Developmental Validation of a Y-STR Multiplex · 2020-03-12 · Lisa Calandro, Cherisse Boland,...

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Developmental Validation of a Y-STR Multiplex

Julio Mulero, Ph.D.Senior Scientist

Human Identification GroupApplied Biosystems

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©2004 Applied Biosystems All Rights Reserved

Components of an Integrated System for Y STR Analysis

AmplificationQuantification

Quantification

Quantifiler ™Y Male Specific

DNA Kit

Amplification

Amplification

AmpFSTR®

Yfiler™ PCR Kit

Electrophoresis

ABI PRISM ®3100

3100-Avant310

Data Analysis

GeneMapper® ID v3.2 or

GeneScan® SoftwareGenotyper ® Software

Estimating Frequency

EstimatingFrequency

Web-Based Haplotype Database

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• Amplification of all loci in a single PCR reaction.

• Employ 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® Sequence Detection Systems for analysis

• No detectable cross reactivity with female DNA.

AmpFSTR® Yfiler™ KitDesign Goals

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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™ Kit17 Y-STR Loci


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Criteria for Selection of Additional Y-STR Markers

• Highly polymorphic– Shown to provide additional power

of discrimination in population studies.

• Well characterized

• Small amplicon size

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Y-STR gene diversity in 148 European-Americans

Y-STR Gene DiversityDYS385 0.823DYS458 0.771DYS456 0.740DYS390 0.732DYS439 0.666DYS448 0.603DYS437 0.584DYS392 0.579DYS19 0.557DYS391 0.533DYS389I 0.521DYS389II 0.501DYS393 0.258

Alan. J. Redd et al., Forensic Science Intl. 130 (2002) 97-111.

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Dye Set G5Em issio n S p e ctra o f 5 -d ye S e t

0

20

40

60

80

100

5 0 0 55 0 6 0 0 6 5 0 7 0 0

W a ve le n g th (n m )

Nor

mal

ize

emis

sion

6 FA M™ d y e V IC™ d y e NED™ d y e

PET™ d y e L IZ ™ d y e

Nor

ma l

ized

em

issi

o n

AmpFSTR® Yfiler™ Kit

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Size DistributionAmpFSTR® Yfiler™ Kit

80 130 180 230 280 330

DYS393

DYS391

DYS439

DYS635

DYS392

DYS456

DYS389I

DYS390

DYS389II

DYS458

DYS19

DYS385 a/b

GATA H4

DYS437

DYS438

DYS448

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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 v.1.1

1 ng Male Control DNA 007AmpFSTR® Yfiler™ Kit

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AmpFSTR® Yfiler™ Kit Developmental Validation

• Species specificity

• Reaction mix buffer• PCR conditions• Primer Concentrations

• AmpliTaq Gold® DNA Polymerase

• Sensitivity

• Mixture studies• Reproducibility

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Human1 ng

Gorilla1 ng

Macaque1 ng

Orangutan1 ng

Chimpanzee1 ng

AmpFSTR® Yfiler™ KitSpecies specificity-primate DNA

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Microbial Pool

Mouse10 ng

Cat10 ng

Dog10 ng

Human1 ng

AmpFSTR® Yfiler™ KitSpecies specificity-non primate

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AmpFSTR® Yfiler™ KitMagnesium Chloride Titration

+10%

+30%

-10%

-30%

Standard

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AmpFSTR® Yfiler™ KitCycle Number

30 cycles

31 cycles

28 cycles

29 cycles

32 cycles

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AmpFSTR® Yfiler™ Kit Annealing Temperature

59°C

60°C

61°C

62°C

63°C

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AmpFSTR® Yfiler™ Kit Effect of Annealing Temperature with 500 ng Female DNA

59°C

60°C

61°C

62°C

63°C

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AmpFSTR® Yfiler™ KitAmplification Parameters

Hold Cycle (x30) Hold

95 oC 94 oC 61 oC 72 oC 60 oC

11 min. 1 min. 1 min. 1 min. 80 min.

• New PCR conditions optimized for the amplificationof male DNA in a high background of Female DNA

• Final extension increased to ensure +A additionin a high background of Female DNA.

• Conditions optimal for recommended DNA input of 0.5-1.0 ng.

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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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Precise Quantification performed with QuantifilerTM Y DNA Quantification kit

AmpFSTR® Yfiler™ KitSensitivity Study

1 ng

500 pg

250 pg

125 pg

62 pg

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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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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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AmpFSTR® Yfiler™ Kit Mixtures of semen and female blood

on white cotton cloth1 ul :100 ul

1 ul : 500 ul

1 ul :1000 ul


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AmpFSTR® Yfiler™ Kit Mixtures of male and female blood

on white cotton cloth1 ul : 100 ul

1 ul : 500 ul

1 ul : 1000 ul


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3:1

Sample A1 ng

10:1

Sample B1 ng

15:1

AmpFSTR® Yfiler™ Kit Mixtures of male-male DNA

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AmpFSTR® Yfiler™ Kit ABI PRISM® 3100, 3100-Avant and 310 Genetic Analyzers

3100Genetic Analyzer

3100-Avant Genetic Analyzer

310Genetic Analyzer

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Size DeviationPrecision

‐1

‐0.5

0

0.5

1

75 125 175 225 275 325 375

Allele Size (bp)

Size Deviation (bp)

DYS19

DYS385

DYS389I

DYS389II

DYS390

DYS391

DYS392

DYS393

DYS437

DYS438

DYS439

DYS448

DYS456

DYS458

DYS635

Y GATA H4

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Genotype Concordance ABI PRISM® 3100, 3100-Avant, and 310 Genetic Analyzers

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• Validation studies completed following DAB and SWGDAM guidelines

• Amplify 17 Y STR loci in a single PCR reaction

• Detects low levels of male DNA in high background of female DNA.

• Designed to perform on Applied Biosystems thermal cyclers and CE platforms.

AmpFSTR® Yfiler™ KitSummary

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Quantification


DNA Kit

Amplification

AmpFSTR®

Yfiler™ PCR Kit

Electrophoresis

ABI PRISM ®3100

3100-Avant310

Data Analysis

GeneMapper® ID v3.2Or

GeneScan® SoftwareGenotyper® Software


EstimatingFrequency


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• GeneMapper® ID Software v3.2

• GeneScan® /Genotyper® Software

AmpFSTR® Yfiler™ KitData Analysis

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New Features of GeneMapper® ID Software v3.2

• Ability to analyze data with tri-, penta- and hexanucleotide repeat motifs

• Improved sample tracking abilities via “Combined Export” functionality

• Size standard peaks are labeled and printable

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AmpFSTR® Yfiler™Allelic ladder

137 alleles

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Analysis of Tri-, Penta- and Hexanucleotide Repeat Motifs

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GeneMapper® ID Software v.3.2 Exporting One-Line-Per-Sample

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Quantification


DNA Kit

Amplification

AmpFSTR®

Yfiler™ PCR Kit

Electrophoresis

ABI PRISM ®3100

3100-Avant310

Data Analysis

GeneMapper® ID v3.2or

GeneScan® SoftwareGenotyper® Software


EstimatingFrequency


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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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AmpFSTR® Yfiler™ KitHaplotype Database

File Upload

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AmpFSTR® Yfiler™ Kit Input Haplotype from Genotyper

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AmpFSTR® Yfiler™ Kit Haplotype Frequency

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Results Screen

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Population StudyOver 2000 samples analyzed and typed at our test sites

Populations include: •Caucasian•African American •Hispanic •Filipino •Asian•Native American

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Concordant with NIST 20plex and 11plex data

Microvariant Example

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duplication

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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Discriminatory capacity* for three U.S. populations

Y-STR marker combination

African American (N=333)

Caucasians (N=254)

Hispanics (N=175)

European minimal haplotype (9) 84.6% 74.8% 85.1%

Eur. Minimal + SWGDAM (11) 91.3% 83.8% 90.3%

Minimal + SWGDAM + DYS437 (12)

91.9% 85.8% 91.4%

AmpFSTR®

Yfiler kit (17)

99.1% 98.8% 98.3%

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

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Number of Unique Haplotypes Observed for Three U.S.

PopulationsY-STR marker

combination

African American (N=333)

Caucasians (N=254)

Hispanics (N=175)

European minimal

haplotype (9)249 162 136

Eur. Minimal + SWGDAM

(11)286 196 146

Minimal + SWGDAM + DYS437 (12)

286 202 148

AmpFSTR®

Yfiler kit (17)

327 248 169

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Integrated System for Y STR Analysis Summary

• Quantifiler™ Y Human Male DNA Quantification Kit– Allows specific quantification of male DNA in mixtures

allowing selection of appropriate STR analysis method• AmpFSTR® Yfiler™ Kit

– Amplifies 17 loci in a single PCR reaction resulting in increased discrimination capacity

• ABI PRISM® Genetic Analyzers– Components of integrated system include validated capillary

electrophoresis instruments• GeneMapper® ID Software v3.2

– Ability to analyze data with tri-, penta- and hexanucleotide repeat motifs

– Expert System candidate software • Web-based haplotype database software

– Estimate haplotype frequency

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AcknowledgementsR&D TeamJulio Mulero, Chien-Wei Chang, Lori Hennessy, Robert Green,Yurah Yen, Rixun Fang, Karen Poulter, Dennis Reeder, Manohar Furtado.

Advanced Manufacturing Development TeamChristie Hou, Yixin Li, Ian D’Silva, Merideth White, Ann Hornby, Beth Ladin.

Technical Marketing/Product MarketingLisa Calandro, Cherisse Boland, Karen Cormier, Stan Weinstein, Marco Calavetta and Mike Rechsteiner.

Oligo Production GroupDana Zugg, Kathy Wang

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•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. Hoffman-La Roche Ltd.

• Applied Biosystems Genotyper, GeneScan, GeneMapper, ABI PRISM, AmpFLSTR, Identifiler, VIC, are registered trademarks and FAM, NED, PET, AB (Design), or Quantifiler, YFiler and Applera iscience and iscience (design) are trademarks of Applera Corporation or its subsidiaries in the U.S. and certain other countries.

•TaqMan and Amplitaq Gold are registered trademarks of Roche Molecular Systems, Inc.

•Windows NT is a registered trademark of Microsoft Corporation.

•Copyright©2004 by Applied Biosystems. All Rights Reserved

Acknowledgements

Yfiler Portal TeamLucas Rix, Charles Lawrence, Maryam Norouzi, Blair Reyburn, Erin Ammon

GeneMapper® ID Software v3.2 Team Ravi Gupta, Ryan Jan, Brenda Takara, Srini Ramachandra

Special thanks to our collaborators, advisors andmore than 15 test sites worldwide!

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Components of Our Integrated System for Y STR Analysis

Quantification


DNA Kit

Amplification

AmpFSTR®

Yfiler™ PCR Kit

Electrophoresis

ABI PRISM ®3100

3100-Avant310

Data Analysis

GeneMapper® ID v3.2or

GeneScan® Software Genotyper® Software

EstimatingFrequency


Developmental Validation of a Y-STR Multiplex · 2020-03-12 · Lisa Calandro, Cherisse Boland,...

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