Evaluation of The QIAGEN Investigator 24plex GO! …...Evaluation of The QIAGEN Investigator®...
Transcript of Evaluation of The QIAGEN Investigator 24plex GO! …...Evaluation of The QIAGEN Investigator®...
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Evaluation of The QIAGEN Investigator® 24plex GO!
Reena Roy, Ph.DForensic Science Program
The Pennsylvania State UniversityUniversity Park, PA 16802
FORENSICSCIENCE 1
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Project Overview
I. Direct Amplification
II. Project Methodology
III.Results
IV.Conclusions 2
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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
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Direct Amplification Research
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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
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http://i.dailymail.co.uk/i/pix/2014/11/21/236246FD00000578-2844764-image-
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Direct Amplification ResearchCrime Scene Substrates
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Evaluation of GlobalFiler™ Express Amplification Kit and PowerPlex® Fusion 6C Systems Using Commonly
Encountered Crime Scene Substrates
(Under Review)
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Developmental Validation Study
Investigator 24plex GO! Kit Validation Report 08/2016
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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
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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
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• 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
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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
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• Blood• Saliva• Nasal Secretions• Semen• Urine
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Body Fluids Commonly EncounteredCrime Scenes
Single source samples
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Simulated Crime Scene Substrates
Substrates commonly encountered at crime scenes can be challenging
Presence of possible inhibitors
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Single sourcebody fluids
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QIAGEN Investigator® 24plex Go!
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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
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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
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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
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Direct Amplification of Saliva (Sputum) and Nasal Secretions
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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
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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
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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
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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
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Samples were also amplified using 12.5 µL reaction volume
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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∞
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Capillary Electrophoresis
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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
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Data Analysis
Fragment analysis was completed using GeneMarker® HID Software v2.9.0 by
SoftGenetics, LLC State College, PA
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Data Analysis
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Consistent and Concordant Results Between and Within Substrates and Donors
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M1 semenLeather in 12.5 µL reaction volume
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M2 semenDenim in 12.5 µL reaction volume
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microFLOQ® Direct Collection Device
Nasal Secretion Pap Smear-KPIC S Peak
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Challenges Encountered GlobalFiler® Express: Brown Leaf
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Saliva
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Challenges EncounteredAllelic Dropout
Dropout D1S1656(Mostly blood Samples)
DropoutLarger Loci
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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)
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0
6
3
0
4
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6
5
0
8
1
0
3
5
2
3
7
0
6
6
0
3
8
0
7
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10
12
0
7
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Sheet1
Complete ProfilePartial ProfileNo Results
Red Fabric930
Leather630
Green Fabric460
Gray fabric650
Denim810
Gauze352
White Fabric370
Straw660
Woodchip380
Grass740
Cigarette10120
Gum740
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Results After First Amplification- Blood 25 µL
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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
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Results of Direct Amplification of BloodstainsNu
mbe
rofA
mpl
ificat
ions
Substrates Substrates
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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
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Quality Sensors: The Coolest FeatureQS1 and QS2
Q peak S peak
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QS1 locus QS2 locus
No DNA Inhibition
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Conclusions
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• 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
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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.
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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
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Marcel Burton
Shayna Gray
Teresa Tiedge
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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
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Thank you!FORENSCE
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Contact Information: [email protected]@psu.edu814-867-2054
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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