The development of a next-generation sequencing panel targeting cannabinoid synthase genes to distinguish between marijuana and hemp.

Hemp and marijuana, both derived from Cannabis sativa L. (C. sativa), are subject to divergent legal regulations due to their different Δ9-tetrahydrocannabinol (Δ9-THC) contents. Cannabinoid synthase genes are considered the key enzymes that determine the chemical composition or chemotype of a particular cultivar. However, existing methods for crop type differentiation based on previous synthase gene theories have limitations in terms of precision and specificity, and a wider range of cannabis varieties must be considered when examining cannabis-based genetic markers. A custom next-generation sequencing (NGS) panel was developed targeting all synthase genes, including Δ9-THC acid synthase, cannabidiolic acid synthase, and cannabichromenic acid synthase, as well as the pseudogenes across diverse C. sativa samples, spanning reference hemp and marijuana, commercial hemp derivatives, and seized marijuana extracts. Interpretation of NGS data revealed a relationship between genotypes and underlying chemotypes, with the principal component analysis indicating a clear distinction between hemp and marijuana clusters. This differentiation was attributed to variations in both synthase genes and pseudogene variants. Finally, this study proposes a genetic cannabis classification method using a differentiation flow chart with novel synthase markers. The flow chart successfully differentiated hemp from marijuana with a 1.3% error rate (n = 147).

Evaluation of metal ions and DNA recovery from the surface of fired and unfired brass ammunition to improve STR profiling.

Interest in recovering DNA from the surface of ammunition evidence for genotyping has increased over the past few years. Numerous studies have examined a variety of methods to maximize DNA recovery from these types of challenging samples, but successful DNA profiling has been inconsistent. Low amounts of DNA and PCR inhibition due to metal ions have been suggested as the leading causes of poor results; however, no study quantitatively examined the presence of metal ions at various stages of the DNA analysis workflow from DNA collection through to amplification. In this study, the effectiveness of six different DNA collection and purification methods commonly used by forensic laboratories to process brass ammunition for DNA evidence was investigated. The amount of copper, zinc, and other metals co-recovered from fired and unfired brass casings during DNA collection (using numerous soaking, swabbing, and direct PCR protocols) was quantified via Inductively Coupled Plasma - Optical Emission Spectrometry (ICP-OES). This same panel of metals was subsequently quantified after DNA lysis and purification steps. Results demonstrated that low amounts of DNA, DNA damage, and degradation are more detrimental to STR typing results than PCR inhibition, as metal ions were successfully removed by all DNA purification methods tested. Furthermore, the use of metal ion chelators increased the amount of DNA recovered and number of reportable STR alleles. This research informs the forensic community on the most effective way to collect and process trace amounts of biological material from brass ammunition and similar evidence.

Comparison of nine extraction methods for bacterial identification using the ONT MinION sequencer.

The Anthrax mailings bioterrorism attack in 2001 revealed the need for universal and rapid microbial forensic analyses on unknown biological evidence. However, the gold standard for bacterial identification includes culturing isolates, which is laborious. Molecular approaches for bacterial identification revolve around 16S ribosomal gene sequencing using Sanger or next generation sequencing (NGS) platforms, but these techniques are laboratory-based and can also be time-consuming. The Oxford Nanopore Technologies (ONT) MinION sequencer can generate long read lengths that span the entire bacterial 16S rRNA gene and accurately identify the species level. This platform can be used in the field, allowing on-site evidence analysis. However, it requires higher quantities of pure DNA compared to other sequencing platforms; thus, the extraction method for bacterial DNA is critical for downstream analysis, which to date are tailored toward a priori knowledge of the species' taxonomic grouping. During an attack, the investigative team may not know what species they are handling; therefore, identifying an extraction method that can handle all bacterial groups and generate clean DNA for the MinION is useful for microbial forensic analysis. The purpose of this study was to identify a "universal" extraction method that can be coupled with ONT MinION sequencing for use in forensic situations for rapid identification. It also evaluated the cloud-based data analysis software provided by ONT, EPI2ME. No "universal" extraction method was identified as optimal for downstream MinION sequencing. However, the DNeasy PowerSoil Kit and Noda et al. Chelex-100 method gave comparable sequencing results and could be used as rapid extraction techniques. This study showed that the ONT 16S Barcoding Kit 1-24 coupled with the 16S FASTQ workflow might not be the best for use in forensic situations where species-level identification needs to be obtained, as most alignments were approximately 89% accurate. In all seven test organisms and nine extraction methods, accurate species identification was only obtained in 63% of the cases.

Development of a novel five dye insertion/deletion (INDEL) panel for ancestry determination.

The use of genetic markers, specifically Short Tandem Repeats (STRs), has been a valuable tool for identifying persons of interest. However, the ability to analyze additional markers including Single Nucleotide Polymorphisms (SNPs) and Insertion/Deletion (INDELs) polymorphisms allows laboratories to explore other investigative leads. INDELs were chosen in this study because large panels can be differentiated by size, allowing them to be genotyped by capillary electrophoresis. Moreover, these markers do not produce stutter and are smaller in size than STRs, facilitating the recovery of genetic information from degraded samples. The INDEL Ancestry Informative Markers (AIMs) in this study were selected from the 1000 Genomes Project based on a fixation index (FST) greater than 0.50, high allele frequency divergence, and genetic distance. A total of 25 INDEL-AIMs were optimized and validated according to SWGDAM guidelines in a five-dye multiplex. To validate the panel, genotyping was performed on 155 unrelated individuals from four ancestral groups (Caucasian, African, Hispanic, and East Asian). Bayesian clustering and principal component analysis (PCA) were performed revealing clear separation among three groups, with some observed overlap within the Hispanic group. Additionally, the PCA results were compared against a training set of 793 samples from the 1000 Genomes Project, demonstrating consistent results. Validation studies showed the assay to be reproducible, tolerant to common inhibitors, robust with challenging casework type samples, and sensitive down to 125 pg. In conclusion, our results demonstrated the robustness and effectiveness of a 25 loci INDEL system for ancestry inference of four ancestries commonly found in the United States.

Optimization of InnoXtract™ extraction and purification system for DNA extraction from skeletal samples.

The InnoXtract™ extraction and purification system is a purification method designed for DNA extraction from low-template samples, specifically rootless hair shafts. Its ability to successfully capture highly fragmented DNA suggests its suitability for use with other challenging sample types, including skeletal remains. However, the lysis and digestion parameters required modifications to successfully optimize the method for this sample type. A two-part digestion was developed utilizing a homebrew digestion buffer (0.5 M EDTA, 0.05% Tween 20, and 100 mM NaCl) and a supplemental lysis with the Hair Digestion Buffer included in the InnoXtract™ kit. Additionally, the magnetic bead volume was modified to improve DNA recovery from these challenging samples. With the altered protocol, the quality and quantity of DNA recovered from InnoXtract™ extracts were comparable to another commercial skeletal extraction method (PrepFiler™ BTA). This modified extraction method successfully purified sufficient amounts of quality DNA from a variety of skeletal samples to produce complete STR profiles. Successful STR typing from surface decomposition, burned, cremated, buried, and embalmed remains indicates the potential of this new method for challenging human identification and missing-person cases.

Evaluation of chloroplast DNA barcoding markers to individualize Papaver somniferum for forensic intelligence purposes.

The opium poppy, Papaver somniferum L., is a forensically important plant due to the medicinal and illegal uses for the milky latex stored in the pods. This latex contains the alkaloids morphine, codeine, and thebaine that are used for their analgesic properties and/or for synthesizing other opioids. However, these compounds are highly addictive and have caused a national opioid epidemic. Two other Papaver species, P. setigerum DC. and P. bracteatum Lindl., are also of forensic interest because they pose both forensic and legal issues. They are largely uncontrolled under the Controlled Substances Act, making these species a common defense strategy. Current morphological and chemical identification methods have been moderately successful but have drawbacks. There is also a lack of sequencing data available. Therefore, exploiting the genome using chloroplast DNA barcoding markers could help to accurately identify these species of interest when plant material is taken. This study screened and assessed the genetic variation both between species and within populations of P. somniferum in nine cpDNA barcode regions (ndhF-rpl32, petA-psbJ, rpl32-trnL, rps16-trnQ, trnE-trnT, trnH-psbA, trnL-trnF, rpl16 intron, and psbE-petL). Published reference genomes from the NCBI GenBank database were aligned and compared for an initial in silico screening. Additionally, ten P. somniferum seed samples from various vendors were sequenced and compared across samples and to published reference data at the various barcode regions of interest. This study showed that the regions trnH-psbA and petA-psbJ have promise for utility in individualization for both inter- and intra-species individualization of P. somniferum.

Evaluation of the trnK-matK-trnK, ycf3, and accD-psal chloroplast regions to differentiate crop type and biogeographical origin of Cannabis sativa.

Cannabis sativa (marijuana and hemp) is one of the most controversial crops worldwide. In the USA, the state-specific legalization of marijuana and recently legalized hemp pose a problem for law enforcement. This study seeks to utilize chloroplast hSTRs, INDEL, and SNPs markers to develop genotyping methods to aid in the differentiation of legal hemp from illicit marijuana and also for tracking the flow of trafficked marijuana. Three polymorphic regions: trnK-matK-trnK, ycf3, and accD-psal, of the C. sativa chloroplast genome were evaluated in order to distinguish crop type and biogeographic origin. A total of nine polymorphic sites were genotyped from five distinct populations (hemp from the USA and Canada, marijuana from Chile and USA-Mexico, and medical marijuana from Chile) with a custom fragment and SNaPshotTM assay. The study also combined genotype results from the same sample set using 21 additional polymorphic markers from previous studies. The effectiveness of these multi-locus assays to distinguish sample groups was assessed using haplotype analysis, phylogenetic analysis, pairwise comparisons, and principal component analysis. Results indicated a clear separation of Canadian hemp using only the nine polymorphic sites developed in this study. The additional 21 markers were able to separate US hemp from both marijuana groups to a significant level (p < 0.05) when assessing average Fixation Indices (FST). This study demonstrated the applicability of these organelle markers for the determination of crop type and biogeographic origin of C. sativa. However, a more extensive database is needed to evaluate the true discriminatory power of these markers.

Characterization of new chloroplast markers to determine biogeographical origin and crop type of Cannabis sativa.

Marijuana (Cannabis sativa) is the most commonly used illicit drug in the USA. Despite its schedule I classification by the federal government, 33 states and the District of Columbia have legalized its use for medicinal or recreational purposes. This state-specific legalization has created a new problem for law enforcement: preventing and tracking the diversion of legally obtained Cannabis to states where it remains illegal. In addition, trafficking of the drug at the border with Mexico remains an issue for law enforcement agencies. C. sativa crops can be classified as marijuana (a drug containing the psychoactive chemical delta-9-tetrahydrocannabinol) or hemp (the non-drug form of the plant). Differentiation between crop types is important for forensic purposes. In addition, investigation of trafficking routes into and within the USA requires genetic association of samples from different seizures, and determining where the crop originated could provide important leads. This project seeks to exploit sequence variations in C. sativa chloroplast DNA (cpDNA) to allow genetic determination of biogeographic origin, discrimination between marijuana and hemp, and association between cases for C. sativa samples. Due to the limited discriminatory ability of common barcoding markers, the authors sought to discover more informative polymorphic regions. By comparing published whole genome cpDNA sequences, 58 polymorphisms and seven hotspot regions were identified. Hemp samples from the USA and Canada, marijuana samples from Mexico and Chile, and medical marijuana samples from Chile were evaluated using two cpDNA hotspot regions, rpl32-trnL and trnS-trnG. Principal component analysis supported some differences between the groups based on their crop type and biogeographic origin.

Massively parallel sequencing of 12 autosomal STRs in Cannabis sativa.

Massively parallel sequencing (MPS) is an emerging technology in the field of forensic genetics that provides distinct advantages compared to capillary electrophoresis. This study offers a proof of concept that MPS technologies can be applied to genotype autosomal STRs in Cannabis sativa. A custom panel for MPS was designed to interrogate 12 cannabis-specific STR loci by sequence rather than size. A simple workflow was implemented to integrate the custom PCR multiplex into a workflow compatible with the Ion Plus Fragment Library Kit, Ion™ Chef, and Ion™ S5 System. For data sorting and sequence analysis, a custom configuration file was designed for STRait Razor v3 to parse and extract STR sequence data. This study represents a preliminary investigation of sequence variation for 12 autosomal STR loci in 16 cannabis samples. Full concordance was observed between the MPS and CE data. Results revealed intra-repeat variation in eight loci where the nominal or size-based allele was identical, but variances were discovered in the sequence of the flanking region. Although only a small number of cannabis samples were evaluated, this study demonstrates that more informative STR data can be obtained via MPS.

Correction to: Nuclear, chloroplast, and mitochondrial data of a US cannabis DNA database.

The original version of this article contained a mistake. In page 10 of the original article, the significant level (p > 0.01) is incorrect. The correct significant level is (p < 0.01). The original article has been corrected.

Nuclear, chloroplast, and mitochondrial data of a US cannabis DNA database.

As Cannabis sativa (marijuana) is a controlled substance in many parts of the world, the ability to track biogeographical origin of cannabis could provide law enforcement with investigative leads regarding its trade and distribution. Population substructure and inbreeding may cause cannabis plants to become more genetically related. This genetic relatedness can be helpful for intelligence purposes. Analysis of autosomal, chloroplast, and mitochondrial DNA allows for not only prediction of biogeographical origin of a plant but also discrimination between individual plants. A previously validated, 13-autosomal STR multiplex was used to genotype 510 samples. Samples were analyzed from four different sites: 21 seizures at the US-Mexico border, Northeastern Brazil, hemp seeds purchased in the US, and the Araucania area of Chile. In addition, a previously reported multi-loci system was modified and optimized to genotype five chloroplast and two mitochondrial markers. For this purpose, two methods were designed: a homopolymeric STR pentaplex and a SNP triplex with one chloroplast (Cscp001) marker shared by both methods for quality control. For successful mitochondrial and chloroplast typing, a novel real-time PCR quantitation method was developed and validated to accurately estimate the quantity of the chloroplast DNA (cpDNA) using a synthetic DNA standard. Moreover, a sequenced allelic ladder was also designed for accurate genotyping of the homopolymeric STR pentaplex. For autosomal typing, 356 unique profiles were generated from the 425 samples that yielded full STR profiles and 25 identical genotypes within seizures were observed. Phylogenetic analysis and case-to-case pairwise comparisons of 21 seizures at the US-Mexico border, using the Fixation Index (F ST ) as genetic distance, revealed the genetic association of nine seizures that formed a reference population. For mitochondrial and chloroplast typing, subsampling was performed, and 134 samples were genotyped. Complete haplotypes (STRs and SNPs) were observed for 127 samples. As expected, extensive haplotype sharing was observed; five distinguishable haplotypes were detected. In the reference population, the same haplotype was observed 39 times and two unique haplotypes were also detected. Haplotype sharing was observed between the US border seizures, Brazil, and Chile, while the hemp samples generated a distinct haplotype. Phylogenetic analysis of the four populations was performed, and results revealed that both autosomal and lineage markers could discern population substructure.

Evaluation of four commercial quantitative real-time PCR kits with inhibited and degraded samples.

DNA quantification is a vital step in forensic DNA analysis to determine the optimal input amount for DNA typing. A quantitative real-time polymerase chain reaction (qPCR) assay that can predict DNA degradation or inhibitors present in the sample prior to DNA amplification could aid forensic laboratories in creating a more streamlined and efficient workflow. This study compares the results from four commercial qPCR kits: (1) Investigator® Quantiplex® Pro Kit, (2) Quantifiler® Trio DNA Quantification Kit, (3) PowerQuant® System, and (4) InnoQuant® HY with high molecular weight DNA, low template samples, degraded samples, and DNA spiked with various inhibitors.The results of this study indicate that all kits were comparable in accurately predicting quantities of high quality DNA down to the sub-picogram level. However, the InnoQuant(R) HY kit showed the highest precision across the DNA concentration range tested in this study. In addition, all kits performed similarly with low concentrations of forensically relevant PCR inhibitors. However, in general, the Investigator® Quantiplex® Pro Kit was the most tolerant kit to inhibitors and provided the most accurate quantification results with higher concentrations of inhibitors (except with salt). PowerQuant® and InnoQuant® HY were the most sensitive to inhibitors, but they did indicate significant levels of PCR inhibition. When quantifying degraded samples, each kit provided different degradation indices (DI), with Investigator® Quantiplex® Pro indicating the largest DI and Quantifiler® Trio indicating the smallest DI. When the qPCR kits were paired with their respective STR kit to genotype highly degraded samples, the Investigator® 24plex QS and GlobalFiler® kits generated more complete profiles when the small target concentrations were used for calculating input amount.

Evaluation of a 13-loci STR multiplex system for Cannabis sativa genetic identification.

Marijuana (Cannabis sativa) is the most commonly used illicit substance in the USA. The development of a validated method using Cannabis short tandem repeats (STRs) could aid in the individualization of samples as well as serve as an intelligence tool to link multiple cases. For this purpose, a modified 13-loci STR multiplex method was optimized and evaluated according to ISFG and SWGDAM guidelines. A real-time PCR quantification method for C. sativa was developed and validated, and a sequenced allelic ladder was also designed to accurately genotype 199 C. sativa samples from 11 U.S. Customs and Border Protection seizures. Distinguishable DNA profiles were generated from 127 samples that yielded full STR profiles. Four duplicate genotypes within seizures were found. The combined power of discrimination of this multilocus system is 1 in 70 million. The sensitivity of the multiplex STR system is 0.25 ng of template DNA. None of the 13 STR markers cross-reacted with any of the studied species, except for Humulus lupulus (hops) which generated unspecific peaks. Phylogenetic analysis and case-to-case pairwise comparison of 11 cases using F st as genetic distance revealed the genetic association of four groups of cases. Moreover, due to their genetic similarity, a subset of samples (N = 97) was found to form a homogeneous population in Hardy-Weinberg and linkage equilibrium. The results of this research demonstrate the applicability of this 13-loci STR system in associating Cannabis cases for intelligence purposes.

Sequence variation of commercially available kratom products at universal DNA barcode regions.

Mitragyna speciosa, commonly known as kratom, is a narcotic plant that is used for its unique mood-enhancing and pain-relieving effects. It is marketed throughout the United States as a 'legal high' and has gained popularity as an alternative to opioids. However, kratom's increasing involvement in accidental overdoses, especially among polydrug users, has prompted warnings from the Drug Enforcement Agency (DEA) and the Food and Drug Administration (FDA). Despite these warnings, kratom remains legal federally, although it is banned in six states. This legal disparity complicates monitoring and enforcement efforts in states where kratom is illegal. Common forensic techniques using morphology or chemical analysis are beneficial in some instances but are not useful in source attribution because most seized kratom is powdered and the alkaloid content of samples can vary within products, making sourcing unreliable. This study focused on developing a DNA barcoding method to access sequence variation in commercial kratom products. It evaluated the utility of one nuclear barcode region (ITS) and three chloroplast barcode regions (matK, rbcL, and trnH-psbA) in assessing sequence variation across commercially available kratom products. Novel polymorphisms were discovered, and the ITS region showed the greatest variation between samples. Among the 15 kratom products tested, only two haplotypes were identified across the four barcoding regions. The findings highlight the potential of DNA barcoding as a forensic tool in the traceability and enforcement against illegal kratom distribution. Nonetheless, the limited haplotypic diversity points to a need for further development and expansion of the M. speciosa DNA sequence database.

Development of a novel five-dye panel for human identification insertion/deletion (INDEL) polymorphisms.

DNA analysis of forensic case samples relies on short tandem repeats (STRs), a key component of the combined DNA index system (CODIS) used to identify individuals. However, limitations arise when dealing with challenging samples, prompting the exploration of alternative markers such as single nucleotide polymorphisms (SNPs) and insertion/deletion (INDELs) polymorphisms. Unlike SNPs, INDELs can be differentiated easily by size, making them compatible with electrophoresis methods. It is possible to design small INDEL amplicons (<200 bp) to enhance recovery from degraded samples. To this end, a set of INDEL Human Identification Markers (HID) was curated from the 1000 Genomes Project, employing criteria including a fixation index (FST) ≤ 0.06, minor allele frequency (MAF) >0.2, and high allele frequency divergence. A panel of 33 INDEL-HIDs was optimized and validated following the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, utilizing a five-dye multiplex electrophoresis system. A small sample set (n = 79 unrelated individuals) was genotyped to assess the assay's performance. The validation studies exhibited reproducibility, inhibition tolerance, ability to detect a two-person mixture from a 4:1 to 1:6 ratio, robustness with challenging samples, and sensitivity down to 125 pg of DNA. In summary, the 33-loci INDEL-HID panel exhibited robust recovery with low-template and degraded samples and proved effective for individualization within a small sample set.

The effectiveness of various strategies to improve DNA analysis of formaldehyde-damaged tissues from embalmed cadavers for human identification purposes.

Formalin-fixed tissues provide the medical and forensic communities with alternative and often last resort sources of DNA for identification or diagnostic purposes. The DNA in these samples can be highly degraded and chemically damaged, making downstream genotyping using short tandem repeats (STRs) challenging. Therefore, the use of alternative genetic markers, methods that pre-amplify the low amount of good quality DNA present, or methods that repair the damaged DNA template may provide more probative genetic information. This study investigated whether whole genome amplification (WGA) and DNA repair could improve STR typing of formaldehyde-damaged (FD) tissues from embalmed cadavers. Additionally, comparative genotyping success using bi-allelic markers, including INDELs and SNPs, was explored. Calculated random match probabilities (RMPs) using traditional STRs, INDEL markers, and two next generation sequencing (NGS) panels were compared across all samples. Overall, results showed that neither WGA nor DNA repair substantially improved STR success rates from formalin-fixed tissue samples. However, when DNA from FD samples was genotyped using INDEL and SNP-based panels, the RMP of each sample was markedly lower than the RMPs calculated from partial STR profiles. Therefore, the results of this study suggest that rather than attempting to improve the quantity and quality of severely damaged and degraded DNA prior to STR typing, a more productive approach may be to target smaller amplicons to provide more discriminatory DNA identifications. Furthermore, an NGS panel with less loci may yield better results when examining FD samples, due to more optimized chemistries that result in greater allelic balance and amplicon coverage.

Comparison of DNA extraction techniques for the recovery of bovine DNA from fly larvae crops.

Forensic entomology aids investigations using insects and is primarily associated with the estimation of post-mortem interval (PMI). Studies have shown that human DNA can be recovered from the crops of fly larvae. While several factors regarding the recovery of human DNA from crops have been studied, DNA extraction methods have not been thoroughly assessed. Determining a method for optimal extraction could aid crime laboratories in implementing DNA extraction from larvae and streamlining future research. Bovine DNA was used as a substitute for human DNA to test several DNA extractions kits. Four DNA extraction kits (Chelex®, PDQeX forensicGEM, EZ1® DNA Investigator, DNeasy® Powersoil® Pro Kit) were evaluated based on the quantity and quality of bovine DNA extracted. Extractions were performed on whole fly larvae and dissected crops. Quantification was performed using real-time PCR (qPCR) on a StepOne™ Real-Time PCR System with SYBR® Green using bovine-specific cytochrome b primers. The quality of extracts was determined by checking for inhibition using commercial qPCR chemistries with an internal PCR control (IPC). When using whole fly larvae, Powersoil® Pro yielded the highest average DNA yield (n = 10, 0.668 ± 0.458 ng/µl), while EZ1® DNA Investigator yielded the highest average with crops (n = 10, 0.605 ± 0.403 ng/µl). Chelex and forensicGEM yielded low amounts of bovine DNA, and its extracts were inhibited, unlike EZ1® and Powersoil® Pro, which have purification steps. Therefore, it is recommended to use EZ1® DNA Investigator coupled with automation on EZ1® Advanced XL to recover DNA from fly larvae crops.

Detection and analysis of DNA mixtures with the MiSeq FGx®.

Recognizing and interpreting mixtures are challenges that occur frequently in forensic casework. Therefore, any new analysis methods that are implemented must handle the challenges of mixed forensic samples. Next generation sequencing offers advantages over capillary electrophoresis in amplicon multiplexing and degraded sample analysis; however, advantages with mixed samples rely heavily on the advancement of user-friendly analysis software. This research analyzed samples with the ForenSeq™ DNA Signature Prep Kit on the MiSeq FGx® and compared them with the GlobalFiler™ STR Kit for capillary electrophoresis. Metrics tested for both chemistries included concordance, limits of detection, and mixture analysis. Data analysis for mixture samples was completed with the MixtureAce™ plug-in and ArmedXpert™ software. Next generation sequencing offered distinct advantages in limits of detection and isoallele heterozygosity but suffered from increased variability in stutter and allele count ratios compared to capillary electrophoresis.

Massively parallel sequencing of Cannabis sativa chloroplast hotspots for forensic typing.

BACKGROUND: Marijuana (Cannabis sativa) is the most commonly used illicit drug in the USA, and the use of DNA barcodes could assist drug trafficking investigations by indicating the biogeographical origin and crop type of a sample and providing a means for linking cases. Additionally, the legality of marijuana in the USA remains complicated with some states fully legalizing marijuana for recreational use while federally marijuana remains completely illegal. Massively parallel sequencing (MPS) offers distinct advantages over capillary electrophoresis (CE), including more comprehensive coverage of target loci, analysis of hundreds of markers simultaneously, and high throughput capabilities. METHODS: This study reports on the development of a MiSeq FGx® assay targeting seven "hotspot" regions in the Cannabis sativa chloroplast genome that are highly polymorphic and informative in attempts to determine biogeographical origin and distinguishing between marijuana and hemp. Sequencing results were compared to previous studies that used CE-based genotyping methods. RESULTS: A total of 49 polymorphisms were observed, 16 of which have not been previously reported. Additionally, sequence data revealed isoalleles at one locus, which were able to differentiate two samples that had the same haplotype using CE-based methods. This study reports preliminary results from sequencing 14 hemp and marijuana samples from different countries using the developed MPS assay. CONCLUSION: Future studies should genotype a more comprehensive sample set from around the world to build a haplotype database, which could be used to provide investigative leads for law enforcement agencies investigating marijuana trafficking.

Evaluation of tetrahydrocannabinolic acid (THCA) synthase polymorphisms for distinguishing between marijuana and hemp.

The Controlled Substances Act (CSA) classifies marijuana (Cannabis sativa) as a Schedule I illicit drug. However, the recent Agriculture Improvement Act of 2018 (U.S. Farm Bill) removed hemp from the definition of marijuana in the CSA, making it a legal crop. As a result, many hemp products are now available, including strains of hemp buds high in other cannabinoids such as cannabidiol (CBD) or cannabigerol (CBG). The genetic inheritance of chemical phenotype (chemotype) has been widely studied, with the tetrahydrocannabinolic acid (THCA) synthase gene at the forefront. Previous studies have speculated that there are two forms of the THCA gene, one that produces an active enzyme (present in marijuana) and one that cannot produce a functional enzyme (present in hemp). A DNA analysis method is desirable for determining crop type in sample types inconducive to chemical analysis, such as immature crops, trace residues, small leaf fragments, seeds, and root material. This study optimized and evaluated a previously reported single nucleotide polymorphism (SNP) assay for determining C. sativa crop type. Furthermore, the presence or absence of 15 cannabinoids, including THC and THCA, was reported in cannabis reference materials and 15 legal hemp flower samples. The SNP assay correctly identified crop type in most samples. However, several marijuana samples were classified as hemp, and several hemp seeds were classified as marijuana. Two strains of legal CBG hemp flowers were also classified as marijuana, indicating that factors other than the genetic variation of the THCA synthase gene should be considered when determining crop type.