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1 Life Technologies™ |
Improved Recovery of Interpretable Results from Challenging Samples Using the GlobalFiler™ Kit
Lisa Calandro, M.P.H. Director, Product Management
2 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
3 Life Technologies™ |
Detection & Analysis
Our Mission: Improve Efficiency. Increase Success Rates.
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
Bode Buccal DNA Collector
Copan NUCLEIC-CARD™
and 4N6FLOQSwabs™
Screening
ParaDNA®
Converge™ Software DNA Workflow
Kinship and Paternity
Direct Search Familial Search
NEW
NEW
NEW
NEW
NEW
Converge™
Software
4 Life Technologies™ |
Poll: What is the Most Time Consuming Step for the Analyst in the Casework Workflow?
A. Sample Screening
B. Sample Extraction
C. Quantification
D. Amplification
E. CE Analysis
F. Data Interpretation
8 Life Technologies™ |
Quantifiler® Kits Product Family
Quan&filer® Duo Kit Quan&filer® Human Kit Quan&filer® Y Human Kit
Three kits – for different applica9ons • Quan9fies DNA from wide variety of sources
• Works with wide variety of DNA input • Sample Extrac9on chemistry compa9ble • Guides selec9on of op9mal AmpFlSTR® PCR Amplifica9on Kit
• Accurate, Sensi9ve & Specific
Next Gen Quan&fica&on
Two new Quan9filer ® Kits in 2013
Flexible workflow so`ware
To integrate with Next Genera0on STR kits
NEW!
Quan&filer® HP Kit Quan&filer® Trio Kit HID RT v1.2 SoAware
9 Life Technologies™ |
Next Gen Quan&filer® Kits More Accurate, Sensi&ve, and Specific
Higher Performance with Challenging Samples • Improved performance with low-‐ra9o male/female mixture samples • IPC op9miza9on to match performance of next genera9on STR kits • Gain key informa9on on sample quality prior to PCR amplifica9on
Enhanced Workflow & Data Analysis • Improved consistency and homogeneity of DNA standard • Customizable quan9fica9on workflows for complex casework sample processing
Fast & Accurate Decision Tool • Shorter 9me to results as compared to original quan9fica9on kits • Increased assay sensi9vity and accuracy • Solid decision making for forensic casework sample processing pathways
Fall 2013
Two new Quantifiler ® Kits in 2013 To integrate with Next Generation STR kits
‘Take Control of your Workflow’
11 Life Technologies™ |
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
12 Life Technologies™ |
A short history of STR Kit Multiplexing
Kit Name Release Date # of Loci # of Dyes*
AmpFlSTR® Blue 1996 3 2
AmpFlSTR® Profiler® 1997 10 4
AmpFlSTR® Identifiler® 2001 16 5
GlobalFiler™ 2012 24 6
*Includes size standard
13 Life Technologies™ |
Poll: What is the main benefit of 6-‐Dye chemistry?
A. Increased discrimination power
B. Improved profile recovery from challenging samples
C. Improved genotyping accuracy
D. Increased concordance with legacy data (previous kits)
E. All of the above
15 Life Technologies™ |
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
16 Life Technologies™ |
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 calcula0ons based on a US Caucasian Database
DYS391
Amel Y indel
All gender markers located in the
same dye channel for ease of
interpretation
17 Life Technologies™ |
• 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?
18 Life Technologies™ |
• 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
19 Life Technologies™ |
Poll: What percentage of evidence profiles do you handle on a regular basis that are mixtures of 2 or more
contributors?
A. <25%
B. 25-40%
C. 40-60%
D. 60-80%
E. >80%
20 Life Technologies™ |
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
22 Life Technologies™ |
Overlay of SE33 Data from 4 individuals
SE33 extremely valuable for mixture interpretation In this case, we have 4 donors and 7 peaks
23 Life Technologies™ |
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
24 Life Technologies™ |
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!
25 Life Technologies™ |
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
27 Life Technologies™ |
6-Dye Configurations
Chemistry/ Standards
GeneMapper® ID-‐X SoAware
v1.4
Service
3730 DNA Analyzer /
31xx Gene&c Analyzers
3500 Gene&c Analyzer
Data Collec&on SoAware
1 2 3
31xx/3730 only All Instruments
28 Life Technologies™ |
GlobalFiler™ Kit Upgrade Roadmap
Product Type 3500 3730 3130 3100
GeneMapper® ID-‐X SoAware v1.4
Data Collec&on SoAware v4.0 N/A
6-‐dye Data Collec&on Module N/A
Service Call/Computer N/A
Instrument Upgrade N/A N/A N/A
30 Life Technologies™ |
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
31 Life Technologies™ |
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
32 Life Technologies™ |
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® Direct Kit & NGM SElect™ Express Kit
~2.5 hours
GlobalFiler™ Express Kit ~80 mins
95°C/ 1 min 94°C/ 20 sec 59°C/ 90 sec 60°C/ 10 min
34 Life Technologies™ |
TS1 TS2 TS3 TS4 TS5
Extrac&on Method Organic or EZ1 Organic Magne9c
Bead Qiagen
Inves9gator Organic
Compara&ve 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
35 Life Technologies™ |
300 Test Site #1
GlobalFiler™ Kit Baseline Quality
300
300
Test Site #2
Test Site #3
Read Region 74 444
Non-template Controls
36 Life Technologies™ |
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
37 Life Technologies™ |
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.
38 Life Technologies™ |
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
39 Life Technologies™ |
[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
40 Life Technologies™ |
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)
41 Life Technologies™ |
GlobalFiler™ Kit Test Site Results
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)
42 Life Technologies™ |
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)
GlobalFiler™ Kit Test Site Results
43 Life Technologies™ |
GlobalFiler™ Kit Test Site Results
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)
44 Life Technologies™ |
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
45 Life Technologies™ |
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
Bouleneck Swab / 50pg
Drawer Swab/60pg
Swab on Safe / 50pg
Wheel Swab /80pg
Cigareue Buu / 40pg
Cigareue Buu / 30pg
Cap Swab / 165 pg
Stone / Undet
No. Alleles R
ecovered
Par&al 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
46 Life Technologies™ |
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…”
47 Life Technologies™ |
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…”
48 Life Technologies™ |
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
50 Life Technologies™ |
Development of a Next Generation Y-STR Multiplex
§ Designed to enable: − Increased discrimination power and potential to discriminate
between close male relatives with expanded marker set − Higher number of alleles recovered at low DNA input amounts − Improved recovery of alleles and intracolor balance in male/
female mixtures containing high female background − Improved resolution capacity for male/male mixtures − Improved recovery of alleles in inhibited samples
51 Life Technologies™ |
50 100 150 200 250 300 350 400
DYS627DYS389IIDYS635DYS389IDYS576
DYS391DYS448
Y GATA H4DYS19DYS458DYS460
DYS518DYS392DYS438DYS390DYS456
DYS449DYS385ab
DYS437DYS570
DYS533DYF387S1ab
DYS481DYS439DYS393
Yfiler® Plus Kit Configura&on
Rapidly Mutating Y-STR Standard Y-STR New Y-STR Markers →
1
3
6
8
10
2
4 5
7
9
11
52 Life Technologies™ |
Y-STR Community
§ Web based Y-STR resource hub and information center developed to: − Provide the most updated information
in the area of forensic Y-STR analysis − Facilitate communication between Life
and its customers − Help forensic scientists around the
world collaborate and stay in touch
http://www.lifetechnologies.com/ycommunity
53 Life Technologies™ |
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