Improved Recovery of Interpretable Results from Challenging Samples Using the GlobalFiler™ Kit - Webinar

1 Life Technologies™ |

Improved Recovery of Interpretable Results from Challenging Samples Using the GlobalFiler™ Kit

Kellie Fenesan Life Technologies


Total Time to Result = ~10-‐58 hrs

Sample Prepara9on

Quan9ta9on/ Normaliza9on Amplifica9on Detec9on Analysis

Time to Result

(hrs/per 6-‐16 samples) 1-‐48 2 3.5 0.75 2-‐3 Time to Result

(hrs/per 13 samples) 1-‐3 2 1.5 0.55 1-‐2

Total Time to Result = ~6-‐9 hrs

Dependent on sample type and method

Improved Efficiency for Casework Samples


Detection & Analysis

Our Mission: Improve Efficiency. Increase Success Rates. Provide More Info.

LIFE Offers Complete Integrated HID Workflow

GeneMapper® ID-X Expert System

Software

Next Generation STR Kits

3500/3500xL Genetic Analyzer

3130/3130xl Genetic Analyzer

Extraction Quantification Amplification

7500 Real-Time PCR Instrument

Next Generation Quantifiler® Kits

PrepFiler™ Forensic DNA Extraction kit

Veriti® 96-Well Thermal Cycler

GeneAmp® PCR System 9700 Cycler

AutoMate Express™

Collection

Copan 4N6FLOQSwab™

& NUCLEIC-CARD™

Screening

ParaDNA®

Converge™ Software DNA Workflow

Kinship and Paternity

Direct Search Familial Search

NEW

NEW

NEW

NEW

Converge™

Software

NEW

NEW


Which Profile Would You Rather Interpret? This One?


Or This One?


The GlobalFiler™ Kit: Going Further for Casework


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


A short history of STR Kit Multiplexing

Kit Name Release Date # of Loci # of Dyes*

AmpFSTR® Blue 1996 3 2

AmpFSTR® Profiler® 1997 10 4

AmpFSTR® Identifiler® 2001 16 5

GlobalFiler™ 2012 24 6

*Includes size standard


Multiplex Configuration Strategy


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

DYS391

Amel Y indel

All gender markers located in the

same dye channel for ease of

interpretation

*


•  Amelogenin Y null alleles can occur

•  Addi9onal Y tes9ng may be required to confirm sample gender

•  Addi9onal gender iden9fica9on markers are on the long arm of the Y chromosome

to minimize the risk of a double dele9on

•  DYS391 is a stable locus with a rela9vely 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


Facts about the SE33 Locus

§  The most discrimination power of any STR locus in commercial kits −  highest Hetobs (0.9353) −  lowest PI value (0.0066) −  highest amount of alleles and

genotypes observed

§  Ideal for mixtures −  more alleles seen and less allele

sharing

§  Widely Used in Europe −  Included in the Applied Biosystems

SEFiler®, SEfiler Plus™ and NGM SElect™ kits for many years

−  Becoming more widely used internationally

- Hill, B et al, "Population Statistics on the Proposed Expanded U.S. Core Loci", poster at the 23rd International Symposium on Human Identification (ISHI) meeting (Nashville, TN), October 16-17, 2012


SE33 Locus – Ideal for Mixtures


Overlay of SE33 Data from 4 individuals

SE33 extremely valuable for mixture interpretation In this case, we have 4 donors and 7 peaks


Maximizing Allele Calls: 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


Achieving New Levels of 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!


GlobalFiler™ Kits: Instrument Compatibility

§ Supported Platforms

§ 3130/3130xL Genetic Analyzer*

§ 3730 DNA Analyzer (GlobalFiler™ Express Kit only)

§ 3500/3500xL Genetic Analyzer

*3100 series may be upgraded to 3130


Instrument Compatibility 3500xL 3130xl


6-Dye Configurations

Chemistry/ Standards

GeneMapper® ID-‐X So`ware

v1.4

Service

3730 DNA Analyzer /

31xx Geneec Analyzers

3500 Geneec Analyzer

Data Colleceon So`ware

1 2 3

31xx/3730 only All Instruments


GlobalFiler™ Kit Upgrade Roadmap

Product Type 3500 3730 3130 3100

GeneMapper® ID-‐X So`ware v1.4

Data Colleceon So`ware v4.0 N/A

6-‐dye Data Colleceon Module N/A

Service Call/Computer N/A

Instrument Upgrade N/A N/A N/A


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


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


Decreased amplification time for casework samples

Op9mized thermal cycling condi9ons shorten amplifica9on 9me to ~80 mins

PCR Condi9ons

29 x

Total Time

Iden9filer® Kit ~3.5 hours

Iden9filer® Plus Kit & NGM SElect™ Kit

~2.5 hours

GlobalFiler™ Kit ~80 mins

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


GlobalFiler™ Kit Test Site Data


TS1 TS2 TS3 TS4 TS5

Extraceon Method Organic or EZ1 Organic Magne9c

Bead Qiagen

Inves9gator Organic

Comparaeve Kit

Iden9filer Plus, PP 16HS, PP Fusion

NGM SElect Iden9filer Plus

Iden9filer Plus NGM SElect

Thermal Cycler 9700 Gold Veri9 Veri9 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


The GlobalFiler™ Kit accommodates up to 15 μL of sample input volume to facilitate improved performance for low level samples.

Reagent Volumes

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

Increased Sensitivity


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 and 15uL 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 7x 2.5 9.5 3.8x

Test Site 2 13.25 15 1.11x 23.75 36.25 1.53x

Test Site 3 0.5 6 12x 8.25 18.5 2.24x

Test Site 4 3.5 5.75 1.64x 13.5 18.25 1.35x

Test Site 5 6.25 14.5 2.32x 21.75 26 1.2x

Total 24 44.75 1.86x 69.75 108.5 1.56x

Allele Recovery: Extended Sample Volume Input

Average alleles per replicate shown for each input volume

•  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


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)



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)




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)


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


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

0

5

10

15

20

25

30

35

40

45

Fat Trace Swab /310pg

Key Swab/ 60pg

Boqleneck Swab / 50pg

Drawer Swab/60pg

Swab on Safe / 50pg

Wheel Swab /80pg

Cigareqe Buq / 40pg

Cigareqe Buq / 30pg

Cap Swab / 165 pg

Stone / Undet

No. Alleles R

ecovered

Pareal Profile Samples

GF Par9al

ID+ Par9al

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

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


GlobalFiler™ Kit Configuration and Workflow

-  A change has been made to the configuration of the GlobalFiler™ Kit workflow to provide longer term expiration dating (18 months at -20ºC)

-  Kits will be received in the standard frozen condition.

-  Prior to first use, the users will: 1)  Thaw reagents (Standard Protocol) 2)  Add 83 uL of Master Mix Additive reagent into Master Mix tube. 3)  Invert the Master Mix tube 10 times. Mark the Master Mix tube

with a + to indicate MM Additive was added. Discard Additive tube.

4)  Master Mix is ready for use and to be stored at 4ºC (Standard Protocol).

Traditional Kit Configuration GlobalFiler™ Kit Configuration

PM

MM

Add

PM

MM

New

New New


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 220bp 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 dura9on (1:20 vs. 2:30 with Iden9filer® 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


Thank You

© 2013 Life Technologies Corpora9on. All rights reserved. The trademarks men9oned herein are the property of Life Technologies Corpora9on or their respec9ve owners. The content provided herein may relate to products that have not been officially released and is subject to change without no9ce. “FOR FORENSIC AND PATERNITY USE ONLY”

http://www.slideshare.net/Lifetech_HID http://www.lifetechnologies.com/globalfiler

Technology

Improved Recovery of Interpretable Results from Challenging Samples Using the GlobalFiler™ Kit - Webinar