Development of a comprehensive genetic tool for identification of Cannabis sativa samples for forensic and intelligence purposes

As submitted by the applicant: This project seeks to explore forensic genetic issues associated with the identification and determination of origin of Cannabis sativa (marijuana). C. sativa is the most commonly used illicit drug in the United States. As a result of its legalization in some states, law enforcement agencies must prevent the diversion of marijuana bought in legalized states from being trafficked to other states where the drug is still illegal. Moreover, significant illegal C. sativa traffic from Mexico exists at the US border. To date no Cannabis short tandem repeat (STR) method following ISFG/SGWDAM recommendations has been reported (i.e., use of sequenced allelic ladder, use of tetra-nucleotide STR markers), no Cannabis STR reference population database for forensic purposes (in Hardy-Weinberg and linkage equilibrium) has been reported, there have been no chloroplast (cpDNA) and mitochondrial DNA (mtDNA) studies that investigate C.sativa haplotypes in US, and massive parallel sequencing (MPS) technology has not yet been applied on C.sativa for forensic purposes. The use of a combined genetic tool (STR, cpDNA and mtDNA) will allow the identification of Cannabis samples, the association of different cases and the determination of origin for forensic and intelligence purposes. This project will involve: a) development and optimization of a new STR multiplex method for C. sativa identification (3500 Genetic analyzer), according to ISFG recommendations, using primer and multiplex STR design software and a gradient PCR approach for optimal Tm , b) validation of STR system according to SWGDAM guidelines, c) case-to-case comparisons performed by phylogenetic analysis using UPGMA method and parsimony analysis (statistically significant differences will be detected using pair-wise genetic-distance comparisons), d) detection of homogeneous subpopulations (low Fst) by phylogenetic analysis and confirmed by bootstrap analysis (95% Cl Fst), e)development of a Cannabis reference STR population database (N=100) with parameters of population genetics (Ho, He, HWE, LD, f) and of forensic interest (allelic frequencies, PD, PE), f) optimization and application of cpDNA and mtDNA markers for determination of Cannabis origin and g)development of a method using MPS technology (AmpliSeq panel online tool, Ion PGM system, Ion Chef) for genetic identification of C. sativa. DNA from 200 Cannabis samples from ten THC-tested cases will be extracted at U.S. Customs & Border Protection (OHS) Houston Laboratory and will be added to our existent DNA databank (N=200). Three peer-reviewed publications in top-tier forensic science journals and oral and poster presentations at forensic scientific meetings will be expected outcomes.

Note: This project contains a research and/or development component, as defined in applicable law.

nca/ncf.

As submitted by the applicant: This project seeks to explore forensic genetic issues associated with the identification and determination of origin of Cannabis sativa (marijuana). C. sativa is the most commonly used illicit drug in the United States. As a result of its legalization in some states, law enforcement agencies must prevent the diversion of marijuana bought in legalized states from being trafficked to other states where the drug is still illegal. Moreover, significant illegal C. sativa traffic from Mexico exists at the US border. To date no Cannabis short tandem repeat (STR) method following ISFG/SGWDAM recommendations has been reported (i.e., use of sequenced allelic ladder, use of tetra-nucleotide STR markers), no Cannabis STR reference population database for forensic purposes (in Hardy-Weinberg and linkage equilibrium) has been reported, there have been no chloroplast (cpDNA) and mitochondrial DNA (mtDNA) studies that investigate C.sativa haplotypes in US, and massive parallel sequencing (MPS) technology has not yet been applied on C.sativa for forensic purposes. The use of a combined genetic tool (STR, cpDNA and mtDNA) will allow the identification of Cannabis samples, the association of different cases and the determination of origin for forensic and intelligence purposes. This project will involve: a) development and optimization of a new STR multiplex method for C. sativa identification (3500 Genetic analyzer), according to ISFG recommendations, using primer and multiplex STR design software and a gradient PCR approach for optimal Tm , b) validation of STR system according to SWGDAM guidelines, c) case-to-case comparisons performed by phylogenetic analysis using UPGMA method and parsimony analysis (statistically significant differences will be detected using pair-wise genetic-distance comparisons), d) detection of homogeneous subpopulations (low Fst) by phylogenetic analysis and confirmed by bootstrap analysis (95% Cl Fst), e)development of a Cannabis reference STR population database (N=100) with parameters of population genetics (Ho, He, HWE, LD, f) and of forensic interest (allelic frequencies, PD, PE), f) optimization and application of cpDNA and mtDNA markers for determination of Cannabis origin and g)development of a method using MPS technology (AmpliSeq panel online tool, Ion PGM system, Ion Chef) for genetic identification of C. sativa. DNA from 200 Cannabis samples from ten THC-tested cases will be extracted at U.S. Customs & Border Protection (OHS) Houston Laboratory and will be added to our existent DNA databank (N=200). Three peer-reviewed publications in top-tier forensic science journals and oral and poster presentations at forensic scientific meetings will be expected outcomes. Note: This project contains a research and/or development component, as defined in the applicable law, and complies with Part 200 Uniform Requirements – 2 CFR 200.210(a) (14). nca/ncf