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

Human Identification, Life Technologies

11/13/2012 2

Agenda

Why a global multiplex?

Multiplex Configuration Strategy

Application-Specific Development and Testing Results − Direct amplification for single source samples − Casework samples

Summary

11/13/2012 3

Why a Global Multiplex?

Missing Persons Investigations

Why?

How?

Increase international data compatibility Increase discrimination power Reduce the likelihood of adventitious matches

Increase the number of loci

Incorporate loci contained in global databases

New CODIS Recommendations

Expanded ESSL

11/13/2012 4

MINIMUM REQUIRED LOCI D13S317 Identifiler® Kit

D7S820 Identifiler® Kit

D5S818 Identifiler® Kit

CSF1PO Identifiler® Kit

D1S1656 NGM SElect™ Kit

D12S391 NGM SElect™ Kit

D2S441 NGM SElect™ Kit

D10S1248 NGM SElect™ Kit

D18S51 Identifiler® & NGM SElect™ Kits

FGA Identifiler® & NGM SElect™ Kits

D21S11 Identifiler® & NGM SElect™ Kits

D8S1179 Identifiler® & NGM SElect™ Kits

VWA Identifiler® & NGM SElect™ Kits

D16S539 Identifiler® & NGM SElect™ Kits

TH01 Identifiler® & NGM SElect™ Kits

D3S1358 Identifiler® & NGM SElect™ Kits

AMEL Identifiler® & NGM SElect™ Kits

D2S1338 Identifiler® & NGM SElect™ Kits

D19S433 Identifiler® & NGM SElect™ Kits

DYS391 Yfiler® Kit

PENTA E

HIGHLY RECOMMENDED LOCI (1) TPOX Identifiler® Kit

(2) D22S1045 NGM SElect™ Kit

(3) SE33 NGM SElect™ Kit

(4) PENTA D

Recommended CODIS Core Loci (April 2011)

New recommendation drops TPOX from the list of required loci

The new list of required loci includes all the markers from the NGM SElect™ & Identifiler® Series Kits

11/13/2012 5

What is GlobalFiler™?

The GlobalFiler™ Kits are our response to the CODIS Core Loci Working Group’s recommendation to expand the CODIS Core Loci from 13 to 20 required loci and 3 optional loci

Originally communicated as a letter to the editor (FSI Genetics) and accepted for publication on April 2011

(revised on Jan 2012)

Authored by Doug Hares from the FBI

Expectation of a single amplification kit

11/13/2012 6

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460

D3S1358VWA

D16S539CSF

TPOXY indel

AmelD8S1179

D21S11D18S51DYS391

*D2S441D19S433

TH01FGA

*D22S1045D5S818

D13S317D7S820

SE33

*D10S1248*D1S1656*D12S391

D2S1338

The GlobalFiler™ Kits Configuration 1 2

3

4 5 6

7 8

9 10

Mini-STRs PI* = 9.2 x 10-12

MiniFiler ™ Kit 8 mini-STRs < 220 bp

PI* = 1.05 x 10-10

11 12

13 14

15

16 17

18

Mini-STR Combination PI*= 5.73 x 10-21

11/13/2012 7

60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460

D3S1358VWA

D16S539CSF

TPOXY indel

AmelD8S1179

D21S11D18S51DYS391

*D2S441D19S433

TH01FGA

*D22S1045D5S818

D13S317D7S820

SE33

*D10S1248*D1S1656*D12S391

D2S1338

The GlobalFiler™ Kits Configuration

Y indel

DYS391

p q

Amelogenin Y-indel/DYS391

11/13/2012 8

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

11/13/2012 9

The GlobalFiler™ Kits Allelic Ladder

343 alleles and 245 virtual bins

11/13/2012 10

Minimizing False Homozygosity Literature reports indicate instances of false homozyosity 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

Locus References

D3S1358 Kline et al. FSI Genetics 5 (2011) 329-332.

vWA Delamoye et al. FSI 143 (2004) 47-52.

D18S51 Delamoye et al. FSI 143 (2004) 47-52.

D19S433 Kline et al. FSI Genetics 5 (2011) 329-332.

TH01 Vanderheyden et al. Int J Legal Med 121 (2007) 297-301.

FGA Ricci et al. JFS 52 (2007) 393-396.

D5S818 Kline et al. FSI Genetics 5 (2011) 329-332.

SE33 Heinrich et al. Int J Legal Med 118 (2004) 361-363.

11/13/2012 11

Maximizing Discrimination

Kit

D8S1

179

D21S

11

D7S8

20

CSF1

PO

D3S1

358

TH01

D13S

317

D16S

539

D2S1

338

D19S

433

vWA

TPO

X

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 2.6 x 10-17

NGM SElect™ Kit X X X X X X X X X X X X X X X X 1.12 x 10-20

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

* 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

11/13/2012 12

GlobalFiler™ Kit Instrument Compatibility 3500xL 3130xl

Variable binning settings for 3130 series instruments adjusted to gain optimal color balance

11/13/2012 13

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

Optimized for use with the 3500/3500xL

11/13/2012 14

GlobalFiler™ Kit Comparison

GlobalFiler™ Express Kit GlobalFiler™ Kit

Master Mix Optimized to overcome inhibitors

found in unpurified single source blood and buccal samples

Optimized to provide optimal sensitivity and intracolor balance for casework

samples

Primer Mix* 2.5X formulation 10X formulation

PCR Protocol PCR protocol optimized for speed of processing for single source samples

PCR protocol optimized for high sensitivity and high performance on

casework samples

Control DNA 007 2 ng/µL 0.1 ng/µL

Allelic Ladder Identical Allelic Ladder

* Both kits use identical primer sequences

11/13/2012 15

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 40 min

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

25-27 x

11/13/2012 17

Optimizing Primer Mix for the GlobalFiler™ Express Kit

Blood on FTA®

Blood on Untreated Bloodstain

Card

Buccal on Bode Buccal Collector™

Buccal on FTA®

Buccal on Copan

4N6FLOQSwab™

Buccal on Omniswab

N=6

11/13/2012 18

GlobalFiler™ Express Kit Population Study

Blood on FTA®

Blood on Untreated Bloodstain

Card

Buccal on Bode

Buccal Collector™

Buccal on FTA®

Buccal on Copan

FLOQSwab™

Blood on FTA®

Blood on Untreated Bloodstain

Card

Buccal on Bode

Buccal Collector™

Buccal on FTA®

Buccal on Copan

FLOQSwab™

Intralocus Balance Intracolor Balance

Sample Type N PCR Cycle Number Success Rate (175 RFU)

Number of Off-Scale Profiles

Blood on FTA® 200 25 100% 4

Blood on Bloodstain Card (903 Paper) 45 25 100% 0

Buccal on FTA® (EasiCollect™ Device) 56 26 96.4% 3

Buccal on Bode Buccal Colector™ 90 26 96.6% 0

Buccal on Copan 4N6FLOQSwab™ 46 24 95.7% 3

N=3552 N=1000 N=761 N=1545 N=774

11/13/2012 19

GlobalFiler™ Express Kit Data Examples

The GlobalFiler™ Express Kit is optimized to deliver high quality results on a wide range of substrates

Prep-n-Go™ Buffer facilitates cell lysis in non-treated paper and swabs.

Bucc

al o

n Co

pan

FLO

QSw

ab™

Bl

ood

on F

TA®

Card

Bucc

al o

n Bo

de B

ucca

l Col

lect

or™

11/13/2012 20

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

Efficient thermal cycling conditions

11/13/2012 21

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

11/13/2012 22

0.0

20.0

40.0

60.0

80.0

100.0

250 125 62 31 15(pg)

Mea

n %

Alle

les

Rec

over

edGlobalFiler™ Kit Performance Example: Sensitivity

The GlobalFiler™ Kit delivers comparable

sensitivity to the NGM SElect™ Kit

N=4 TM

TM

NGM SelectTM Kit GlobalFilerTM Kit

11/13/2012 23

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

50.0

250 125 62 31 15(pg)

Alle

les

Rec

over

edGlobalFiler™ Kit Performance Example: Sensitivity

The GlobalFiler™ Kit amplifies more alleles

than the NGM SElect™ Kit.

N=4

GlobalFiler™ Kit amplifies 43 alleles in a full profile of the control DNA 007 NGM Select™ Kit amplifies 34 alleles in a full profile of the control DNA 007

TM

TM

NGM SelectTM Kit GlobalFilerTM Kit

11/13/2012 24

GlobalFiler™ Kit Performance Example: Inhibitor Tolerance

The GlobalFiler™ Kit buffer has been optimized to deliver enhanced inhibition tolerance compared to the NGM Select™ Kit

Humic Acid Inhibition Hematin Inhibition

0102030405060708090

100

0 120 150 180

(ng/uL)

0

10

20

30

40

50

60

70

80

90

100

0 300 340 380

(uM)

TM

TM

NGM SelectTM Kit GlobalFilerTM Kit

11/13/2012 25

GlobalFiler™ Kit Performance Examples

The GlobalFiler™ Kit maintains the high levels of intracolor balance, intralocus balance and stutter percentage established with previous next generation kits such as the Identifiler® Plus Kit

Stut

ter P

erce

ntag

e (%

)

Intr

aloc

us B

alan

ce

GlobalFiler™ Kit

GlobalFiler™ Kit

Identifiler® Plus Kit

Identifiler® Plus Kit

500 pg 1 ng

Intr

acol

or B

alan

ce (%

)

Blue – Identifiler® Plus Kit Red – GlobalFiler™ Kit

11/13/2012 26

Prototype GlobalFiler™ Kit vs. Identifiler® Plus Kit Test Site data: Bone Sample; Organic Extraction

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

Identifiler® Plus Kit: 50 RFU PAT (3130xl) GlobalFiler™ Kit: 175 RFU PAT (3500xL)

11/13/2012 27

Prototype GlobalFiler™ Kit vs. NGM SElect™ Kit Test Site data: Cigarette Butt, Organic Extraction

NGM SElect™ Kit: Partial Profile (18 alleles) GlobalFiler™ Kit: Full Profile (43 alleles)

NGM SElect™ Kit: 50 RFU PAT (3130xl) GlobalFiler™ Kit: 175 RFU PAT (3500xL)

11/13/2012 28

300 Test Site #1

Prototype GlobalFiler™ Kit Baseline Quality

300

300

Test Site #2

Test Site #3

Read Region 74 444

Non-template Controls

11/13/2012 29

GlobalFiler™ Kit Feature Summary

GlobalFiler™ Express Kit GlobalFiler™ Kit

Amplifies the 20 ‘required’ and 3 ‘recommended’ loci in the new CODIS Core Loci recommendations

Amplifies 3 Y chromosomal markers to ensure accurate gender determination

Improves genotyping accuracy and automation through expanded allelic ladders and virtual bin sets

Compatible with the Veriti® & 9700 thermal cyclers and the 3500 & 3130 series genetic analyzers

Developed specifically for the direct amplification of single-source samples

Developed specifically for the amplification of casework samples

Delivers rapid throughput on blood and buccal samples on treated paper, untreated paper and

swabs

Delivers increased sensitivity, improved intralocus and intracolor balance, enhanced performance on

inhibited and degraded samples

Amplification Time ~ 40 min Amplification Time ~ 80 min

11/13/2012 30

Acknowledgements R&D

Dennis Wang, Lori Hennessy, Siddhita Gopinath, Wilma Norona, Matthew Ludeman, Robert Lagace, Chien-Wei Chang, Chang Zhong

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

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

Product Management Karen Cormier, Bob Kays, Larry Joe, Jacki Benfield, Guido Sandulli

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

11/13/2012 31

Thank You

© 2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners.

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.