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Amy Liberty HIDS 2017 - Vienna

Extending your Quantification Workflow for Upfront Decision Support to Streamline Sample Processing

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•  Introducing the Applied Biosystems™ QuantStudio™ 5 Real-Time PCR Instrument and HID Real-Time Software v1.3

•  Enabling customer flexibility with a Virtual Standard Curve

•  Applied Biosystems™ Quantifiler™ Trio Quantification Kit: A sample assessment solution

•  Innovative workflow solution: The Y-screen Assay using the Quantifier Trio Quantification Kit

•  Quantification Decision Support for NGS

Overview

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What’s next? 2020

25+ years of Innovation and partnership in Forensics

1995 310

5-dye Technology Mobility Modifiers

2001

6-dye Technology

2012

Degradation index 2013

2015 NGS Workflow

NGS HID Panels 2014

2016 Ion S5™ System

?

2000-2007 31xx-series

2010 3500-series

Blue & Green

Kits 1996

2013 Ion PGM™

GeneScan/Genotyper™ GeneMapper ID-X™ Converge™

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Innovation In DNA Quantification

Quantifiler™ Human and Y Kits

2004

Quantifiler™ Duo Kit 2008

Quantifiler™ Trio Kit

2014

2017 QuantStudio™ 5 Real-Time

PCR instrument

2003 7000 Real-time PCR

Instrument

2007 7500 Real-Time PCR instrument

HID Real-time SW v1.1 HID Real-time SW v1.2 HID Real-time SW v1.3

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•  Fewer off-scale, overamplified, or underamplified samples

•  Normalized profiles result in reliable data interpretation

•  Designed to reduce the need for downstream reanalysis, which enables long-term cost savings

•  Provides key sample information to enable forensic workflow decisions

•  Enables conservation of sample through targeted downstream assay decisions

The Value of DNA Quantification

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•  Virtual standard curve capability enables 10-17% savings per run

•  Trusted HID design and analysis software Streamlined

•  Interactive touch screen provides on-instrument capabilities

•  Easy and intuitive interface Interactive

• White LED light sources with median 5-year life span • Less frequent maintenance, with dye calibrations

recommended every 2 years • Validated system

Reliable

•  Multiplexing capability for up to six dyes, enabling future quantification technology Innovative

QuantStudio™ 5 Real-Time PCR Instrument

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•  Real-time PCR analysis software version designed specifically to meet the needs of the HID customers

•  Developed to complement Quantifiler Kits, the 7500 System, the QuantStudio™ 5 System, and the HID workflow

•  Streamlined plate set-up, run, and automated analysis tools

•  Virtual standard curve functionality

HID Real-Time PCR Analysis Software v1.3 Overview

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Broadening Your Software Capabilities

The Virtual Standard Curve feature as part of the HID Real Time SW enhancement allows you the option to define your own standard curve values and use those values to calculate DNA quantitation. User can also use the traditional standard curve run on the plate.

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•  Streamlined and Time Saving •  Simple User Interface allowing users to define their own standard curve values

for all targets.

•  Analyst time saving as well as assist in workflow decision making. •  Efficiency and Cost Savings

•  The virtual standard curve feature allows for samples to be run in the place of standards, increasing throughput

•  Flexibility •  Adaptable analysis settings allowing for permutations of running and analyzing

with traditional or virtual standard curves.

•  Accuracy •  Mitigates concerns of variability introduced due to operators on

standard curve.

Virtual Standard Curve Benefits

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Accurate results in less time for your most challenging samples.

•  Greater correlation with downstream results when used in conjunction with

latest generation STR Kits

•  Increased robustness of reactions help deliver better correlation of results to the latest generation STR kits

•  Evaluate sample quality (degradation and inhibition) and quantity in a single reaction.

•  Improved IPC helps deliver results that correlate to dropout in the STR kit reaction •  Accurate negative results and quality index deliver confidence in downstream processing

decisions

Quantifiler Trio Kit: A Sample Assessment Solution

Quan%filer  Trio  Kit  

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Quantifiler Trio Kit: A Sample Assessment Solution

Quantifiler Trio Results Indicates Processing Options*

Male quantity (Y target) = undetected Insufficient male DNA

Stop processing or prioritize other case samples

High Male:Female ratio Unlikely to obtain informative male autosomal STR profile Perform Y-STR analysis

IPC CT shift Significant PCR inhibitors present Dilute and/or re-purify sample

Degradation Index (DI) > 1 or blank DNA degradation likely

1)   Concentrate sample 2)   Increase DNA target and/or sample

input volume for STR rxn 3)   Consider NGS

•  Enables rapid sample assessment for more informed casework lab processing decisions

* These are general guidelines that may not apply to all samples depending on the inhibitors present, the varying quantity of contributor DNA in mixed samples and the STR kit used. Perform validation studies to determine interpretation guidelines and procedures for your laboratory.

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Why are Sexual Assault Samples Challenging?

Time-consuming

•  Among the most difficult samples encountered by forensic labs

•  At least 4-6 hours to review evidence in a single SAK

•  A high percentage of cases for many laboratories

Sample Quality

•  Small quantity of male DNA with relatively high quantity of female DNA

•  30-50% of SAKs yield no male DNA1

•  PCR inhibitors or DNA degradation

Labor-intensive

•  Microscopic sperm search

•  Serological assay Interpretation issues

•  Differential extraction

1 (2014 Voice of Customer Survey, Thermo Fisher Scientific)

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Y-Screen for swabs Uses the Quantifiler™ Trio kit as an upfront DNA confirmatory swab screening method to detect the quality and presence/absence of amplifiable male DNA

•  Streamlined solution that can reduce number of backlogged samples and better manage incoming cases

•  Empowers analysts with qualitative data to confidently identify samples with amplifiable male DNA

•  Quickly eliminates labor-intensive differential extractions of samples where no male DNA is present

Y-screen Assay Using the Applied Biosystems™ Quantifiler™ Trio Kit

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Sample preparation

and incubation

•  Take small cutting from swab •  Place in LySep column •  Add Sodium Hydroxide •  Incubate for 10 minutes at 80ºC with

shaking

Centrifuge and dilute

•  Centrifuge column •  Add acetic acid •  Perform 1/5 dilution with TE-4 buffer

Quantification using

Quantifiler Trio

•  Perform standard Trio setup and plate run

Y-screen Assay Protocol

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Traditional Workflow

Differential extraction

Low concentration

Degraded How good/bad?

Inhibited

M:F ratio

Autosomal STR

Y STR

Mini STR

Y or no Y?

High incidence of

no male profile at final data analysis

Screening Extraction Quantitation STR Data Analysis

Manual slide search

Serological assays

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+ Male DNA

- Male

DN

A

Y-screen SAK Workflow

Y-Screen DNA Assay Quantifiler Trio

Differential extraction

Low concentration

Degraded

Inhibited

M:F ratio

Autosomal STR

Y STR

Mini STR

Y or no Y?

High incidence of

no male profile at final data analysis

Improved results with confidence

Screening Extraction Quantitation STR Data Analysis

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Y-screen Protocol Options

Reduce Backlogged Samples

Streamline Use of Differential Extraction

Empower Confident Decision Making

•  Screen swab evidence in SAKs quickly for the presence/ absence of male DNA

•  If male DNA is present, process swab for STR

•  If no male DNA is present, write report, no further processing

•  Eliminate differential extractions for negative Y-Screen and presumptive testing samples

•  Process only the main

SAK swabs with differential extraction, and use Y-Screen for other swab evidence

•  Add a DNA confirmatory test to complement presumptive screening tests

•  Better inform analyst decisions on the presence/absence of male DNA in sexual assault kit swab samples

Sexual assault kit DNA screening prior to standard sexual assault sample processing workflow

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•  “Direct to DNA” for the process of foregoing traditional serology

•  “Although the detection and characterization of body fluids in a forensic laboratory has not changed considerably over time, DNA testing has dramatically increased in sensitivity.”

•  “Processing the swabs from a SAK for DNA first allows for potential reduction in false negative serology results of swabs that are not moved on for DNA analysis because they fell below the serological test’s limit of detection.”

New SWGDAM Approved Guidelines (December 5, 2016)

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Y-Screen Test Site Commentary

“Predicted downstream STR results better than traditional screening

assays”

“It tells you for sure what not

to move through”

“Protocol was quick and easy to

use – very straightforward”

“There weren’t any samples that failed to give a positive Y-Screen result that went on to give a

usable profile”

“Could eliminate 1/3 to 1/4 of our

samples upfront so it

could save us a great

amount of time”

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Y-screen HID Professional Services Validation Solution

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Applied Biosystems™ Quantifiler™ Trio Kit: A Sample Assessment Solution

Quantifiler Trio Results Indicates Processing Options*

Male quantity (Y target) = undetected Insufficient male DNA

Stop processing or prioritize other case samples

High Male:Female ratio Unlikely to obtain informative male autosomal STR profile Perform Y-STR analysis

IPC CT shift Significant PCR inhibitors present Dilute and/or re-purify sample

Degradation Index (DI) > 1 or blank DNA degradation likely

1)   Concentrate sample 2)   Increase DNA target and/or sample

input volume for STR rxn 3)           ‐‑‒–—―‖‗‘’‚‛“”„‟†‡•‣․‥…‧

SGN redisnoC ⁞⁝⁜⁛⁚⁙⁘⁗⁖⁕⁔⁓⁒⁑⁐⁏⁎⁍⁌⁋⁊⁉⁈⁇⁅⁆⁄⁃⁂⁁⁀‿‾‽‼※‹›‸‷‶‵‴″′‱‰ 

•  Enables rapid sample assessment for more informed casework lab processing decisions

* These are general guidelines that may not apply to all samples depending on the inhibitors present, the varying quantity of contributor DNA in mixed samples and the STR kit used. Perform validation studies to determine interpretation guidelines and procedures for your laboratory.

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Gain key quantity and quality information from valuable samples so compromised that may be below the threshold of detection by traditional CE

The Degradation Index (DI) gives upfront decision support on how to recover the most information from highly compromised DNA samples

Applied Biosystems™ Quantifiler™ Trio easily integrates your current forensic workflow into Next Gen Sequencing (NGS) with the Ion Chef™ and Ion S5™ Systems.

Quantification and NGS

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CE + NGS = Integrated Forensics Workflow

DNA Extraction

Quantification By qPCR

50-70% of Casework

Mixtures Degraded

Investigative leads

STR by CE

Converge Platform

Analysis GMID-X

Brings CE + NGS data together

Precision ID NGS System

NGS will not displace CE for the foreseeable future

Position NGS for difficult samples

Integrate NGS into current workflow

Create integrated NGS workflow

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Extending Quantification in Your Integrated HID Workflow

Detection & Analysis

Improve Efficiency. Helps Increase First-Pass Success Rates.

GeneMapper™ ID-X Expert System

Software

GlobalFiler™ STR Kits

3500/3500xL Genetic Analyzer

3130/3130xl Genetic Analyzer

Extraction Quantification Amplification

7500 Real-Time PCR Instrument

Quantifiler™ HP and Trio Kits

PrepFiler™ Forensic DNA Extraction kit

ProFlex™ Thermal Cycler

AutoMate Express™DNA

Extraction System

Collection

Copan NUCLEIC-CARD™

and 4N6FLOQSwabs™

Precision ID Panels

Next Gen Sequencing

Y-Filer™ Plus Kit

Ion Chef™ System and Ion S5™ Systems

Converge™ Software

QuantStudio™ 5 Real-Time PCR

Instrument +

HID Real Time Software1.3 w/VSC

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