development and validation of a novel 13-loci str...
TRANSCRIPT
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Development and Validation of a Novel 13-loci STR Multiplex Method for Cannabis sativa DNA
ProfilingRachel Houston, BS; Sheree Hughes-Stamm, PhD; David Gangitano, PhD
Department of Forensic ScienceSam Houston State University
Huntsville, TX, USA
69th Annual AAFS, New Orleans, 2017
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FSF Emerging Forensic Scientist Award Paper Presentation
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Disclosure
• There is no real or apparent conflicts of interest related to the content of this presentation
• Products used: • DNeasy® Plant Mini Kit• Type-IT® Microsatellite kit• SYBR™ Green Master Mix• Big Dye Direct® Cycle Sequencing Kit• Centri-Sep™ purification columns
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Marijuana Background
• Family: Cannabaceae
• Genus: Cannabis
• Species: Cannabis sativa
• Diploid genome (2n = 20)• 9 pairs of autosomes• Pair of sex chromosomes
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Significance
• Marijuana is the most commonly used illicit drug in United States
Statistics of Drug Use in United States (U.S. Department of Health and Human Services, 2013)
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Marijuana Legalization
•Recreational use:
• 8 states & D.C.
•Medical use:
• 20 states
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Illegal Trafficking
http://www.huffingtonpost.com/2013/06/21/marijuana-accounts-for-va_n_3480127.html USA Today; El Paso Intelligence Center, National Seizure System, as of March 20, 2015.
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Purpose and Goals
• Provide forensic DNA community a comprehensive analytical tool to genetically identify C. sativasamples:
1. Presence of clones2. Association between group of samples
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DNA Based Individualization
• Polymorphic STR markers first described (Gilmore and Peakall (2003); Alghanim and Almirall (2003); Hsieh et al. (2003))
• Marijuana DNA STR multiplex and database (Howard et al. (2008))
• CS1 marker study (Miller Coyle et al. (2003))
• 15 loci - STR tool (Köhnemann et al. (2012))
• Proposed new tetranucleotide markers (Valverde et al. (2014))
• Previous research: 13 loci – STR tool (Houston et al. (2016))
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Improvements Upon Previous Research
• Based upon previous STR multiplex:1. Discard poorly performing loci 2. Incorporate six new tetranucleotide markers3. Optimization 4. Developmental validation 5. Internal validation
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Materials and Methods
• Sampling (3 cases – 101 samples) – Reference Population
• DNA Extraction (DNeasy® Plant Mini Kit)
• DNA Quantitation (real-time PCR)
• 13 STR Multiplex
• Validation Studies
• Statistical Analysis
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13 STR Multiplex
Primer Selection
and Optimization
Allele Sequencing and Ladder
Design
Validation Studies
STR Genotyping
Primer Selection and Optimization
Allele Sequencing and Ladder
Design
Validation Studies
STR Genotyping
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Primer Selection
Type Marker
Trinucleotide ANUCS305, B05, D02, C11, H06
Tetranucleotide 9269, 4910, 5159, 9043, 1528, 3735
Pentanucleotide ANUCS501
Hexanucleotide CS1
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Multiplex Optimization
• Multiplex Manager Software v1.2:• Evaluate primer-primer interactions • Optimal loci layout
• Annealing temperature determination:• Individual loci: 65˚C – 55˚C • Multiplex annealing temperature: 57˚C
• Primer titration and cycle number: • Type-IT® Microsatellite PCR Kit (QIAGEN)
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Final 13-plex
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13 STR Multiplex
Primer Selection
andOptimization
Allele Sequencing and Ladder
Design
Validation Studies
STR Genotyping
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Sequencing/Allelic Ladder
2 – 8 alleles per marker
Big Dye Direct® Cycle Sequencing Kit
Centri-Sep™ purification columns
Geneious Pro Software
Allelic Ladder Design
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Alle
lic L
adde
r
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13 STR Multiplex
Primer Selection
and Titration
Allele Sequencing and Ladder
Design
Validation Studies
STR Genotyping
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Validation Studies
•Developmental Validation (SWGDAM): • Species specificity • Sensitivity/Stochastic effects • Precision and accuracy• Concordance study
• Internal Validation: • Stutter ratio• Peak height ratio• Inter-loci balance
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Species Specificity
•Cross-reactivity observed in one species:• Humulus lupulus (Hops) • Generated non-specific peaks (previously reported)
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Sens
itivi
ty0.5 ng
1.0 ng
0.25 ng
0.13 ng
0.06 ng
0.03 ng
0.02 ng
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Sensitivity Cont.
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Precision & Accuracy
-1
-0.5
0
0.5
1
50 100 150 200 250 300 350
Siz
e D
evia
tion
(bp)
Allele Size (bp)
ANUCS501926949105159ANUCS3059043B0515283735CS1D02C11H06
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Concordance
•100% concordance with loci previously amplified: 1. ANUCS3052. ANUCS5013. B05 4. D025. H066. C11 7. CS1
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Stutter
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Peak Height Ratio
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Het
eroz
ygou
s Pe
ak H
eigh
t Rat
io
Loci
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Inter-loci Balance
0
2000
4000
6000
8000
10000
12000
14000
Peak
Hei
ght (
RFU
s)
Average Peak Height
Inter-loci balance range: 0.50 (5159) – 1.671 (B05)
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13 STR Multiplex
Primer Selection
and Titration
Allele Sequencing and Ladder
Design
Validation Studies
STR Genotyping
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Posi
tive
Con
trol
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STR Results
•All samples (N=101) successfully amplified:• Mixtures (N=5) discarded • 2 duplicate genotypes within same seizure found
•95 distinguishable DNA profiles:• 100% full profiles
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Statistical Analysis
•Population genetic statistics and parameters of forensic interest:
• Allele Frequencies (PowerStats v.1.2)
• Random Match Probability (PowerStats v.1.2)
• Hardy-Weinberg Equilibrium (GDA)• Linkage Disequilibrium (GDA)
• Power of Discrimination (PowerStats v.1.2)
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Conclusions
1. High quality profiles with template input as low as 125 pg
2. Negligible cross-reactivity with the 13 STR markers
3. STR success rates improved from previous multiplex(100% vs. 64%)
4. No departures from Hardy-Weinberg
5. One departure from Linkage Equilibrium detected due togenetic drift
6. Combined power of discrimination of the multiplex is 1 in55 million
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Potential Impact
• Provide the forensic community with a genetic tool for identification of C. sativa samples 1. Authenticate legal Cannabis products 2. Link cases (as intelligence tool) 3. Link and identify illegal growers/distributers
• Complement previously established profiling programs for intelligence purposes for organizations, such as Homeland Security/CBP and DEA
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Phylogenetic Analysis
Genetic Distance = Fst
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Phylogenetic Analysis
Genetic Distance = Fst
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References
1. Sakamoto K, Akiyama Y, Fukui K, Kamada H, Satoh S. Characterization; Genome Sizes and Morphology of Sex Chromosomes in Hemp (Cannabis sativa L.). Cytologia. 1998 Oct; 63: 459 – 64.
2. S. Gilmore and R. Peakall. Isolation of microsatellite markers in Cannabis sativa L. (marijuana). Molecular Ecology Notes (2003) 105-7.
3. Alghanim HJ, Almirall JR. Development of microsatellite markers in Cannabis sativa for DNA typing and genetic relatedness analyses. Anal Bioanal Chem. 2003 Aug;376(8):1225-33.
4. Hsieh HM, Hou RJ, Tsai LC, Wei CS, Liu SW, Huang LH, Kuo YC, Linacre A, Lee JC. A highly polymorphic STR locus in Cannabis sativa. Forensic Sci Int. 2003 Jan 9;131(1):53-8.
5. Howard C, Gilmore S, Robertson J, Peakall R. Developmental validation of a Cannabis sativa STR multiplex system for forensic analysis. J Forensic Sci. 2008 Sep;53(5):1061-7.
6. Miller Coyle H, Shutler G, Abrams S, Hanniman J, Neylon S, Ladd C, Palmbach T, Lee HC. A simple DNA extraction method for marijuana samples used in amplified fragment length polymorphism (AFLP) analysis. J Forensic Sci. 2003 Mar;48(2):343-7.
7. Köhnemann S, Nedele J, Schwotzer D, Morzfeld J, Pfeiffer H. The validation of a 15 STR multiplex PCR for Cannabis species. Int J Legal Med. 2012 Jul;126(4):601-6.
8. Valverde L, Lischka C, Erlemann S, de Meijer E, de Pancorbo MM, Pfeiffer H, Köhnemann S. Nomenclature proposal and SNPSTR haplotypes for 7 new Cannabis sativa L. STR loci. Forensic SciInt Genet. 2014 Nov;13:185-6.
9. Houston R, Hughes-Stamm S, Gangitano D. Evaluation of a 13-loci STR multiplex system for Cannabis sativa genetic identification. Int J Legal Med. 2016; 130:635-47.
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Acknowledgements
Sam Houston State University: Dr. David Gangitano Dr. Sheree Hughes-Stamm
Funding: NIJ GRF (2015-R2-CX-0030)
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FSF Emerging Forensic Scientist Award Paper Presentation