Quantifiler™ Trio: Decision-support to help streamline Sexual Assault sample processing.

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Sheri Olson, Thermo Fisher Scientific Bode West Meeting March 2015, Coronado, CA

Quantifiler™ Trio: Decision-support to help streamline Sexual Assault sample processing.


NEW Quantifiler Trio Workflow Solutions

•  Y Screen for Swabs •  Quantifiler Trio as an upfront DNA confirmatory swab screening method to detect

presence / absence of male DNA prior to standard SAK sample processing. •  To help reduce backlogged samples •  Allow forensic analysts to feel confident in decision making about sperm / male DNA •  Eliminate labor intensive differential extraction for samples where no male DNA is present

•  Extremely Degraded Samples - NGS •  Quantifiler Trio easily integrates your current forensic workflow into Next Gen Sequencing

(NGS) with the HID-Ion Personal Genome Machine™ System •  The Degradation Index (DI) gives upfront decision support on how to recover the most

information from highly compromised DNA samples •  Gain key quantity and quality information from valuable samples so compromised that may

be below the threshold of detection by traditional CE

Expected Spring 2015


Quantifiler Trio Kit: A Sample Assessment Solution

Quantifiler Trio Data

Indicates* Processing Options*

Male Y Quantity = undetected Insufficient male DNA Stop processing or prioritize other

case samples

Low Male:Female ratio Unlikely to obtain informative 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)  Select STR kit with high # of miniSTRs, e.g. GlobalFiler™, NGM SElect™, MiniFiler™ & Yfiler™ Plus kits

•  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.


Quantifiler® Trio Kit: Supporting Lab Workflows If you are having trouble viewing this email, click here.

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Enhancing the Sexual Assault Workflow Sexual assault samples are among the most difficult sample types encountered by forensic DNA laboratories, frequently suffering from multiple challenges including small quantity of male DNA, relatively high quantity of female DNA and presence of PCR inhibitors.

Join this webinar to learn about powerful new tools for DNA sample assessment and amplification, including the Quantifiler® Trio, Yfiler® Plus and GlobalFiler® Kits, that can significantly improve the workflow for obtaining interpretable profiles from sexual assault and other challenging casework samples.

Jack Ballantyne from the National Center for Forensic Science at University of Central Florida will present data from in-depth studies demonstrating the successful use of these next generation tools to obtain informative profiles from challenging sexual assault sample types, including extended interval post coital samples collected up to 9 days after intercourse.

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REMINDER: Attend this live webinar and pose your critical questions to the panel of experts during the live Q&A session during the broadcast.

Please forward this announcement to your colleagues or friends who will benefit from the educational content of this webinar. Your colleagues may register at no charge.

Presented by: In Association with:

Thursday, January 30, 2014

11:00 a.m. Eastern Time USA

Space is limited.

REGISTER FREE

MODERATOR:

Rebecca Waters Editor-in-Chief Forensic M agazine

SPEAKERS:

Allison Holt, Scientist, Human Identif ication Life Technologies

Jack Ballantyne Professor University of Central Florida (UCF)

Lifetechnologies.com/quantifiler

FORENSIC MAGAZINE 2014 READERS’ CHOICE

AWARD WINNER

Category: Forensic Lab Equipment

Quantifiler Trio Applied Biosystems


Sexual Assault Cases - Challenges

• Samples from SAKs are among the most difficult samples types encountered by many forensic laboratories

•  (~4-6 hours or more to review evidence in 1 SAK)

• Typical challenges with sexual assault samples: •  Small quantity of male DNA with relatively high quantity of female DNA •  Presence of PCR inhibitors or DNA degradation

• Labor intensive & time consuming processing •  Microscopic sperm search •  Interpretation issues with serological assays •  Differential extraction

• For many laboratories sexual assault cases represent a high percentage of cases •  30-50% of SAKs can yield no presence of male DNA1

•  1 2014 Voice of Customer Survey, Thermo Fisher Scientific


Traditional Sexual Assault Lab Workflow

Screening Extract Quant STR Data Analysis

Low concentration

How much?

Degraded How good/bad?

Inhibited How good/bad?

M:F ratio Is there Y?

Autosomal STR

Y STR

Mini STR

Differential Extraction

Manual Slide Search

Serological Assays

Y or no Y?

High incidence of

no male profile at final data analysis


Quantifiler Trio: Sexual Assault Case Example

Minor IPC CT shift

The ratio of male to female DNA is calculated by the software as follows: In this example: MTFR = .635/.635 : (62.705 - .635)/.635 = 1:97.695

Quantity Human DNA -

Quantity Male DNA

Quantity Male DNA

Quantity Male DNA

Quantity Male DNA MTFR = :

Low M:F ratio flag (user defined) Degradation Index

Profiling with Yfiler* or Yfiler

Plus Kit* expected to help

yield more probative results

Autosomal STR profile: no minor male detected; lower peak heights for HMW loci

*For Forensic or Paternity Use Only


Alternative Sexual Assault Lab Workflow

Screening Extract Quant STR Data Analysis

Low concentration

Degraded

Inhibited

M:F ratio

Autosomal STR

Y STR

Mini STR

Differential Extraction

Manual Slide Search

Serological Assays

Y or no Y?

High incidence of

no male profile at final data analysis

Y-Screen DNA Assay

Quantifiler Trio

Enhance screening

Stop or Go?? Stop or Go

Improved Results with Confidence


Y-Screen Protocol Options using Quantifiler Trio

Assist in reducing backlogged samples

Streamline samples for differential

extraction

Provide confidence about how to

process sample

•  Quickly screen key swab evidence in SAKs for presence / absence of Male DNA

•  If male present,

Process swab for STR

•  If no male present, Write report and no further processing is done

•  Possibly eliminate the need to go to differential extraction for any negative in Y Screen & presumptive testing

•  Process main SAK

swabs to differential extraction only and use Y Screen for other swab evidence

•  To add a DNA confirmatory test to ‘complement’ upfront presumptive tests used for screening

•  Allow forensic analysts to make more informed decisions on presence / absence of male/sperm DNA in SAK swab samples

SAK DNA screen prior to standard SA sample processing workflow.


Voice of Customer Commentary on Y-Screen Idea

• “Through the valida0on of GlobalFiler and Quan0filer Trio, we feel confident to stop with Trio. We go even as far with the poten0al to select a value above zero -‐ at which you don’t get STR results. We would like to just move forward to a differen0al if the Y-‐screen is posi0ve. It would be great to stop at quant” •  (~180 SAKs per month; 43% no male present in all SAKs)

• “A Y-‐Screen would free up 0me of processing every piece of evidence. And give confidence in results without fear of a ‘Go-‐back’. It would help op0mize the process between screening and DNA group”. •  (~12 SAKs per month)

• “Yes we would be VERY interested in a Y-‐Screen. This type of DNA test would greatly help op0mize our process and reduce ~50% of our SAK processing 0me. This is the first 0me you (Thermo) have come to us with an idea that does not involve us buying a new kit -‐ we can re-‐purpose one we have already validated, very cool”. •  (Process everything; ~50% no male present in all SAKs)


Y-Screen Proposed Protocol

• SAK Swab evidence 1.  Cut off a small piece of each cotton swab sample type

-  Save rest of swab for differential extraction 2.  Place swab cuttings in heated sperm lysis buffer column for 5 min. 3.  Centrifuge column 4.  Add neutralizing solution and perform simple dilution of sample in TE 5.  Add 2uL of lysate into Quantifiler® Trio real-time PCR reaction

- Run standard 60 min protocol -  5 point standard curve

Process swabs from 5 - 20* SAKs in less 2 hours *dependent on number of swabs and replicates


Correlation of Y-Screen Assay to Differential Extraction Results (Internal Feasibility Study) • 12 buccal swabs

•  Cotton Puritan swabs used •  Spotted with equivalent of 1, 0.5, 0.2, 0.05 uL semen •  3 replicates of each

• Cut small piece of each swab for direct lysis • Processed the rest of the swab with standard differential

protocol using AutoMate Express™ sample cleanup • Performed PCR amplification using the following kits on all

differential extraction samples: •  Identifiler Plus, GlobalFiler and YFiler Plus


Results: QuantifilerTrio and Male Target

•  All samples tested gave a detectable Y target quantity using Y-Screen and Y target quantity after differential extraction

•  Samples 10-12: direct lysis & sperm fraction Y target CT values = 35-39 •  Male: Female Ratio for Sperm Fraction Samples

•  10- 1: 2.5 •  11- 1: 1.7 •  12- 1: 1.3

(uL)


Results: Identifiler Plus, Samples with 0.05 uL Semen (10-20 pg male DNA added to STR reaction)

•  On average, 1 locus from male donor (+ amel) was recovered. •  For 0.2 uL semen samples, full male profiles (with some contaminating

alleles from female donor) were obtained.


Results: YFiler Plus, Samples with 0.05 uL Semen (10-20 pg male DNA added to STR reaction)

• On average, 2 loci from male donor were recovered.


Results: Test Site 1 – No Male DNA

•  For 9 samples, no male DNA was detected with the Y-Screen protocol •  Serology = weak positive; No clear correlation across serology results

•  Y-Screen = No Male DNA

•  STR profiles following differential extraction did not have a male contributor •  GlobalFiler gave full profiles in two samples where Identifiler gave partial

•  Quantifiler Trio gave results in sub-pico gram detection

•  DI values 0.5 to 4.7

•  Y-Screen compared to Serology + Slide Screen


Results: Test Site 1 – Male DNA present •  Y-Screen compared to Serology + Slide Screen

•  For 7 samples, male DNA was detected with the Y-Screen protocol •  Slide Screen = negative to 4+; Serology = weak to strong positive;

-  No clear correlation across serology results •  Y-Screen = Male DNA

•  STR profiles following differential extraction did have a male contributor •  Both GlobalFiler and Identifiler gave concordant full male profiles

•  Quantifiler Trio gave results in sub-pico gram detection •  DI values 0.8 to 1.5

•  Test site in process of running Yfiler Plus data


Results: Test Site 2 Samples Sample ID Sample Description Extraction

method Y-Screen SA Quant

Y-Screen Y Quant

YFiler and/or IDPlus results for Male Fraction or Standard DNA extraction

Mixture 1 F1+M1 SF 1:10,000 differential + - Sperm fraction not run Mixture 2 F1+M1 SF 1:1000 differential + + Full profile Mixture 3 F1+M1 SF 1:100 differential + - No alleles Mixture 4 F1+M1 SF 1:10 differential + + Full profile Mixture 5 F2+M5 unknown dilution differential + + Full profile Mixture 6 F3 blood+M1 SF neat* differential + + Full profile Mixture 7 F4 blood 1:10+M3 SF

neat* differential + + Full profile

Mixture 8 F4 blood + M4 SF 1:10* differential + + Full profile Mixture 9 F3 blood + M4 SF 1:250* differential + + Full profile Mixture 10 F3 blood + M3 SF 1:100* differential + + Full profile Touch 1 M1 touch + F1 arm Non-

differential + + Mixed partial profile w/ IDPlus. Most loci

have 1 contributor Touch 2 M1 saliva + F1 arm Non-

differential + + Full profile

Touch 3 M2 touch +M6 arm Non-

differential + + Mixed partial profile w/ IDPlus.

Touch 4 M2 saliva + M6 arm Non-differential

+ + Full profile

SF1 M2 SF 1:10,000 differential - - 2 alleles with YFiler SF2 M4 SF 1:10 differential + + Full profile SF3 M3 SF neat** differential + + Full profile SF4 M4 SF neat** differential + + Full profile SF5 M4 SF 1:10** differential + + Full profile SF6 M4 SF 1:50** differential + + Full profile

100% Concordance between Y-Screen and STR Results for 20 Mock Samples


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”


Lab Considerations for Y-Screen Implementation

• Swab sample cuttings – how and where to cut on swab •  Must sample from a good area with uniform cutting protocols •  May want to take multiple samples from same swab

• Protocol only supports swab samples • Sample amount and homogeneity issues with swabs

•  This can impact both the IPC and DI as quality indicators for Y-Screen; these values may have wider ranges than assessing standard casework quantification

•  Cannot make direct correlation from Y-Screen quant values and post-extraction quant values

• Highly compromised samples may cause dropout •  Working with test sites on understanding a few swab samples (3) that gave

inconclusive results across serology and no Y screen •  Additional test methods being evaluated


Next Steps

• Continue work with test sites • Waiting on data return from test sites •  Looking for more compromised samples to better understand possible

failure modes -  Low sample amount on swab - Are there swab sample types that cannot be processed - Older samples -  Impact of spermicide - Body fluid mixtures on swabs

•  If you are interested in participating, contact your local HID FAS.

• Anticipated Y-Screen User Bulletin Spring 2015


Conclusion •  Y-Screen has shown in preliminary testing (internal and external) to

be a useful DNA confirmatory screening tool when used complementary to other presumptive screening methods •  Confirms conclusive and inconclusive serology results •  Aids in detecting male/sperm DNA when slide searching was negative or

questionable

•  Adds valuable DNA confirmatory screening test as insurance about sample quantity for best downstream autosomal and Y results.

•  Quick and easy to set up with chemistry and technology that is already familiar to forensics labs •  Quantifiler Trio •  7500 Real-Time PCR System •  HID Real-Time PCR Analysis Software v1.2


THANK YOU!!

Presenter Name: Sheri J. Olson Title: Senior Global Product Manager Email: [email protected] GlobalFiler Kits, Identifiler Kits, NGM SElect Kits, Minifiler Kits and Yfiler Kits: For Forensic or Paternity Use Only. For Research, Forensic, or Paternity and Cell Line Authentication. When used for purposes other than Human Identification, the instrument and the modules of the cited software are for Research Use Only. Not for use in diagnostic procedures. ©2015 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. The content provided herein may relate to products that have not been officially released and is subject to change without notice.


Questions for Workshop

• SAK Swabs •  What swab sample types are the most problematic to correlate Serology and

STR results? •  How accurate is serology results for swab cutting? •  How homogeneous is the male sample across the swab?

• Screening •  What is the most informative screening process / technology in your lab

currently? Why? •  If you could detect male DNA with Y-screen, would you / could you remove one

of the serology steps?

• Validation •  How would this be implemented in your lab? •  What would it take to validate?

Quantifiler™ Trio: Decision-support to help streamline Sexual Assault sample processing.

Technology

Transcript of Quantifiler™ Trio: Decision-support to help streamline Sexual Assault sample processing.