 11 13 13 12 13 15 19 17 18 12 23 C327 14 11,14 13 29 24 11 13 13 12 13 15 19 16 16 12 24 C63 14 11,14 13 29 24 11 13 13 12 13 15 17 16 17 11 23 C177 14 11,14 13 29 24 11 13 13 12 12 15 21 17 19 12 24 C198 14 11,14 13 29 24 11 13 13 12 12 15 18 16 17 12 23 C276RL 14 11,14 13 29 24 11 13 13 12 12 15 19 15 19 12 23 C294 14 11,14 13 29 24 11 13 13 12 12 15 19 15 17 11 24 C236 14 11,14 13 29 24 11 13 13 12 12 14 19 15 18 12 23 C85RL 14 11,14 13 29 24 11 13 13 12 12 14 19 16 16 11 24 C12 14 11,14 13 29 24 11 13 13 12 11 15 19 17 18 12 23 C194 14 11,14 13 29 24 11 13 13 12 11 15 19 17 17 12 23 C197 14 11,14 13 29 24 11 13 13 12 11 15 19 15 16 >13 24 C318 14 11,14 13 29 24 11 13 13 12 11 15 19 16 17 12 23 C345 14 11,14 13 29 24 11 13 13 12 11 15 19 15 18 12 23 C66RL 14 11,14 13 29 24 11 13 13 12 11 15 19 16 19 12 23 C205 14 11,14 13 29 24 11 13 13 12 11 14 19 15 17 12 23 C158 14 11,14 13 29 24 11 13 13 12 10 15 19 16 20 13 23 European Minimal Haplotype # of different 0 haplotypes SWGDAM 6 Yfiler TM Kit 20

AmpF STR 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

AmpF STR Yfiler Kit Haplotype Database User Interface Options 1. Drop-down allele selections 2. Custom entry (recommended for microvariants) 3. Automatic upload of allele tables http://www.appliedbiosystems.com/yfilerdatabase/

AmpF STR Yfiler Kit Haplotype Database

Microvariant Example Concordant with NIST 20plex and 11plex data

DYS456 DYS389I DYS390 DYS389II DYS458 Duplication of DYS389I region DYS19 DYS385 a/b Duplication of DYS389II region DYS393 DYS391 DYS439 DYS635 DYS392 Y GATA H4 Duplication of DYS439 region DYS437 DYS438 DYS448 Duplication of DYS437 region

Example of Microvariant Not Amenable to Database Search 3 alleles

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

Yfiler Kit Haplotype Database File Upload

Input Batch Haplotypes from GeneMapper ID Software v3.2

Haplotype Search Result

Yfiler Kit Haplotype Database Results Print Screen

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

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