extending your quantification workflow for upfront ... · • analyst time saving as well as assist...
TRANSCRIPT
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The world leader in serving science
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
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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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