Solutions for Challenging Casework Samples in Sexual Assault Cases and Cases with Degraded and/or Inhibited Samples

1 The world leader in serving science

HID Field Application Specialist Team 2014 Seminar Series

Solutions for Challenging Casework Samples in Sexual Assault Cases and Cases with Degraded and/or Inhibited Samples

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Quantifiler® Trio and HP Quantification Kits

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 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 delivers results that correlate to dropout in the STR kit reaction •  Accurate negative results and new quality index deliver confidence

in downstream processing decisions

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Quantifiler® Trio and HP Quantification Kits

ü Better sample assessment ü More informed processing

decisions ü Faster time-to-result

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•  All 17 loci amplified by the Yfiler® kit •  10 new Y-STR loci including 7 Rapidly Mutating1 Y-STRs

for ability to discriminate between close male relatives •  11 mini STRs below 220bp

Plus in Power of Discrimination

Plus in Performance

•  Improved sensitivity & results with challenging casework samples •  Improved recovery of alleles and intracolor balance in male/female

mixtures containing high female background •  Improved resolution capacity for male/male mixtures •  Expanded allelic ladder •  Shorter amplification times •  Direct amplification protocol with single-source samples

1.  Rapidly Mutating Y-STRs have mutation rates above 1 x 10-2 per marker per generation. 17 Yfiler® Y-STRs show mutation rates between 1 x 10-3 and 1 x 10-4 per marker per generation. The content provided herein may relate to products that have not been officially released and is subject to change without notice.

Yfiler® Plus: The First 6-Dye, 27 Marker Y-STR Multiplex

Available now!

http://www.lifetechnologies.com/us/en/home/industrial/human-identification/y-str-products.html

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Yfiler® Plus Kit Configuration 50 100 150 200 250 300 350 400

DYS627DYS389IIDYS635DYS389IDYS576

DYS391DYS448

Y GATA H4DYS19DYS458DYS460

DYS518DYS392DYS438DYS390DYS456

DYS449DYS385ab

DYS437DYS570

DYS533DYF387S1ab

DYS481DYS439DYS393

Rapidly Mutating Y-STR Standard Y-STR New Y-STR Markers →

1

3

6

8

10

2

4 5

7

9

11

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The GlobalFiler™ Kit: Going Further for Casework Go Faster! •  Up to 5x faster amplification time than

previous generation kits Go Further! •  Increased discrimination power by up to

9 orders of magnitude •  Enhanced performance on inhibited and

low-level samples Go Global! •  Maximizes global database compatibility

GlobalFiler® Kits – Next Generation STR Chemistry

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To learn more or to request a demo, please visit: lifetechnologies.com/globalfiler

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The GlobalFiler® Kits: 6-Dye Configuration Only 2 loci require primer redesign

* Preliminary calcula.ons based on a US Caucasian Database

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13 14

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16 17

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MiniFiler ™ Kit 8 Mini-STRs

PI* = 1.05 x 10-10

1 2

3

4 5 6 7 8

9 10

GlobalFiler™ Kit 10 Mini-STRs

PI* = 9.2 x 10-12 97% of alleles in

allelic ladder <400bp

18 Mini-STRs Combination

PI*= 5.73 x 10-21

All gender markers located in

the same dye channel for ease of interpretation


Sexual Assault Cases

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Sexual Assault Cases - Challenges

• Sexual assault samples are among the most difficult samples types encountered by many forensic laboratories

• Typical challenges with sexual assault samples: •  small quantity of male DNA with relatively high quantity of female DNA •  presence of PCR inhibitors •  DNA degradation

• Labor intensive & time consuming processing •  microscopic sperm search •  differential extraction

• For many laboratories sexual assault cases represent a high percentage of cases

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Statistics About Sexual Assault in the U.S.

Rapists are more likely to be a serial criminal than a serial rapist •  In the US there is an average of 237,868 victims (age 12 or older) of rape and

sexual assault each year.1

•  One victim every 2 minutes

•  46% of rapists who were released from prison were re-arrested within 3 years of their release for another crime. •  18.6% for a violent offense. •  14.8% for a property offense. •  11.2% for a drug offense. •  20.5% for a public-order offense.

•  Processing sexual assault cases efficiently not only helps to bring closure to the victim and their family, but could also prevent or solve other crimes.

1 Source Rainn website

Source: Rainn webite

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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 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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Sexual Assault Case Example

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

Minor IPC CT shift

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 yield more probative

results

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

*For Forensic or Paternity Use Only

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Analysis Summary

•  Automatically QC’s standard curve, NTC and unknown sample •  Can identify standards and

samples that meet/do not meet defined threshold

•  Hyperlinks to flagged samples for quick data review

•  Customizable plate, table and plot views designed to streamline data review

Standard Curve

NTC

Unknowns

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•  Reproducible Quan1filer® Trio results with samples that previously gave ‘undetermined’ results with Quan1filer® Duo • Improved performance with low-‐ra1o M:Fmixtures

Does Quan5filer Trio show improved sensi5vity?

• Nega1ve quant results = nega1ve or unusable STR result

• Accurately iden1fied low level male samples appropriate for Yfiler® Plus

Do the quan5fica5on results correlate well with the STR results?

• Yfiler® Plus kit* demonstrated higher sensi1vity (17/25 locus profiles with 31/62 pg of male DNA) compared to Yfiler® Kit*

• Full male profiles were obtained with 1:1000 and 1:4000 male/female admixtures. (>100pg of male DNA detected)

•  ‘‘usable profiles’’ with F:M ra1os ranging from 333:1 100,000:1. (mg quan11es of background female)

Improved recovery without the need for addi5onal pre-‐amplifica5on work?


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To Learn More…

View the on-demand webinar via the LifeTechnologies.com portal: Enhancing the Sexual Assault Workflow: Testing of Next Generation Assessment and Y-STR Systems. Poster presented at the 25th World Congress of the ISFG. View and download via SlideShare:

Forensic Science International: Genetics Supplement Series 4 (2013) e228-e229


Cases with Degraded and/or Inhibited Samples

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Degraded and/or Inhibited Samples - Challenges

•  Forensic casework samples are frequently affected by DNA degradation and may contain PCR inhibitors

•  Quantifiler® HP and Trio kits have been designed with: ~5x higher inhibitor tolerance for better correlation with recent STR kit and a Large Autosomal (LA) target to help assess the level of DNA Degradation [Degradation Index (DI)] = SA quant / LA quant

•  Now you can evaluate the DI in conjunction with the IPC in the HID Real-Time PCR Software to determine the overall sample quality and the optimal downstream processing approach!

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Quantifiler® Trio & HP Kits: Sample Assessment Solutions

*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.

When the quantity for the small or large autosomal target is undetermined, the Degradation Index is not calculated and the Degradation Index field in the Well Table will be blank

IPC CT Flag Triggered

Degradation Index

Interpretation* Processing Options*

No

1 to 10

1)  Indicates slight/moderate degradation

2)  Inhibition possible, but not significant

1)  Concentrate sample 2)  Increase DNA target conc and/or STR sample input

volume 3)  Select STR kit with high # of miniSTRs, e.g. GlobalFiler®,

NGM SElect™, MiniFiler® & Yfiler® Plus kits*

>10 or blank

1)  Indicates significant degradation

2)  Inhibition possible, but not significant

Same as above

Yes

<1 Not likely, PCR inhibitors should affect LA target also

>1 or blank

Indicates degradation and/or inhibition (severity of IPC CT shift can help assess inhibition level)

1)  Dilute and/or re-purify sample 2)  Concentrate sample 3)  Increase DNA target conc and/or STR sample input

volume 4)  Select STR kit with high # of miniSTRs


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Degradation Study by Scientific Police Service, Forensic Genetics Laboratory, Rome Correla5on of DI and % of Markers*

Called (GlobalFiler® Kit 1.0 ng DNA input)

100 forensic adjudicated casework samples (oral swab, blood, touch DNA, bone, sperm, hair) *at least one allele is above the validated Analytic Threshold

% of M

arkers

% of m

arkers

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Customer Test Site Data Inhibited + Degraded (Medium) Non-probative Semen Stain on Pants

Quality Assessment

DI IPC CT

35 34.43 – Shifted 8 CT

8000

NGM SElectTM Kit* - 1/86 dilution (1 ng total input)

Degradation Index IPC CT Flag High Quantity


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Validation Stability Studies Degraded DNA Study - Medium Degradation

Quality Assessment

Degradation Index (DI) IPC 40 No Shift

MiniFiler™ Kit*

Iden5filer® Plus* GlobalFiler® Kit* 6000

4400

16000

NGM SElect™ Kit* 10000

*For Forensic or Paternity Use Only.

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Validation Stability Studies Humic Acid Inhibited Samples – Purify to Improve Recovery

6000

2500

160 ng/uL IPC CT 30.48

240 ng/uL IPC CT =undet

IPC CT Flag

IPC CT Flag

GlobalFiler® Kit Results*


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Next Generation Quantification Designed to provide maximum accuracy, sensitivity and workflow efficiency

Higher Performance with Challenging

Samples

• Improved performance with low-‐ra1o male/female mixture samples

• Short-‐amplicon and long-‐amplicon assay targets assess DNA degrada1on

• IPC op1miza1on to match performance of next genera1on STR kits

Enhanced Workflow & Data Analysis

• Improved consistency and homogeneity of DNA standard

• Integrated quan1fica1on and STR setup tools to support complex casework sample processing

Fast & Accurate Decision Tool

• Shorter 1me to results compared to original quan1fica1on kits (1 hour)

• Increased assay sensi1vity and accuracy

• Addi1onal sample quality info + data review and reac1on setup enable faster, beber decisions and results

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Are you currently facing any of these challenges?

• Partial or no profiles from highly degraded or trace samples • No suspect and no STR profile matches in DNA database • Laborious sequencing of mtDNA • Difficult mixture interpretations • Unknown tissue/body fluid origin • Complex kinship cases and/or familial searches

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HID-Ion Torrent™ solutions can help resolve these challenges

Get more information from your challenging samples resulting in more investigative leads •  Ion Torrent™ next gen sequencing

technology with Ion AmpliSeq™ panels make it possible to sequence hundreds of SNPs in less than 24 hr

•  SNPs are great for analysis of highly degraded DNA •  Utilizing Ion AmpliSeq™ technology

for streamlined sequencing analysis of whole or degraded mtDNA genome

•  Potential customized solutions for

SNP, STR, RNA, and mtDNA analysis

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Two HID-Ion Torrent™ Solutions are Available Now!

HID-Ion AmpliSeq™ Identity Panel, consists of 124 autosomal markers, which provide discrimination of individuals similar to STR genotype match probabilities used by forensic analysts (between 1 x 10-31 and 6 x 10-35). Lifetechnologies.com/HID/identity

HID-Ion AmpliSeq™ Ancestry Panel, consists of 165 autosomal markers, which generate biogeographic ancestry information from your casework samples to guide your investigation process or help stratify subjects for your research projects. Lifetechnologies.com/HID/ancestry

Simplicity •  Ready-to-use panel •  1 ng of input gDNA

recommended •  suitable for degraded

samples Scalability •  designed for low-,

medium-, or high-throughput samples

Speed •  Total sample-to-result

duration is less than 24 hours

Plug & Play- no need to change your current CE workflow

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Solutions for Challenging Casework Samples in Sexual Assault Cases and Cases with Degraded and/or Inhibited Samples