The Evolution of Direct Amplification: From Sample to Result in 2 Hours*
Lisa Calandro, M.P.H. Director, Product Management Human Identification, Life Technologies
* Typical workflow for 24 samples
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Traditional Single Source Sample Workflow
Time to Result
(hrs/per 24 samples) ~1-3.5 ~0-2 ~4 ~1.5 ~1
Total Time to Result = ~7-12 hrs
Collection Sample Preparation
Quantitation/ Normalization Amplification Detection Analysis
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Development of Direct Amplification on Treated Paper Substrates
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Evaluation of Direct Amp Capability of the Identifiler® Kit
Control DNA 007 25 μL Reaction Volume
1.2 mm Blood on FTA® Disc
No full profiles under direct amp conditions
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Effect of Reducing Disc Size on Identifiler® Kit Direct Amp Performance
0.5 mm blood on FTA® disc
1.2 mm blood on FTA® disc
Control DNA 007 (1 ng)
0.75 mm blood on FTA® disc
But: − No automated
0.5 mm punch head options
− 0.5 mm disc size too small for buccal samples
− 0.5 mm discs very hard to handle
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Identifiler® Kit Direct Amp Performance Evaluation
Possible explanations for poor performance − Insufficient DNA?
− Excess PCR inhibitors?
0.75 mm disc ~ 22.08 ng DNA
0.5 mm disc ~ 9.81 ng DNA
1.2 mm disc ~ 56.52 ng DNA Height = 1mm
1 μL blood ~ 50 ng DNA
Sufficient DNA still available Inhibitor concentration reduced by >5-fold
0.5 mm disc ~ 9.81 ng DNA
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Optimization of Reaction Buffer for 1.2 mm Discs
Component 1
Response Component 2
Use of Design of Experiments (DOE) Approach
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Optimization of Identifiler® Direct Kit Master Mix Buccal sample on 1.2 mm FTA® disc before DOE
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Optimization of Identifiler® Direct Kit Master Mix Buccal sample on 1.2 mm FTA® disc after DOE
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− 3 x liquid blood samples − 80 μL of blood onto FTA® Classic (passive diffusion) − Sample 1.2 mm discs from the center to the edge of the blood stain − Perform replicates for each position
Effect of Punch Position on Sample Peak Heights
1 3 2 4 5
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Direct Amplification from Treated Paper
FTA® Classic Card
EasiCollect® System
Identifiler® Direct Kit
Identifiler® Kit
Exam
ple
1 Ex
ampl
e 2
Identifiler® Direct Kit
Identifiler® Kit
Blood on FTA® samples
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Identifiler® Direct Kit Validation: External Test Site Results
Sample Type
PCR Success Rate Interpretation Success Rate Number of
Samples Tested Range of Success rates
Mean Success Rate
Range of Success rates
Mean Success Rate
VTS Study
Blood 99.4 % 99.4 % 95.7 – 98.8% 97.3 % 414
Buccal 91.8 – 99.4% 97.1 % 84.2 – 95.5% 90.9 % 653
CTS Study
Blood 100% 100 % 98.8 – 100% 99.8 % 437
Buccal 98.7 – 100% 99.0 % 91.7 – 100% 94.7 % 703
1st Pass Success Rate Definition − All profile peaks higher than specified threshold − Off scale peaks produce no artifacts which interfere with profile
interpretation > OL-labelled pull-up peaks <20% of highest peak of the marker
> No split, double called peaks
> Stutter peaks < 20% of marker or < marker stutter cut off, whichever is higher
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Expansion of the Direct Amplification Workflow to Non-FTA Substrates
Untreated Paper
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Identifiler® Direct Kit development and optimization performed on FTA® substrates only
Laboratories using untreated paper or swab substrates and/or alternative marker sets looking to recognise time and cost savings of the Direct Amplification workflow
Expansion of the Direct Amplification Workflow to Non-FTA® Substrates
Goal: Enable direct amplification of non-FTA® buccal samples
Minimal additional workflow steps
High quality, well balanced profiles
No introduction of artifacts
Prep-n-Go™
Buffer
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Direct Amp Workflow: Untreated Paper Substrates
Collect Samples on Untreated Paper
One New Step
Electrophorese
Amplify
Punch 1.2 mm disc
Add PCR reagents
Add 2 µL Prep-n-Go™ Buffer Untreated paper
protocol development
performed on Bode Buccal Collector™
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Identifiler® Direct Kit Amplification of Bode Buccal DNA Collector™ Samples lysed with Prep-n-Go™ Buffer
2 different individuals amplified for 26 PCR cycles Well-balanced profiles within each dye color
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Performance Study Results Summary
Test Site Number of
Samples Tested
PCR Cycle
Number
CE Platform
PCR Success Rate Interpretation Success Rate
Number of Full Profiles
First Pass Success Rate
Number of Full Profiles
First Pass Success Rate
1 82* 27 3130xl 81/82 98.8% 78/82 95.1%
2 80 26 3130xl 78/80 97.5% 78/80 97.5%
3 84* 26 3130xl 83/84 98.8% 79/84 94.0%
4 84* 26 3130xl 80/81 98.8% 76/81 93.8%
5 84* 26 3730 83/84 98.8% 83/84 98.8%
6 84 26 3500 82/84 97.6% 74/84 88.1%
Life Technologies 84 26 3130xl 82/84 97.6% 79/84 94.0%
Total 582 569/582 97.8% 547/582 94.0%
* Used real offender samples
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Performance Study: Bode Buccal DNA Collector™ Samples lysed in Prep-n-Go™ Buffer
Peak Heights Intracolor Balance
All amplifications performed using the Identifiler® Direct Kit
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Identifiler® Direct Kit w/ Prep-n-Go™ Buffer Identifiler® Direct Kit w/Other Buffer
1200 1200
Lysis Buffer Performance Comparison
No allelic drop-out using Prep-n-Go™ Buffer
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Expansion of the Direct Amplification Workflow to Non-FTA Substrates
Buccal Swabs
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Pros − Easy to use − Inexpensive − Multiple types available
Buccal Swabs
Cotton Swab
Foam Swab
T-Swab
Flocked Swab
Omni Swab
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Cons − High performance variability
among swab types − Donor variation − Sample collection techniques
and storage conditions − Limited opportunities for
automation increasing labor requirements and the risk of contamination
Buccal Swabs
Donor Mouth Conditions Collection Technique
Long-Term Storage Swab Age
Drying & Transport Conditions
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Flocked Swab Swab Structure Comparison
Cotton Swab
2 km microfiber 6 m microfiber
Sample stays entrapped in the
fiber wand
Sample is released quickly and in
higher amounts
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4N6FLOQSwabs™ by Copan
Distributed by FLOQSwabs™ consist of short
Nylon® fibers that are arranged in a perpendicular fashion
Excellent recovery of DNA
Maximum efficiency in collection capacity
Available with different anatomical and ergonomic designs
Certified free of Human DNA, Dnase and RNase
ETO-treated (Ethylene Oxide)
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Direct Amp Workflow: Identifiler® Direct Kit
Collect Samples on Buccal Swab
Lyse swab in 400 μL Prep-n-Go™ Buffer
Add PCR Reagents
Transfer Lysate
New steps
Electrophorese
Amplify
Incubate in oven adaptor at 99°C for 20 minutes
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Performance of Aged Swabs: Identifiler® Direct Kit & Prep-n-Go™ Buffer
4N6FLOQSwabs™
Puritan Swabs
Omni Swabs
Mean time between collection and analysis: 90 days; Amplification: 26 cycles
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Expansion of the Direct Amplification Workflow to Other STR Marker Sets
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Direct Amplification with the NGM™ & NGM SElect™ Kits NGM SElect™ Kit NGM™ Kit
Amplification of buccal samples on Bode Buccal Collector™ treated with Prep-n-Go™ Buffer
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Utilizes the same primer sequences as the NGM™ and NGM SElect™ Kits
Includes a new master mix optimised specifically to support direct amplification of swab and treated/untreated paper substrates
Delivers rapid cycling times through the introduction of a new fast-capable enzyme (< 1 hr)
May be amplified using the Veriti® 96-Well or 9700 (silver or gold-plated silver block) thermal cyclers − Veriti® 96-Well Thermal Cycler (standard) now supported for use with all
existing AmpFSTR® kits
Direct Amplification for the Expanded ESSL
AmpFSTR® NGM SElect™ Express Kit
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Direct Amp Workflow: NGM SElect™ Express Kit
Collect Samples on Buccal Swab
Lyse swab in 400 μL Prep-n-Go™ Buffer
Add PCR Reagents
Transfer Lysate
New steps
Electrophorese
Amplify
Heated lysis optional
depending upon swab age/type
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AmpFSTR® NGM SElect™ Express Kit Example Profiles: Treated Paper
Blood on FTA® Classic Card
Buccal on Copan NUCLEIC-CARD™
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AmpFSTR® NGM SElect™ Express Kit Example Profiles: Untreated Paper
Blood on 903 Paper
Buccal on Bode Buccal Collector™
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AmpFSTR® NGM SElect™ Express Kit Example Profiles: Swabs
Buccal on Copan Flocked Swab
Buccal on Whatman Omniswab
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Test Site
Sample Type
Cycle #
CE Platform N
Profile Assessment
Partial Profile
No
Prof
ile
Off
Scal
e Pr
ofile
s
First Pass Success Rate
50 RFU
175 RFU
450 RFU
50 RFU 175 RFU 450 RFU
# % # % # %
Repr
oduc
ibili
ty 3 Copan 25 3500xL 60 1 1 1 1 1 58 97 58 97 58 97
4 Copan (2 weeks) 26 3100 60 0 4 N/A 1 37 58 97 58 97 NA NA
5 Copan (Fresh) 25 3130xl 60 0 0 N/A 0 8 60 100 60 100 NA NA
Perf
orm
ance
3 Omniswab 27 3500xL 40 1 2 3 1 0 37 93 36 90 35 88
4 Prionics (2 months) 26 3100 40 0 2 N/A 1 31 39 98 39 98 NA NA
5 Omniswab 26 3130xl 42 0 1 N/A 0 7 42 100 42 100 NA NA
Test Site Results
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Factors Influencing Direct Amplification Results
Choice of kit
Choice of sample type/substrate Sample Transfer Efficiency
Punch Size and Position
Cycle Number
Data Analysis Parameters
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Maximising Direct Amplification Results
Swab Substrate Handling − Ensure swabs are fully dried before storing − Ensure swabs are stored correctly to prevent excessive degradation over
time
Swab Lysate Handling − If using a 96-well deep well plate for lysis, remove lysate from swab heads
when aliquotting for storage and discard the plate containing the swab heads to reduce contamination risk
− Avoid taking cell debris or precipitation from the bottom of the lysate tube when transferring swab lysate to amplification or storage plates/tubes
− Follow the kit-specific instructions for lysis and amplification > Heated lysis may improve performance dependent upon direct amplification kit, swab
age and type
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Maximising Direct Amplification Results
Thermal Cycling Platform − Life Technologies kits optimised for use on the 9700 with silver or gold-
plated silver block and the Veriti® 96-Well thermal cycler only > Not supported for use on the 9700 with Aluminium block or
> Not supported for use on the Veriti 96-well Fast thermal cycler
Choice of Cycle Number − Choose a cycle number that prevents allele drop-out and minimizes off-
scale alleles − Use of elevated cycle numbers may cause presence of artifacts
Optimisation of Software Analysis Settings − Use of settings appropriate to single source samples will reduce editing
requirements and facilitate expert system analysis
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Optimising Data Analysis Parameters for Single-Source Samples
Use of a global cut-off filter can reduce significantly the amount of editing required for single-source sample data
Peak Quality settings may also be adjusted to better reflect the characteristics of single-source samples
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Optimized Parameters = More Efficient Analysis U
nopt
imize
d Optim
ized
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Time to Result
(hrs/per 24 samples) ~1-3.5 ~0-2 ~4 ~1.5 ~1
Maximized Efficiency for Single Source Sample Processing
Time to Result
(hrs/per 24 samples) 0 0 0.75 0.55 0.1
Total Time to Result = ~7-12 hrs
Collection Extraction Quantitation/ Normalization Amplification Detection Analysis
Total Time to Result = ~2 hrs
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*Kits currently in development
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For Forensic or Paternity Use Only.