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

1/10/2013 2

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

1/10/2013 3

Development of Direct Amplification on Treated Paper Substrates

1/10/2013 4

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

1/10/2013 5

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

1/10/2013 6

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

1/10/2013 7

Optimization of Reaction Buffer for 1.2 mm Discs

Component 1

Response Component 2

Use of Design of Experiments (DOE) Approach

1/10/2013 8

Optimization of Identifiler® Direct Kit Master Mix Buccal sample on 1.2 mm FTA® disc before DOE

1/10/2013 9

Optimization of Identifiler® Direct Kit Master Mix Buccal sample on 1.2 mm FTA® disc after DOE

1/10/2013 10

− 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

1/10/2013 11

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

1/10/2013 12

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

1/10/2013 13

Expansion of the Direct Amplification Workflow to Non-FTA Substrates

Untreated Paper

1/10/2013 14

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

1/10/2013 15

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™

1/10/2013 16

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

1/10/2013 17

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

1/10/2013 18

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

1/10/2013 19

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

1/10/2013 20

Expansion of the Direct Amplification Workflow to Non-FTA Substrates

Buccal Swabs

1/10/2013 21

Pros − Easy to use − Inexpensive − Multiple types available

Buccal Swabs

Cotton Swab

Foam Swab

T-Swab

Flocked Swab

Omni Swab

1/10/2013 22

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

1/10/2013 23

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

1/10/2013 24

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)

1/10/2013 25

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

1/10/2013 26

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

1/10/2013 27

Expansion of the Direct Amplification Workflow to Other STR Marker Sets

1/10/2013 28

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

1/10/2013 29

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

1/10/2013 30

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

1/10/2013 31

AmpFSTR® NGM SElect™ Express Kit Example Profiles: Treated Paper

Blood on FTA® Classic Card

Buccal on Copan NUCLEIC-CARD™

1/10/2013 32

AmpFSTR® NGM SElect™ Express Kit Example Profiles: Untreated Paper

Blood on 903 Paper

Buccal on Bode Buccal Collector™

1/10/2013 33

AmpFSTR® NGM SElect™ Express Kit Example Profiles: Swabs

Buccal on Copan Flocked Swab

Buccal on Whatman Omniswab

1/10/2013 34

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

1/10/2013 35

1/10/2013 36

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

1/10/2013 37

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

1/10/2013 38

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

1/10/2013 39

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

1/10/2013 40

Optimized Parameters = More Efficient Analysis U

nopt

imize

d Optim

ized

1/10/2013 41

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

1/10/2013 42

*Kits currently in development

1/10/2013 43

Thank You © 2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners

For Forensic or Paternity Use Only.