Development of a “Global” STR Multiplex for Human Identification Analysisafdaa.org/.../uploads/2013/01/GlobalFiler-AFDAA-2-7-13-.pdf · 2013. 2. 8. · Development of a “Global”

1Life Technologies™ |

Development of a “Global” STR

Multiplex for Human Identification Analysis

Jonathan TabakLife Technologies


Agenda

Introduction

Multiplex Configuration Strategy

Application-Specific Development and Testing Results

− Direct amplification for single source samples

− Casework samples

Summary


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

LocusCODIS

(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

12

3

4

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

12

3

45

6

7 8

Fewer Mini-STRs poor performance for

degraded DNA

Primer Redesigns less concordant


The GlobalFiler™ Kits: 6-Dye Configuration

12

3

45 6

78

9 10

GlobalFiler™ Kit10 Mini-STRs

PI* = 9.2 x 10-12

MiniFiler ™ Kit8 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 inallelic 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

AmelAll 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


The GlobalFiler™ Kits: Allelic Ladder

New virtual bins added to

improve genotyping efficiency

343 alleles246 virtual bins589 total alleles

31 alleles added to 9

GlobalFiler™ Kit marker ladders


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

D8

S1

17

9

D2

1S

11

D7

S8

20

CS

F1

PO

D3

S1

35

8

TH

01

D1

3S

31

7

D1

6S

53

9

D2

S1

33

8

D1

9S

43

3

vWA

TP

OX

D1

8S

51

D5

S8

18

FG

A

D1

0S

12

48

D2

2S

10

45

D2

S4

41

D1

S1

65

6

D1

2S

39

1

SE

33 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 TraceabilityPackaging 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 KitGlobalFiler ™ Express PCR Amplification Kit

The GlobalFiler™ Kits

A high performance, 6-dyechemistry 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 KitGlobalFiler™

Express Kit

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

PCR Conditions95°C/ 1 min94°C/ 3 sec60°C/ 30 sec60°C/ 8 min

25-27 x


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 µL10X Primer Set 2.5 µLSample 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 min94°C/ 20 sec59°C/ 90 sec60°C/ 10 min

29 x

Optimized thermal cycling conditions shorten amplification conditions by > 1 hr


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

− 66 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 ApplicationsJennifer Schroeder, Lisa Calandro, Jeff Sailus, Nicola Oldroyd

Quality AssuranceLiwei Qi, Yurah Yen

MarketingAriana Wheaton, Jonathan Tabak, Shereen Darwish, Lisa Schade

Oligo DevelopmentSarah Ruhe, Kathy Wang, Hong Ji

UK ManufacturingCatherine Duke

Project ManagementWendy Coffey, Linda Kauffman


Thank You

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Refer to product page on the Life Technologies website for Limited Use License.

The content provided herein may relate to products that have not been officially released and is subject to change without notice.

FOR FORENSIC AND PATERNITY USE ONLY

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Documents

Development of a “Global” STR Multiplex for Human Identification Analysisafdaa.org/.../uploads/2013/01/GlobalFiler-AFDAA-2-7-13-.pdf · 2013. 2. 8. · Development of a “Global”