Evaluation of The QIAGEN Investigator® 24plex GO!

Reena Roy, Ph.DForensic Science Program

The Pennsylvania State UniversityUniversity Park, PA 16802

FORENSICSCIENCE 1

Project Overview

I. Direct Amplification

II. Project Methodology

III.Results

IV.Conclusions 2

Direct Amplification Procedure

4. Capillary Electrophoresis6. STR Profile

Compare STR profile to reference profile Compare STR profile to profiles in database

Prepare statistics to support inclusion or exclusion

5. Detection of DNA Fragments

Statistics

1. DNA Extraction 2. DNA Quantification 3. DNA Amplification

3

Direct Amplification Research

4

RapidHIT and Similar System

• Previous research with RapidHIT System

• This and similar instruments are extremely expensive and reagents are costly

• Pursue direct amplification manually with various direct amplification kits

Image obtained from http://i.dailymail.co.uk/i/pix/2014/11/21/236246FD00000578-2844764-image- 1_1416609761474.jpg

FORENSICSCIENCE 5

http://i.dailymail.co.uk/i/pix/2014/11/21/236246FD00000578-2844764-image-

Direct Amplification ResearchCrime Scene Substrates

6

Evaluation of GlobalFiler™ Express Amplification Kit and PowerPlex® Fusion 6C Systems Using Commonly

Encountered Crime Scene Substrates

(Under Review)

Developmental Validation Study

Investigator 24plex GO! Kit Validation Report 08/2016

7

An Internal Validation Study

• Evaluate the Investigator 24plex GO! Kit and determine if it is a suitable kit for direct amplification of body fluids

• Various types of substrates

• Direct amplification of four body fluids commonly encountered at crime scenes in two different reaction volume (25 µL and 12.5 µL)

• Substrates remain in the reaction mixture – Inhibition

• Determine an amplification protocol for each body fluid

8

QIAGEN Investigator® 24plex GO!

• Direct Amplification Kit• 22 autosomal polymorphic loci and two Internal PCR Controls, QS1 and QS2• One Y chromosome marker- DYS391

24plex GO! LociTHO1, D3S1358, vWA, D21S11

TPOX, DYS391, D1S1656, D12S391,SE33

D10S1248, D22S1045, D19S433, D8S1179, D2S1338

D2S441, D18S51, FGA

QS1, D16S539, CSF1PO, D1S317, D5S818, D7S820, QS2

FORENSICSCIENCE 9

• QS1-74 bp and QS2- 435 bp-BTP

• Three primer system-same target for both QS1 and QS2 • Random algorithm

To address the issue of sequence similarity and non-specific binding these sensory loci were designed with synthetic DNA template

• The template sequence differs from all known DNA sequence, in particular bears no resemblance to human DNA

• Chance of non-specific binding in the context of multiplex PCR amplification is very low

Internal Quality Sensor Loci

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• “Limited” in the Investigator 24plex GO!

• Means that at a maximum of 25 cycles, the peak heights will reach a maximum height

• After 25 cycles average peak height will not increase

• Peak heights should typically be equal

• Are not affected by sample degradation

Quality Sensor Loci

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Quality Sensor Loci Interpretation

FORENSICSCIENCE 12

Allele Peaks QS1 QS2 InterpretationPresent Present Present Successful ProfileAbsent Present Present No DNAAbsent Absent Absent Failed amplification

Ski-slope profile Present Dropdown Inhibitors presentSki-slope profile Present Present Degraded DNA

Provide information on inhibition or degradation in amplified product

• Blood• Saliva• Nasal Secretions• Semen• Urine

FORENSICSCIENCE 13

Body Fluids Commonly EncounteredCrime Scenes

Single source samples

Simulated Crime Scene Substrates

Substrates commonly encountered at crime scenes can be challenging

Presence of possible inhibitors

FORENSICSCIENCE 14

Single sourcebody fluids

QIAGEN Investigator® 24plex Go!

15

Investigator® 24plex GO! Lysis Buffer

Experimental Design With and Without Buffer Using Reference Samples

With STR GO! Lysis BufferProfiles were cleaner and had less

artefacts. Peak morphology and height were consistent

Without STR GO! Lysis BufferSamples had more noise,and non-specific binding

Alleles detected had lower peak height

FORENSICSCIENCE 16

Direct Amplification of Simulated Crime Work Flow

Cutting or punch of crime scene substrate

Deposition of body fluid on

substrate

Substrate with single body fluid

placed inside tube

STR GO! Lysis Buffer added to

tube

Reaction mix added to tube

Incubate samples at room temperature (RT)

Samples placed in Applied Biosystems

Veriti™ Thermal Cycler

Samples left to dry overnight

17

Direct Amplification of BloodFrozen for Four years

Amplification Setup (25 µL)

SubstratesTotal volume

of blood deposited on

each substrate

24plex GO!Lysis Buffer

Incubation time in 24plex GO!

Lysis Buffer at RT

Total volume of master mix (Fast Reaction and

Primer Mix) added

1.2 mm punch or cutting

0.2 µL 5 µL 20-30 mins 20 µL

Amplification Setup (20 µL)

Substrates24plex

GO!Lysis Buffer

Total volume of master mix (Fast

Reaction and Primer Mix) added

1.2 mm punch None 20 µL

Four Donors- Two deceased males and two deceased females (M1, M2, F1, and F2)

Recommended Protocol for Blood on FTAPaper

Protocol for Blood on Simulated Crime Scene Substrates

Substrates remained in amplification reagents during thermal cycling

FORENSICSCIENCE 18

Direct Amplification of Saliva (Sputum) and Nasal Secretions

FORENSICSCIENCE 19

Amplification Reaction (25 µL)

Substrates

Total volume of saliva or nasal secretion deposited on each

substrate

Volume of24plex GO!

Lysis Buffer

Incubation time in 24plex GO! Lysis Buffer at RT

Total volume of master mix added (Fast Reaction and

Primer mix)

1.2 mm punch or cutting 0.5 µL 5 µL 20-30 mins 20 µL

Four Donors- Two males and two females (M1, M2, F1, and F2)Nasal Secretion: Collected on Kimwipes® and Extracted with water

Substrates remained in amplification reagents during thermal cycling

Direct Amplification of Semen

Amplification Reaction (12.5 µL)

Substrate G2Buffer

Volume of Dithiothreitol

(1M DTT)

Volume ofProteinase K

Incubationtime at 70˚C

Incubationtime at 56˚C

Total volume of semen mixture deposited on

each substrate

24plexGO! LysisBuffer

Total volume of master mix added (Fast Reaction and Primer mix)

1.2 mm punch or cutting

200 µL 20 µL 10 µL(>600 mAU/ml,

solution)

120 mins

90mins

0.2 µL 2.5 µL 10 µL

Donor-Commercial Source

FORENSICSCIENCE 20

Direct Amplification of SemenWithout G2 Buffer

Amplification Reaction (12.5 µL)

Substrate Volume of Dithiothreitol

(1M DTT)

Volume ofProteinase K

Incubationtime at 56˚C

Incubationtime at 70˚C

Total volume of semen mixture deposited on

each substrate

24plexGO! LysisBuffer

Total volume of master mix added (Fast Reaction and Primer mix)

1.2 mm punch or cutting

20 µL 10 µL(>600 mAU/ml,

solution)

120 mins

120mins

0.5 µL 2.5 µL 10 µL

Four Donors-CommercialSource

FORENSICSCIENCE 21

Reduced Reaction Volume AmplificationBlood and Saliva

Amplification Reaction (12.5 µL)

SubstratesVolume of 24plex

GO!Lysis Buffer

Incubation time in 24plex GO! Lysis

Buffer at RT

Total volume of blood or saliva deposited on

each substrate

Total volume of master mix (Fast Reaction and

Primer Mix) added

1.2 mm punch 2.5 µL 20-30 mins 0.2 µL (Blood)0.5 µL (Saliva)

10 µL

FORENSICSCIENCE 22

Samples were also amplified using 12.5 µL reaction volume

Thermal Cycling Protocols

Cycling protocols for Investigator® 24plex GO!

representing the temperature, time, and number of cycles for

each run

Temperature Time Number of Cycles98°C64°C72°C

30 sec40 sec5 sec

3 cycles

25 µL Reaction volume24 Cycles (Blood and Nasal Secretions)

25 Cycles (Saliva)

12.5 µL Reaction volume23Cycles (Blood)24Cycles (Saliva)25Cycles (Semen)

96°C61°C72°C

10 sec40 sec72 sec

68°C60°C10°C

2 min2 min∞

FORENSICSCIENCE 23

Capillary Electrophoresis

FORENSICSCIENCE 24

DNA fragment analysis on 3130xl genetic analyzer– 16 capillary– 24plex GO!- 6 dye system

Color MatrixStandardBlue (B) 6-FAMGreen(G) BTGYellow (Y) BTYRed (R) BTR2Purple (P) BTPOrange (O) BTO

Volume of Formamide 12.0 µL

Volume of BTO SizeStandard

1.0 µL

24plex GO! LociTHO1, D3S1358, vWA, D21S11

TPOX, DYS391, D1S1656, D12S391,SE33D10S1248, D22S1045, D19S433, D8S1179, D2S1338

D2S441, D18S51, FGAQS1, D16S539, CSF1PO, D1S317, D5S818, D7S820, QS2

Data Analysis

Fragment analysis was completed using GeneMarker® HID Software v2.9.0 by

SoftGenetics, LLC State College, PA

FORENSICSCIENCE 25

Data Analysis

25

Consistent and Concordant Results Between and Within Substrates and Donors

FORENSICSCIENCE

M1 semenLeather in 12.5 µL reaction volume

27

M2 semenDenim in 12.5 µL reaction volume

microFLOQ® Direct Collection Device

Nasal Secretion Pap Smear-KPIC S Peak

28

Challenges Encountered GlobalFiler® Express: Brown Leaf

29

Saliva

Challenges EncounteredAllelic Dropout

Dropout D1S1656(Mostly blood Samples)

DropoutLarger Loci

30

Allelic Dropout on D1S1656 and D2S1338

D2S1338

Semen22% dropout

Blood

No allelic dropout observed with semen on D1S1656 No allelic dropout observed with blood, saliva, and nasal secretion on D2S1338

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Chart1

Red FabricRed FabricRed Fabric

LeatherLeatherLeather

Green FabricGreen FabricGreen Fabric

Gray fabricGray fabricGray fabric

DenimDenimDenim

GauzeGauzeGauze

White FabricWhite FabricWhite Fabric

StrawStrawStraw

WoodchipWoodchipWoodchip

GrassGrassGrass

CigaretteCigaretteCigarette

GumGumGum

Complete Profile

Partial Profile

No Results

Number of Samples

M1, M2, F1, and F2- Semen (25 µL)

9

3

0

6

3

0

4

6

0

6

5

0

8

1

0

3

5

2

3

7

0

6

6

0

3

8

0

7

4

0

10

12

0

7

4

0

Sheet1

Complete ProfilePartial ProfileNo Results

Red Fabric930

Leather630

Green Fabric460

Gray fabric650

Denim810

Gauze352

White Fabric370

Straw660

Woodchip380

Grass740

Cigarette10120

Gum740

Results After First Amplification- Blood 25 µL

FORENSICSCIENCE 32

1 Red Fabric 2 Leather 3 Green Fabric4 Gray Fabric5 Denim6 Gauze7 White Fabric8 Green Straw9 Wood Chip

10 Leaf11 Grass

12A Cigarette 12B Cigarette

13 Gum

Results of Direct Amplification of BloodstainsNu

mbe

rofA

mpl

ificat

ions

Substrates Substrates

FORENSICSCIENCE 33

0

2

4

6

8

10

12

M1, M2, F1, and F2- Blood (25 µL)

0

2

4

6

8

10

12

M1, M2, F1, and F2- Blood (12.5 µL)

Num

bero

fAm

plific

atio

ns

Quality Sensors: The Coolest FeatureQS1 and QS2

Q peak S peak

FORENSICSCIENCE 34

QS1 locus QS2 locus

No DNA Inhibition

Conclusions

FORENSICSCIENCE 35

• When substrates were not present and only the body fluids remain in the reagents, complete profiles were observed --Reference samples• complete profiles first amplification

• Complete, consistent and concordant profiles were obtained from all body fluids deposited on 13 simulated crime scene substrates when using both 25 µL or 12.5 µL reaction volumes

• The S peak on QS2 locus occasionally dropped out indicating inhibition in the sample, even when a complete profile was generated

• Specific loci seemed more susceptible to allelic dropout depending on the body fluid

• The results of this research indicate that the Investigator® 24plex GO! is a valuable tool for direct amplification of blood, saliva, nasal secretion, and semen which can be easily incorporated in forensic laboratories

References1. QIAGEN. Investigator 24Plex GO! Technical Manual. 2016.

2. QIAGEN. Developmental Validation of the Investigator® 24plex GO! Kit. Validation Report. 2016. file:///C:/Users/burtonm3/Downloads/HB-1959-002_1103863_AT_HID_VR_Inv24plexGO_0816_WW%20(3).pdf. Accessed on August, 2016.

3. Amanda Dargay and R. Roy; Direct Y-STR Amplification of Body Fluids Deposited on Commonly Found Crime Scene Substrates. Journal of Forensic and Legal Medicine. 39, 50-60 (2016).

4. Aamer Alshehhi and R. Roy; Generating Rapid DNA Profiles from Crime Scene Substrates Commonly Encountered in the United Arab Emirates. Journal of Forensic Research. (2015).

5. Gigl, K., Dargay, A and Roy, R; Direct Amplification of Blood Deposited on Substrates Commonly Encountered at Crime Scenes Using thePowerPlex® 18D and PowerPlex® Fusion Systems. Profiles in DNA (Promega Corporation Web site. http://www.promega.com/resources/profiles-in-dna/2016/direct-amplification-of-blood-on-substrates-commonly-encountered-at-crime-scenes/ Updated 2016.

6. Hallie Altshuler and R. Roy; Evaluation of Direct PCR Amplification Using Various Swabs and Washing Reagents. J Forensic Sci, 60 (6), 1542-1552 (2015).

7. Hall, D.E. and Roy, R. (2014) An evaluation of direct PCR amplification. Croat. Med. J. 55, 655–61.

36

AcknowledgementsSpecial thanks to: Qiagen

John Pickert –QIAGENAmber McManus -QIAGENDr. Mark Guilliano-QIAGENMary Jones Dukes

Graduate StudentsMarcel BurtonShayna GrayTeresa Tiedge

Kayla Hendricks- SoftGeneticsDr. Teresa Snyder-Leiby-SoftGeneticsDr. Tom Andrew-OCME, NHThe Pennsylvania State University Forensic Science Program

FORENSICSCIENCE 37

Marcel Burton

Shayna Gray

Teresa Tiedge

Before climbing Kilimanjaro

Taj M

ahal

, Ind

ia

Mongolia

Serengeti

Thatching Huts in a Masai Village

BhutanYoga in Thailand

Trekking in Cambodia

Turkey

Greece

South AfricaCubaBotswana China

Monk in Myanmar

GobiNepal

Thank You!!

Namibia

Masai Mara

Petroglyphs, Namibia

Chile

China

Afar, Ethiopia

Mursi and Me

Thank you!FORENSCE

39

Contact Information: reena@psu.edurxr34@psu.edu814-867-2054

39

mailto:reena@psu.edumailto:rxr34@psu.edu

Previous Validation on Inhibitors

Specifically…Humic acid and Indigo Carmine

Investigator 24plex GO! Kit Validation Report 08/2016

Slide Number 1Project Overview Direct Amplification ProcedureDirect Amplification Research � RapidHIT and Similar System� Direct Amplification Research�Crime Scene SubstratesDevelopmental Validation StudyAn Internal Validation StudyQIAGEN Investigator® 24plex GO!Internal Quality Sensor LociQuality Sensor LociQuality Sensor Loci InterpretationSlide Number 13Simulated Crime Scene SubstratesQIAGEN Investigator® 24plex Go!Investigator® 24plex GO! Lysis BufferDirect Amplification of Simulated Crime �Work FlowDirect Amplification of Blood�Frozen for Four yearsDirect Amplification of Saliva (Sputum) and Nasal SecretionsDirect Amplification of SemenDirect Amplification of Semen �Without G2 BufferReduced Reaction Volume Amplification�Blood and Saliva Thermal Cycling ProtocolsCapillary ElectrophoresisData AnalysisData AnalysisConsistent and Concordant Results �Between and Within Substrates and Donors� ��microFLOQ® Direct Collection Device � ��Challenges Encountered Challenges Encountered�Allelic Dropout� Allelic Dropout on D1S1656 and D2S1338 Results After First Amplification- Blood 25 µL�Results of Direct Amplification of Bloodstains �Quality Sensors: The Coolest Feature�QS1 and QS2ConclusionsReferencesAcknowledgementsSlide Number 38Thank you!Previous Validation on Inhibitors