improving your casework workflow with a next generation quantification solution

1 Life Technologies™ |

Improving your Casework Workflow with a Next Generation Quantification Solution Allison Holt R&D Scientist


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


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


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*


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


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


Prototype Sensitivity – Data Consistency

1.2 pg/µL

20 ng/µL

DNA (ng/µL)

Sensitive and Reproducibl

e


Prototype Sensitivity – Data Consistency

1.2 pg/µL 20 ng/

µL

DNA (ng/µL)

Sensitive and Reproducibl

e


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


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


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


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


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


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


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


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


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


HID RT v1.2 Software


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


Setup a Run Assign Samples – Plate Layout view


Data Analysis Analysis Summary – QC standards to samples


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


STR Amplification Set-up Tool

Dilu4on Se\ngs STR Reac4on Se\ngs


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


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


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


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


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”


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

improving your casework workflow with a next generation quantification solution

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