the globalfiler(tm) kit: going further for casework

1 Life Technologies™ |

The GlobalFiler™ Kit: Going Further for Casework Jennifer Schroeder Forensic Specialist Bode Technology 10th Annual Advanced DNA Technical Workshop March 24-28, 2013 San Diego, CA


What is the GlobalFiler™ Kit?

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

INTERPOL HIT EXEMPLIFIES FUTURE!OF INTERNATIONAL MATCHING!

DNA Gateway

Suspect arrested for rape in 2009 in Austria DNA profile sent to Interpol Gateway and matches profile in CODIS

3 rapes in 2004 linked by Cal DOJ CODIS Suspect idenEfied – flees country – alias assumed Profile sent to Interpol in 2009

Ali Achekzai extradited to California Convicted on all counts

Laguna Beach/San Diego, California


Multiplex Configuration Strategy


What do you think today?

“Large STR multiplexes are not well suited for casework. Sensitivity and robustness can suffer with the addition of more loci.”

A.  True

B.  False


5-Dye Configuration Containing All Required and Highly Recommended Loci

1 2

3

4

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

1 2

3

4 5

6

7 8

Fewer Mini-STRs à poor performance for

degraded DNA

Primer Redesigns à 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 calcula.ons based on a US Caucasian Database


What is an Indel? •  Indels are short length polymorphisms, consisEng of the presence (INserEon)

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

incorporated into the mulEplex

DeleEon InserEon


•  Amelogenin Y null alleles occur

•  AddiEonal Y tesEng may be required to confirm sample gender

•  AddiEonal gender idenEficaEon markers are on the long arm of the Y chromosome

to minimize the risk of a double deleEon

•  DYS391 is a stable locus with a relaEvely narrow allele range •  The Y-‐indel is very short therefore more likely to perform even if DYS391 drops out

in degraded samples

p q

Amelogenin Y-‐indel/DYS391

Why Include Additional Y Markers?


•  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


What percentage of evidence profiles you handle on a regular basis that are 2 or 3 contributor mixture

profiles, with at least one female contributor?

A.  <25%

B.  25-40%

C.  40-60%

D.  60-80%

E.  >80%


The GlobalFiler™ Kits: Configuration

DYS391 Amel

All gender markers located in the

same dye channel for ease of

interpretation

Y indel


The GlobalFiler™ Kits: Allelic Ladder

New virtual bins added to

improve genotyping efficiency

343 alleles 246 virtual bins 589 total alleles

31 alleles added to 9

GlobalFiler™ Kit marker ladders


Maximizing Discrimination Kit

D8S

1179

D21

S11

D7S

820

CSF

1PO

D3S

1358

TH01

D13

S317

D16

S539

D2S

1338

D19

S433

vWA

TPO

X

D18

S51

D5S

818

FGA

D10

S124

8

D22

S104

5

D2S

441

D1S

1656

D12

S391

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: −  Deliver a new level of discrimination −  Reduce the risk of adventitious matches −  Facilitate international DNA profile comparisons

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


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


Improved Reagent Traceability Packaging Improvements for the GlobalFilerTM 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: Workflow Concepts


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


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 Overview


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 sensiEvity was observed with the GlobalFiler™ Kit compared to the IdenEfiler® Plus Kit

GlobalFiler™ Kit Identifiler® Plus [DNA] D8

D21 D7 CSF D3 TH01 D13 D16

D2(S1) D19 vW

A TPO

X D18 Am

el D5 FGA D1 D10 D12

D2(S4) D22 DY3 SE33 Yind D8

D21 D7 CSF D3 TH01 D13 D16 D2 D19 vW

A TPO

X D18 Am

el 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 sensiEvity than the IdenEfiler® 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

IdenZfiler® Plus Kit

IdenZfiler® 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 generaEon 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


GlobalFiler™ Kit Test Site Data


TS1 TS2 TS3 TS4 TS5

ExtracZon Method Organic or EZ1 Organic MagneEc

Bead Qiagen

InvesEgator Organic

ComparaZve Kit

IdenEfiler Plus, PP 16HS, PP Fusion

NGM SElect IdenEfiler Plus

IdenEfiler Plus NGM SElect

Thermal Cycler 9700 Gold VeriE VeriE 9700 Gold 9700 Gold

CE Instrument 3500 3500 3500xL 3500xL 3500

GlobalFiler™ Kit Test Site Overview


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 Performance Example: Sensitivity

GlobalFiler™ Kit [DNA] D8

D21

D7

CSF

D3

TH01

D13

D16

D2(S1)

D19

vWA

TPOX

D18

Amel

D5

FGA

D1

D10

D12

D2(S4)

D22

DY3

SE33

Yind

1 ng

500 pg

250 pg

125 pg

62.5 pg

31.25 pg

15.63 pg

Increased allele recovery with low input samples.


Sensitivity Results <50pg of DNA using 10uL Sample Input Volume

TS 1 TS2 TS3 TS4 TS 5

46.88 pg 4 5 16 13 33 38 36 38 18 15 15 26 19 12 16 26 29 35 30 10

31. 25 pg 4 3 1 2 25 24 24 22 6 8 7 12 20 10 6 18 21 18 22 26

23.43 pg 4 2 4 4 12 21 15 12 3 11 7 3 4 5 6 8 10 17 12 19

15.62 pg 0 0 1 1 15 12 14 12 0 1 1 0 1 6 5 2 6 4 7 8

•  Variability in allele recovery from site to site


[DNA] = 1.5 pg/uL [DNA] = 3.1 pg/ uL

N = 4 Sample Input Volume Increase in

Recovery

Sample Input Volume Increase in Recovery 10 uL 15 uL 10 uL 15 uL

Test Site 1 0.5 3.5 7 2.5 9.5 3.8

Test Site 2 13.25 15 1.11 23.75 36.25 1.53

Test Site 3 0.5 6 12 8.25 18.5 2.24

Test Site 4 3.5 5.75 1.64 13.5 18.25 1.35

Test Site 5 6.25 14.5 2.32 21.75 26 1.2

Total 24 44.75 1.86 69.75 108.5 1.56

Allele Recovery: Extended Sample Volume Input

•  Test sites ran the same samples varying only the volume of DNA added to the reaction. •  Adding an additional 5uL of DNA to the reaction delivered approximately 1.5 -2.0 times more alleles in low concentration samples


Test Site Sample Success Rates and Allele Recovery GlobalFiler™ Kit vs. Identifiler® Plus Kit

TS1 TS3 TS4

Kit GF ID+ GF ID+ GF ID+

Full Profile 8 8 13 11 5 4

ParEal Profile -‐ -‐ 2 4 9 9

No Profile -‐ -‐ -‐ -‐ -‐ 1

Total Alleles Recovered 337 240 578 393 461 212

40% 47% 117%

•  More full profiles recovered with GlobalFiler resulting in an increased number off alleles called

• 10 samples with partial profiles in both kits: Total alleles recovered is greater with GlobalFiler than with Identifiler Plus.

Profile GF / ID+ GF ID+ % Inc alleles ParZal / ParZal (10

samples) 302 107 182%


Alleles Recovered Using the GlobalFiler™ and Identifiler® Plus Kits : Partial Profiles

0

5

10

15

20

25

30

35

40

45

Fat Trace Swab /310pg

Key Swab/ 60pg

Booleneck Swab / 50pg

Drawer Swab/60pg

Swab on Safe / 50pg

Wheel Swab /80pg

Cigareoe Buo / 40pg

Cigareoe Buo / 30pg

Cap Swab / 165 pg

Stone / Undet

No. Alleles R

ecovered

ParZal Profile Samples

GF ParEal

ID+ ParEal

The increased robustness of the GlobalFiler Kit delivered 182% more alleles than Identifiler Plus with partial profiles

• Deliver more information per sample • Increases the likelihood of a meaningful database match


GlobalFiler™ Kit Test Site Results

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

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


GlobalFiler™ Kit: Full (40 alleles) Identifiler® Plus Kit: Full (29 alleles)


Bone Sample; Magnetic Bead Extraction (1ng) Identifiler® Plus Kit: 50 RFU PAT (3130xl) GlobalFiler™ Kit: 175 RFU PAT (3500xL)



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

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


GlobalFiler Kit: Full (40 alleles) Identifiler® Plus Kit: Partial (21 alleles)

Door Handle Swab, Qiagen Investigator (310pg) Identifiler® Plus Kit: 50 RFU PAT (3130xl) GlobalFiler™ Kit:175 RFU PAT (3500xL)



Identifiler® Plus: Partial (21 alleles) GlobalFiler Kit: Partial (34 alleles)


Hair Root, Magnetic Bead (460pg) Identifiler® Plus Kit: 50 RFU PAT (3130xl) GlobalFiler™ Kit: 175 RFU PAT (3500xL)



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

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


GlobalFiler Kit: Full (42 alleles)

Sample Type: Unknown GlobalFiler Kit: 175 RFU PAT (3500) PowerPlex Fusion:175 RFU PAT (3500)

PowerPlex® Fusion Kit: Partial (33 alleles)



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


After hearing more about the GlobalFiler System, how do you feel now when you

think about implementing and analyzing a 24 locus multiplex?

A.  Terrified, I can’t imagine how difficult it might be to use

B.  Apprehensive, I need a lot of questions answered before agreeing to try it

C.  Interested, I’d like to try it sooner rather than later

D.  Very excited, when can I get my hands on it


GlobalFiler™ Kit: Test Site Feedback

“The baseline is perfect, no artifact was observed in

negative controls. Sample profiles have showed good

quality data with a good balance PHR.”

“The fact that we are able to use up to 15µL of DNA per PCR reaction improves the

sensitivity … very interesting for the amplification of poor DNA samples (<50pg/

µL).”

“Having 50% of the PCR products with a size less than or equal to 220pb allows better amplification of degraded DNA. In addition, with a volume of 15µL of DNA per PCR reaction, we are in the best conditions for amplification of degraded DNA with an amount of

DNA lower than100pg/µL.”

“Seems to withstand well to inhibitors…”


GlobalFiler™ Kit: Test Site Feedback

“Thanks to the integration of DYS391 and the Y-indel we can

no longer have any doubt concerning the analysis of the Y

chromosome in the case of amelogenin mutation.”

“Using a 6th dye allows to separate the markers analyzed and thus avoid allele overlap between neighboring markers.”

“The use of this kit should reduce the sample rate that has to be re-amplified whether with the same PCR kit or with another kit, while improving performance…”

“PCR duraEon (1:20 vs. 2:30 with IdenEfiler® Plus)

is also an advantage especially in the context of

custody…”


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, Siddhita Gopinath, Wilma Norona, Maohew Ludeman, Robert Lagace, Chien-‐Wei Chang, Chang Zhong, Nigel Beard

§  Tech Transfer Larry Wang, Anthony Maohews, Vanessa Chan, Yenyoung Lei

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

§  Product Management Karen Cormier, Jacki Benfield

§  Forensic Science ApplicaEons Jennifer Schroeder, Lisa Calandro, Jeff Sailus

§  Quality Assurance Liwei Qi, Yurah Yen

§  MarkeEng 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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the globalfiler(tm) kit: going further for casework

Technology

highlyd21s11 x x x x

fga x x x x

d1s1656 x x x

d22s1045 x x x genet

d19s433 x x x x e52e54

codisd12s391 x x x core

dys391 x

codis tpox x x x core