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Improving your Casework Workflow with a Next Generation Quantification Solution presentation given at the 2013 Life Technologies Forensic Seminar Series
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1 Life Technologies™ |
Improving your Casework Workflow with a Next Generation Quantification Solution Allison Holt R&D Scientist
2 Life Technologies™ |
Quan%filer® Duo Kit Quan%filer® Human Kit Quan%filer® Y Human Kit
Three kits – for different applica4ons
Quan4fies DNA from wide variety of sources Works with wide variety of DNA input Sample Extrac4on chemistry compa4ble
Quantifiler® Kits Product Family
Quan%filer® HP Kit Quan%filer® Trio Kit HID RT v1.2 So=ware
Two new Quan4filer® kits in 2013 +
Flexible workflow soLware Integrate with Next Genera4on STR Kits
3 Life Technologies™ |
Next Gen Quantifiler® HP & Quantifiler® Trio Kits More Accurate, Sensitive, and Specific
Higher Performance with Challenging Samples • Improved performance with low-‐ra4o male/female mixture samples • IPC op4miza4on to match performance of next genera4on STR kits • Gain key informa4on on sample quality prior to PCR amplifica4on
Enhanced Workflow & Data Analysis • Flexible protocols for improved sample throughput • Improved consistency and homogeneity of DNA standard • Customized workflow templates plus data analysis and STR setup tools
Fast & Accurate Decision Tool • Shorter 4me to results compared to original quan4fica4on kits • Increased assay sensi4vity and accuracy • Solid decision making for forensic casework sample processing pathways
Fall 2013
Take Control of your Workflow
4 Life Technologies™ |
Next Gen Quantifiler® Kit Has Faster Protocol
50°C 2 min. 95°C 10 min. 95°C 15 sec 60°C 1 min
Quantifiler ® Human or Duo
Quantifiler ® Human Plus or Trio
95°C/2 min 95°C/ 9 sec 60°C/ 30 sec
Sample
*Protocol & thermal cycling conditions are approximate
Amp Time ~ 90 min Amp Time 60 min*
5 Life Technologies™ |
Streamlined Workflow Solutions
• Flexible options for DNA standard curves decrease reaction setup time and more samples per plate if a reduced number of standards are used
• Preconfigured Quantifiler ® Templates streamline sample setup – simply add/import sample names
• Decreased Cycle Amplification Time for faster time
to result • Integrated STR setup tools enable faster STR setup
6 Life Technologies™ |
Multicopy Targets
Multicopy Targets selected: 1. Maximum sensitivity
2. Increased reproducibility
3. Awareness of homogeneity in populations • Extensive bioinformatics analysis • Tested with population database samples
7 Life Technologies™ |
Prototype Sensitivity – Data Consistency
1.2 pg/µL
20 ng/µL
DNA (ng/µL)
Sensitive and Reproducibl
e
8 Life Technologies™ |
Prototype Sensitivity – Data Consistency
1.2 pg/µL 20 ng/
µL
DNA (ng/µL)
Sensitive and Reproducibl
e
9 Life Technologies™ |
Sensitivity Sample 007 DNA
§ At 1.25 pg/µL, partial profile is obtained with NGM® Select and all detectors in Quantifiler® Trio give positive assay results.
§ At 1.25 pg/µL, autosomal target CTs are ~36.
Quantifiler® Results STR Results
10 Life Technologies™ |
Sensitivity Sample 9947A DNA
§ At 1.25 pg/µL, partial profile is obtained with NGM® Select and all detectors in Quantifiler® Trio give positive assay results.
Quantifiler® Results STR Results
11 Life Technologies™ |
Internal Positive Control Correlates to Next Generation STR Kits
§ Master mix and primers engineered to correlate better with next generation STR chemistries § Inhibitor tolerance is 5X higher then Quantifiler® Duo
-2
0
2
4
6
8
10
12
14
16
0 20 40 60 80 100 120
CT S
hift
(CT S
ampl
e-C
T Cle
an D
NA)
Humic Acid Concentration (ng/uL)
CT Shift Due to Humic Acid Inhibition
Autosomal
Y Target
IPC
• 007 at 100 pg/µL • Inhibitor Concentrations in
the Quantifiler® Assays • 20-100 ng/µL humic
acid • Identifiler® Plus Reactions
with 10 µL sample volume
• NOTE: multiply 5x inhibitor concentration of quantitation reaction to obtain the relative inhibitor concentration for STR reactions
Identifiler® Plus Results (Alleles detected) Full 23/29 No profile
12 Life Technologies™ |
Internal Positive Control Correlates to Next Generation STR Kits
CT Shift Due to Hematin Inhibition
-2
0
2
4
6
8
10
12
14
16
0 25 50 75 100 125 150 175
Hematin Concentration (uM)
CT
Shift
(CT
Sam
ple -
CT C
lean
DNA
) AutosomalY TargetIPC
Identifiler® Plus Results (Alleles detected)
Full Profile No profile
• 007 at 100 pg/µL • Inhibitor Concentrations in the
Quantifiler® Assays • 50–150 µM hematin
• Identifiler® Plus Reactions with 10 µL sample volume
• NOTE: multiply 5x inhibitor concentration of quantitation reaction to obtain the relative inhibitor concentration for STR reactions
§ Master mix and primers engineered to correlate better with next generation STR chemistries § Inhibitor tolerance is 5X higher then Quantifiler® Duo
13 Life Technologies™ |
Improved Performance Male/Female Mixtures
§ Male DNA detected with increasing amounts of background female DNA § IPC Ct remained constant (26.3- 26.7)
0
0.5
1
1.5
2
2.5
3
0:1 1:0 1:1 1:5 1:10 1:20
Qua
ntity
(ng/
uL)
Ratio (male:female)
Y Target ConsistencySamples have 100 pg/uL Male DNA
Y Target
Autosomal Target
14 Life Technologies™ |
Multiple Targets Balanced and Consistent
0.001
0.010
0.100
1.000
10.000
100.000
1:0 1:800 1:1000 1:1200 1:1400
Mean Q
uant Valu
e (ng/uL) N = 3
Mixture Sample
SA
LA
Y
• Well balanced targets with non-degraded DNA • Y target remains constant in background of high female DNA
15 Life Technologies™ |
Quality Index (QI) Comparison of Large Autosomal (LA) Target to Small Autosomal (SA) Target
• Difference between the two targets assists users in identifying possible degradation
0.0001
0.0010
0.0100
0.1000
1.0000
10.0000
100.0000
Ctrl Low Med Hi
DNA Qua
n%ty (n
g/uL) b
y Target
DNA Degrada%on Condi%on
Small Autosomal
Large Autosomal
16 Life Technologies™ |
Light Degradation QI = 2.6 26 alleles (full profile)
Medium Degradation QI = 23 16 alleles
High Degradation QI = 300 6 alleles
AmpFℓSTR® NGM™ PCR Amplification Kit
Quality Index (QI) Artificially degraded human genomic DNA
17 Life Technologies™ |
Blood QI = 0.95
Sexual assault trace QI = 4.5
Saliva on fabric QI = 31.3
Saliva on fabric QI = 115
NGM Select™ Kit profiles
Quality Index (QI) Casework samples (not artificially degraded)
External test site data
Assists user in deciphering inhibition vs. degradation
18 Life Technologies™ |
HID RT v1.2 Software
19 Life Technologies™ |
Setup a Run Define Samples, Sample Type and View Targets
Standards, targets, run method and experiment properties have been
pre-defined for each Quantifiler® template
20 Life Technologies™ |
Setup a Run Assign Samples – Plate Layout view
21 Life Technologies™ |
Data Analysis Analysis Summary – QC standards to samples
22 Life Technologies™ |
Data Analysis Analysis Summary
§ Customizable View designed to streamline data review
§ Overview of standard curve, NTC and unknown sample quality flags − View samples that
meet/do not meet defined threshold
§ Hyperlinks to flagged samples for quick data review
1
2
3
23 Life Technologies™ |
STR Amplification Set-up Tool
Dilu4on Se\ngs STR Reac4on Se\ngs
24 Life Technologies™ |
STR Amplification Set-up Tool
§ Select the AmpFℓSTR® Kit(s) for downstream analysis from the STR Kit Library
§ Each kit template is pre-populated with default reaction set-up parameters and dilution scheme (customizable)
§ Software determines sample dilution and volume added to the STR reaction based on Quantifiler® results
25 Life Technologies™ |
Lab Configurable Software Solutions
§ Default Quantifiler ® and AmpFℓSTR® Templates can be optimized based on your workflow – simply add/import samples
§ Default Analysis Settings can be defined based on your SOP
− Choose which Data Quality Flags you wish to use as well as threshold values
− Dilution Calculations – define overage, min/maximum volumes, dilution factors
− STR Reaction Setup – target input amounts, reaction component and sample volumes.
§ Data Display and Export – define Table, Plot and Plate views and Export formats
26 Life Technologies™ |
Be Part of the Change!
Contact your local Human Identification support and sales representative
Become a Test Site
• Support our rigorous development process • Give us feedback and help us build the kits that meet your needs • Be the first to evaluate the Quantification kits with your challenging samples
27 Life Technologies™ |
Acknowledgement
§ R&D: − Robert Green, Allison Holt, Mary Ma,
Pius Brzoska, Emanuel Langit, Sharon Wootton, Julio Mulero
§ Oligo − Sarah Ruhe, Emma Duimstra, Hong
Ji § Product Management:
− Sheri Olson
§ Forensic Science Applications: − Jacki Benfield, Jeff Sailus, Jill
Muehling § Tech Transfer
− Ellen Kuo , Yenyoung Lei § Project Management
− Ada Wong § Manufacturing Sciences
− John Dektar, Yurah Yen, Ryo Hasegawa
§ Marketing − Shareen Fousekis, Jonathan Tabak
§ Manufacturing − Marilena Philippidou, Catherine
Duke
28 Life Technologies™ |
Thank You!!
© 2013 Life Technologies Corpora%on. All rights reserved.
The content provided herein may relate to products that have not been officially released and is subject to change without no%ce.
“FOR FORENSIC AND PATERNITY USE ONLY”
29 Life Technologies™ |
Streamlined Workflow Solutions
8 Standards
Manual Run setup
90 min Amp
Manual STR setup
Flexible Standards Op%ons
Streamlined Run setup -‐
Quantifiler® Templates
~60 min Amp
Integrated, Automated STR
setup
Quantifiler® Trio and Human Plus
Quantifiler® Duo and Human