Development of a “Global” STR Multiplex for Human Identification Analysis

1 Life Technologies™ |

Development of a “Global” STR Multiplex for Human Identification Analysis

Lisa Schade Life Technologies


What is GlobalFiler™?

1. D.R. Hares, Expanding the CODIS core loci in the United States, Forensic Sci. Int. Genet. 6 (2012), e52-e54.

2. D.R. Hares, Addendum to expanding the CODIS core loci in the United States, Forensic Sci. Int. Genet. 6 (2012), e135.

The GlobalFiler™ Kits are our response to the worldwide expansion of forensic locus sets, recently captured in the CODIS Core Loci Working Group’s recommendation to expand the CODIS Core Loci from 13 to 20 “required” loci and 3 “highly recommended” loci

Locus CODIS (Current) Europe CODIS

(Proposed) GlobalFiler™ Kit

D13S317 X X X

D7S820 X X X

D5S818 X X X

CSF1PO X X X

D1S1656 X X X

D12S391 X X X

D2S441 X X X

D10S1248 X X X

D18S51 X X X X

FGA X X X X

D21S11 X X X X

D8S1179 X X X X

VWA X X X X

D16S539 X X X X

TH01 X X X X

D3S1358 X X X X

AMEL X X X X

D2S1338 X X X X

D19S433 X X X X

DYS391 X X

TPOX X X X

D22S1045 X X X

SE33 X X X


MINIMUM REQUIRED LOCI

Amelogenin Identifiler® & NGM SElect™ Kits

D13S317 Identifiler® Kit



CSF1PO Identifiler® Kit

D1S1656 NGM SElect™ Kit




D18S51 Identifiler® & NGM SElect™ Kits

FGA Identifiler® & NGM SElect™ Kits



VWA Identifiler® & NGM SElect™ Kits


TH01 Identifiler® & NGM SElect™ Kits


AMEL Identifiler® & NGM SElect™ Kits



DYS391 Yfiler® Kit

HIGHLY RECOMMENDED LOCI

(1) TPOX Identifiler® Kit

(2) D22S1045 NGM SElect™ Kit

(3) SE33 NGM SElect™ Kit

• Original recommendations (April 2011) included 21 loci “required” and 4 “highly recommended” for inclusion by manufacturers1

• Revised recommendations (January 2012) removed the Penta E and Penta D loci2

• The revised list includes all markers commonly used in U.S. and Europe, already found in the NGM SElect™ & Identifiler® Series Kits

• GlobalFiler™ Kits include all required and recommended loci within a single multiplex

1. D.R. Hares, Expanding the CODIS core loci in the United States, Forensic Sci. Int. Genet. (2011), doi:10.1016/j.fsigen.2011.04.01

2. D.R. Hares, Addendum to expanding the CODIS core loci in the United States, Forensic Sci. Int. Genet. (2012), doi:10.1016/j.fsigen.2012.01.003

CODIS Loci Recommendations


Customer Feedback

“With so many loci, isn’t there a risk of losing

sensitivity and having more drop out?”

“Our lab is dealing with more low level trace and touch type samples, and analysis

can be a nightmare, especially with mixtures. With even more loci, I think that

difficulty could increase exponentially.”

“We are already facing big backlogs and budget shortfalls. Having to purchase

and implement new instrument platforms is an enormous additional

hurdle.”

*Source: 2011 HID Future Trends in Forensic DNA Analysis Technology Seminar Series, Technical Lead Mtgs.


Multiplex Configuration Strategy


5-Dye Configuration Containing All Required and Highly Recommended Loci

1 2

3

4

Multiple loci > 400 bp poor performance for degraded DNA compromised sizing and resolution

1 2

3

4 5

6

7 8

Fewer Mini-STRs poor performance for

degraded DNA

Primer Redesigns less concordant


The GlobalFiler™ Kits: 6-Dye Configuration 1 2

3

4 5 6

7 8

9 10

GlobalFiler™ Kit 10 Mini-STRs

PI* = 9.2 x 10-12

MiniFiler ™ Kit 8 Mini-STRs

PI* = 1.05 x 10-10

11 12

13 14

15

16 17

18

18 Mini-STRs Combination

PI*= 5.73 x 10-21

97% of alleles in allelic ladder <400bp

Only 2 loci require primer redesign

* Preliminary calculations based on a US Caucasian Database


The GlobalFiler™ Kits: Configuration

p q

Amelogenin DYS391

DYS391 Amel All gender

markers located in the same dye

channel for ease of interpretation

Y indel

/ Y indel Deletion Insertion


• Indels are short length polymorphisms, consisting of the presence (INsertion)

or absence (DELetion) of a short sequence (1-50 nucleotides) • In the GlobalFiler™ Kits, an Indel marker on the Y chromosome was

incorporated into the multiplex

Deletion Insertion

What is an Indel?


Gender identification redundancy helps remove the need for laboratories to maintain a second gender confirmation test protocol. In addition, the Y-indel position (<90 bp) in the GlobalFiler™ Kit helps improve data recovery in degraded samples.

♂

Improved Accuracy of Gender Determination


• 343 alleles total • 31 new alleles added to 9

autosomal STR loci • 246 virtual bins • 589 distinct alleles can be assigned

automatically using GeneMapper® ID-X v1.4 software

• The expanded panel and virtual bins will minimize OL allele calls and facilitate more accurate and efficient automated genotyping

More Genotyping Accuracy: The GlobalFiler™

Kits Allelic Ladder


The GlobalFiler™ Kits Allelic Ladder Improvements

D19S433

GlobalFiler™ Kit range: 6-19.2

NGM SElect™ Kit range: 9-17.2

GlobalFiler™ Kit range: 11-28

Identifiler® Kit range: 15-28

D2S1338


• Literature reports indicate instances of false homozygosity in the Identifiler®/NGM SElect™ Kits as a result of primer site SNP mutations

• Eight new SNP-specific primers have been included in the GlobalFiler™ Kits to minimize the occurrence of null alleles in future data sets − A series of SNP-specific primers already exist in the

Identifiler®/NGM SElect™ Kits to account for other primer site mutations

GlobalFiler™ Kit Loci with New SNP-Specific Primers

D3S1358

vWA

D18S51

D19S433

TH01

FGA

D5S818

SE33

More Genotyping Accuracy: Minimizing False Homozygosity


Maximizing Discrimination

Kit

D8S1

179

D21S

11

D7S8

20

CSF1

PO

D3S1

358

TH01

D13S

317

D16S

539

D2S1

338

D19S

433

vWA

TPOX

D18S

51

D5S8

18

FGA

D10S

1248

D22S

1045

D2S4

41

D1S1

656

D12S

391

SE33

PI

Identifiler® Kit X X X X X X X X X X X X X X X 5.01 x 10-18

NGM SElect™ Kit X X X X X X X X X X X X X X X X 2.35 x 10-21

GlobalFiler™ Kit X X X X X X X X X X X X X X X X X X X X X *7.12 x 10-26

GlobalFiler™ Mini’s X X X X X X X X X X *9.20 x 10-12

* Preliminary calculations based on a US Caucasian Database

The GlobalFiler™ Kits will: − Deliver a new level of discrimination − Reduce the risk of adventitious matches − Facilitate international DNA profile comparisons


Source - www.prb.org/

The GlobalFiler™ Kits offer a power of discrimination ~1014 times greater than the total population that has ever lived on Earth!

GlobalFiler™ Kit PI = 7.12 x 10-26 = 1 in 1.4 x 1025

Total Historical Human Population = 1.1 x 1011

An Unprecedented Power of Discrimination


3130 Series Genetic Analyzers

GeneMapper® ID-X Software v1.4

GeneAmp® 9700 Thermal Cycler

with Silver or Gold-Plated

Silver 96-Well Block

Veriti® Thermal Cycler

3500 Series Genetic Analyzers

3730 Genetic Analyzer (GlobalFiler™ Express Kit

only) The GlobalFiler™ Kits utilize the DS36 (J6) Matrix

Standard Set

GlobalFiler™ Kits: Instrument Compatibility


Instrument Compatibility 3500xL 3130xl


Instrument Compatibility 3500xL 3730


Improved Reagent Traceability Packaging Improvements for the GlobalFiler Kits

New “Peel and Stick” kit labels can be removed from kit sleeves and placed on component boxes or recorded in laboratory logbooks

Improves ability to link kit components with kit lot numbers


Application Specific Development & Testing


Kit Development Strategy

GlobalFiler™

PCR Amplification Kit GlobalFiler ™ Express PCR Amplification Kit

The GlobalFiler™ Kits

A high performance, 6-dye chemistry for casework

samples

An ultra-high throughput, 6-dye chemistry for single

source samples


The Diverse Demands of Forensic Testing

− Predictable − Standardized collection − High yield, high quality DNA − Single source − Fresh

− Unpredictable − Highly variable substrates − Low yield, low quality DNA − Mixed source − Aged

The Samples

− Medium - high throughput − High 1st pass success rate − Minimal sample transfer and

processing steps − Eliminate rate limiting steps − Automated processing

− Low - medium throughput − Maximum results from

compromised samples − High sensitivity − Maximum specificity − Simplified interpretation

Key Workflow Requirements

Databasing Casework


GlobalFiler™ Express Kit Workflows


Optimizing Reaction Time for the GlobalFiler™ Express Kit

Protocols developed on Veriti® thermal cyclers using 100% ramp speed

Performance verified on the 9700 thermal cyclers using MAX mode

Identifiler® Direct Kit

NGM SElect™ Express Kit

GlobalFiler™ Express Kit

Total Time 2 hr 35 min 45 min 39 - 40 min

PCR Conditions 95°C/ 1 min 94°C/ 3 sec 60°C/ 30 sec 60°C/ 8 min

25-27 x


Optimizing Reaction Volume for the GlobalFiler™ Express Kit

Success Rate 25 µL volume

15 µL volume

Blood on FTA® 10/10 10/10

Bode DNA Collector™ 19/19 19/19

Copan 4N6FLOQSwab™ 11/11 11/11

Bode Buccal DNA Collector™

Buccal on Copan FLOQSwab™ Blood on FTA®

Performance and profile quality were maintained at 15 μL with a shorter and faster amplification time

15 μL 25 μL

15 μL 25 μL 15 μL 25 μL


GlobalFiler™ Express Kit Population Study Sample Type N PCR Cycle

Number 1st Pass Success Rate

(175 RFU)

Blood on FTA® 200 25 100%

Blood on Bloodstain Card (903 Paper) 45 25 100%

Buccal on FTA® (EasiCollect™ Device) 56 26 96.4%

Buccal on Bode Buccal Collector™ 90 26 96.6%

Buccal on Copan 4N6FLOQSwab™ 46 24 95.7%

Test Site Sample Type N PCR Cycle Number

1st Pass Success Rate (175 RFU)

A Blood & Buccal on FTA® 80 27 90.0%

B Buccal on FTA® 86 26 93.0%

C Buccal on FTA® 80 27 97.5%

D Buccal on FTA® 64 26 93.8%

E Buccal on FTA® and Swabs 40 26/25 97.5%

Total 350 94.0%

GlobalFiler™ Express Kit External Testing


GlobalFiler™ Kit: Casework Design Considerations

Casework Kit Performance Requirements:

High sensitivity for trace/low level DNA samples Good intracolor balance for mixture samples Sufficient tolerance to PCR inhibitors Maintain high species specificity Efficient thermal cycling conditions


Optimization of GlobalFiler™ Kit Reaction Conditions

Reagent Volumes

3.3X Master Mix 7.5 µL 10X Primer Set 2.5 µL Sample Input ≤15.0 µL 25.0 µL

Greater flexibility of sample input volume to facilitate analysis of low level samples

Identifiler® Plus & NGM SElect™ Kits GlobalFiler™ Kit

Total Time 2 hr 25 min 1 hr 20 min

PCR Conditions

95°C/ 1 min 94°C/ 20 sec 59°C/ 90 sec 60°C/ 10 min

29 x

Optimized thermal cycling conditions shorten amplification conditions by > 1 hr


GlobalFiler™ Kit Performance Example: Sensitivity

Greater sensitivity was observed with the GlobalFiler™ Kit compared to the Identifiler® Plus Kit

GlobalFiler™ Kit Identifiler® Plus [DNA]

D8 D21 D7 CSF D3

TH01 D13 D16

D2(S1) D19 vW

A TPOX D18

Amel

D5 FGA D1

D10 D12

D2(S4) D22 DY3 SE33 Yind

D8

D21 D7 CSF D3

TH01 D13 D16 D2

D19 vW

A TPOX D18

Amel

D5 FGA

1 ng

500 pg

250 pg

125 pg

62.5 pg

31.25 pg

15.63 pg

The GlobalFiler™ Kit exhibits slightly greater sensitivity than the Identifiler® Plus Kit


GlobalFiler™ Kit Performance Example: Inhibitor Tolerance

The GlobalFiler™ Kit buffer has been optimized to deliver enhanced inhibition tolerance compared to the Identifiler® Plus Kit

Humic Acid Inhibition Hematin Inhibition

Identifiler® Plus Kit GlobalFiler™ Kit


GlobalFiler™ Kit Performance on Casework Samples

Intralocus Balance

GlobalFiler™ Kit

GlobalFiler™ Kit

Identifiler® Plus Kit

Identifiler® Plus Kit

500 pg 1 ng

Intracolor Balance (%)

The GlobalFiler™ Kit maintains the high levels of intracolor and intralocus balance established by other Applied Biosystems next generation STR kits


Identifiler® Plus Kit GlobalFiler™ Kit

Species Specificity Stutter Percentage 70bp 470bp

10 ng Cat DNA

10 ng Dog DNA

10 ng Horse DNA

10 ng Bacteria DNA

400 RFU

GlobalFiler™ Kit Performance on Casework Samples

Reduced mean stutter percentage for the majority of loci and high specificity delivered by optimized buffer chemistry and established primer design methods

simplify interpretation of GlobalFiler™ Kit data

007 DNA


300 Test Site #1

GlobalFiler™ Kit Baseline Quality

300

300

Test Site #2

Test Site #3

Read Region 74 444

Non-template Controls


GlobalFiler™ Kit Test Site Results

Test Site #1: T-Shirt Swab, Organic Extraction Identifiler® Kit: 50 RFU PAT (3130 xl) GlobalFiler™ Kit: 175 RFU PAT (3500xL)

Identifiler® Kit: No Profile GlobalFiler™ Kit: Full Profile (39 alleles)



Identifiler® Plus Kit: Partial Profile (21 alleles) GlobalFiler™ Kit: Partial Profile (28 alleles)

Test Site #2: Bone Sample; Organic Extraction Identifiler® Plus Kit: 50 RFU PAT (3130xl) GlobalFiler™ Kit: 175 RFU PAT (3500xL)



NGM SElect™ Kit: Full Profile (26 alleles) GlobalFiler™ Kit: Full Profile (36 alleles)

Test Site #2: Cigarette Butt, Organic Extraction NGM SElect™ Kit: 50 RFU PAT (3130xl) GlobalFiler™ Kit: 175 RFU PAT (3500xL)



NGM SElect™ Kit: Partial/Mixture (24 alleles) GlobalFiler™ Kit: Partial/Mixture (37 alleles)

Test Site #2: Underwear, Organic Extraction NGM SElect™ Kit: 50 RFU PAT (3130xl) GlobalFiler™ Kit: 175 RFU PAT (3500xL)


Test Site Results: 900 Year Old Bone Sample

Medieval Austrian, Agnes of Waiblingen was the daughter of Henry IV, Holy Roman Emperor and was married to Leopold III, one of the most important Babenberg Margraves, who ruled the Austrian territory

Born in 1072, she died in

1143 (aged 71) and is reported to have had >20 children


Development of a Next Generation Global Multiplex Go Faster!

− Up to 5x faster amplification time than previous generation kits > GlobalFiler™ Express Kit: ~40 minutes; GlobalFiler™ Kit: ~80 minutes > Enables sample to answer in <2 hours

Go Further! − 24 loci, including 10 mini-STRs, increasing discrimination power by up to 9

orders of magnitude − Enhanced buffer system and sensitivity for even better performance on

inhibited and low-level samples Go Global!

− The only kit containing all markers recommended for inclusion by the CODIS Core Loci Working Group, including all markers commonly used in Europe

− Maximizes global database compatibility


Acknowledgements External Test Sites

− 57 worldwide and growing!

R&D Julio Mulero, Lori Hennessy, Siddhita Gopinath, Wilma Norona, Matthew Ludeman, Robert Lagace, Chien-Wei Chang, Chang Zhong, Nigel Beard

Tech Transfer Larry Wang, Anthony Matthews, Vanessa Chan, Yenyoung Lei

Software Allan Minn, Gloria Lam, Sharada Vijaychander, Tony Hatch, Makesh Kumar, Ravi Gupta, Doug Greig, Babu Yama

Product Management Karen Cormier, Larry Joe, Jacki Benfield

Forensic Science Applications Jennifer Schroeder, Lisa Calandro, Jeff Sailus, Nicola Oldroyd

Quality Assurance Liwei Qi, Yurah Yen

Marketing Ariana Wheaton, Jonathan Tabak, Shereen Darwish, Lisa Schade

Oligo Development Sarah Ruhe, Kathy Wang, Hong Ji

UK Manufacturing Catherine Duke

Project Management Wendy Coffey, Linda Kauffman


Thank You

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Development of a “Global” STR Multiplex for Human Identification Analysis

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