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Integrated Solutions For Sexual Assault Analysis

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Differential Extraction

Separation of non-sperm DNA and sperm DNA

Differential wash workflow

Identify stains and take sample

that contains

Epithelial/non-sperm cells

Sperm cells

Digestion of non-sperm cells

Washing and separation

of non-sperm cells

Digestion of sperm cells

Non sperm/Epithelial DNA Sperm Fraction

Sperm DNA

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Pyrosequencing for Sperm identification in forensic casework

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Epigenetics for tissue identification

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Epigenetic marker analysis CpG methylation degree

Each tissue has its own marker pattern

Requires sensitive analysis of multiple, consecutive CpGs

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Methylation analysis for tissue identification

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Analysis of tissue specific DNA methylation using Pyrosequencing

Methylation analysis

o CpG methylation works as epigentic switch to control tissue specific gene regulationo Each tissue has its own DNA methylation pattern o Loci were identified that can be used to distinguish between the following sample types:

o Bloodo Salivao Semeno Epithelial tissue

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Methylation analysis for tissue identification

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Analysis of tissue specific DNA methylation using Pyrosequencing

The determination of tissue-specific DNA methylation patterns in forensic biofluids using bisulfite modification and pyrosequencing. Madi, T., Balamurugan, K., Bombardi, R., Duncan, G, and McCord, B. Electrophoresis 2012, 33, 1736–1745

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Find out more…

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Application Note: San Francisco Police Department, Florida International University

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QIAGEN’s workflow for sexual assault analysis

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Main goals

o Automate differential separation of sexual assault samples to accelerate your analysiso Simplify isolation of challenging samples for best possible yield and quality of DNAo Obtain detailed and accurate information on the male DNA in your sampleso Perform rapid and reliable downstream analysis with the Investigator assays

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Differential wash, lysis and DNA purification

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Automated “manual” processing

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The QIAcube differential wash workflow

Add wash buffer

Pellet sperm

Repeat 3x

Add sperm lysis buffer

Sperm fraction

Non-sperm fraction

Load mixture, Epi lysed

Pellet sperm

Transfer non-spermfraction to 2 ml tubes

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6 samples <1hr12 samples ~1.5hr

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QIAcube workflow for differential lysis

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Up to 48 differential wash samples per day, 2 operators

EZ1 Adv XL QIAcube for automateddifferentiall wash

• Fully automated differential fraction separation and sperm pellet lysis • Up to 12 samples/run• The QIAcube can be used up front of other automation workflows• Designed to run on the QIAcube, the protocol is directly compatible with QIAGEN’s EZ1,

QIAsymphony or QIAamp DNA Investigator Kit chemistry for downstream DNA purification.

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Find out more…

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Customer poster: Alabama Department of Forensic Sciences

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Assay setup

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Assay setup

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Quantiplex Pro RGQ reaction setup

Template Files

QProtocols

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DNA Quantification

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Rotor-Gene Q system

o Outstanding thermal and optical performance

o Fast cycling (40 cycles in 40 minutes)

o Up to 6 fold multiplexing

o High resolution melting and fast PCR

o Tube & disc formats (1-100 samples)

o Minimal maintenance and calibration

o Versatile software package

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Investigator Quantiplex Pro RGQ Kit

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Key features

• New male target

◦ Superior sensitivity with new male target sequence and small target length

◦ Detection threshold up to 1:2,500,000 (male:female)

• New Degradation Index System

◦ Unique design for small and large autosomal targets

◦ Unique design for small Y and large Y targets

◦ Assess degradation status specifically of male DNA in female:male mixtures

◦ Minimize the risk that your Y-STR fails due to degraded or partially degraded male DNA

• Internal Control for inhibitor monitoring

◦ Correlates very well with the STR results

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Investigator Quantiplex Pro RGQ Kit – Performance Data

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Sensitivity

• Dynamic range from 200 ng/µl down to 0.5 pg/µl

• Accurate quantification of low amounts of DNA

Sensitive detection of traces, robust at high template input

Amplification PlotStandard Curve

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Investigator Quantiplex Pro RGQ Kit – Performance Data

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Mixture analysis

• 0.2 pg/µl male DNA was spiked to up to 200 ng/µl female DNA• Male and female DNA was robustly and accurately quantified

400.000:1

Reliable quantification of male DNA in the presence of excess female DNA

0.5pg/µl male DNA + 100ng/µl female DNA (1:200.000)0.25pg/µl male DNA + 100ng/µl female DNA (1:400.000)0.2pg/µl male DNA + 200ng/µl female DNA (1:1.000.000)

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Investigator Quantiplex Pro RGQ Kit – Performance Data

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Degraded samples

• Male genomic DNA was sheared with a Covaris™ S220 Focused-ultrasonicator to an average fragment size of 1500 bp, 500 bp, 400 bp , 300 bp, 200 bp and 150 bp

• 4.6 ng used as template for quant

Investigator 24plex QS Kit with 150 bp sampleInvestigator Quantiplex Pro RGQ Kit

Reliable prediction of sample integrity and accurate DNA degradation detection

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Investigator Quantiplex Pro RGQ – detects degraded male DNA

Comparison data

The QuantiplexPro RGQ system for DNA degradation allows for detection of degraded male DNA even in a high background of female DNA – competitor kits fail to detect degraded male DNA in female DNA background.

Each sample contains 0.1 ng/μl of degraded male DNA mixed with 100 ng/μl of non-degraded female DNA.

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Investigator Quantiplex Pro RGQ Kit – Performance Data

Internal control

• Humic acid up to 40 ng/μl (final concentration in PCR) was added to reactions amplifying 1 ng of male DNA

◦ Corresponds to 250 ng/µl in STR if maximum template volume of 15 µl is applied

• Robust quantification results up to critical inhibitor concentrations

• The Quantiplex Pro IC correlates to the STRs and reflects the Quality Sensor of STR kits

0 ng/µL

150 ng/µL

200 ng/µL

250 ng/µL

Investigator 24plex QS

The Internal Control reports the presence of inhibitor with a CT shift, while quantification remains reliable up to a final humic acid concentration of 40 ng/μl

Quantiplex Pro RGQ

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STR Analysis

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Investigator STR QS Kits

Investigator Quality Sensor

Argus X-12 QS24plex QS Kit 24plex GO! Kit ESSplex SE QS• 23 markers of CODIS

expansion set• BT6 matrix• FRM 2.0

• 23 markers of CODIS expansion set

• BT6 matrix• FRM 2.0

• 17 markes of ESS set

• BT5 matrix• FRM 2.0

• 12 XChr markers, D21S11 and AM

• BT5 matrix• FRM 2.0

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Investigator STR Kits

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Kit features

• 24 plex QS/GO! kits : 23 markers recommended by CODIS Working Group

• Innovative Quality Sensors for more information and workflow optimization

• 6 color setup with new BT6 matrix

• New Reaction Mix FRM 2.0

◦ Even higher convenience and stability

Up to 16h stability of PCR mixed at RT

◦ Convenient manual and automated pipetting

◦ Even faster reaction speeds

60 minutes for casework, ca. 45 min for GO!

◦ Very high inhibitor tolerance

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Investigator Quality Sensor

Internal Performance Control – more information for better results

• The kits include two Quality Sensors that will provide you with more information about a profile to improve your workflow and save unnecessary re-runs of a sample

• The two internal Quality Sensors are included in the Primer Mix and are amplified simultaneously with the STR markers

• The Quality Sensor gives one small (appr. 70 bp) and one large (435 bp) amplicon• Artificial sequence, with no similarity to known sequences, and tested against various human and non

human DNAs• You will be able to distinguish: ◦ Successful amplification◦ No DNA ◦ Failed PCR amplification◦ Inhibited DNA◦ Degraded DNA

The Quality Sensor provides additional information to improve quality of results and reduce costs

Investigator ESSplex SE QS Kit

Investigator 24plex QS and GO! Kits

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Quality Sensor (QS) explained

Normal non-degraded / non-inhibited sample

No DNA

Failed PCR Amplification

QS confirms failed PCR. Re-run will most likely lead to better results.

QS confirms a successful PCR without DNA. Re-run will not lead to better results.

The Quality Sensor confirms that your sample is not degraded or inhibited

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Quality Sensor (QS) explained

Inhibited DNA sample

Degraded DNA

QS indicates a degraded sample. Re-run will not improve profile.

QS indicates PCR inhibition. An inhibited profile may be rescued by re-running the PCR with a higher sample dilution.

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Summary

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