development of a “global” str multiplex for human identification analysis
TRANSCRIPT
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
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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
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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.
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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
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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
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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
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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)
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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
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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
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