Forensic Applications of DNA Sequencing to Combat Drug Trafficking and Biothreats



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Nonhuman forensic genetics encompasses a variety of fields that focus on using the genome to assess variations within species and between species for legal purposes. Technological advances in sequencing technologies and data analysis have allowed for a better understanding of these variations and have helped develop assays that can be used as investigative tools. This dissertation sought to apply sequencing technologies for use in both microbial forensics by assessing the utility of a third-generation sequencing platform, the Oxford Nanopore Technologies (ONT) MinION portable sequencer for taxonomic identification of bacterial pathogens of interest; and in forensic plant science by applying Sanger sequencing to assess the genetic variation of two forensically relevant species, Papaver somniferum (opium poppy) and Mitragyna speciosa (kratom), for the development of comprehensive tools for species identification and biogeographical source attribution.

Microbial forensics focuses on identification of pathogenic agents used in bioterrorism and biocrime for the purpose of source attribution to an individual(s). In 2001, the Anthrax letters bioterrorism attack revealed the need for more robust, universal, and rapid microbial forensic analysis on unknown biological evidence. Traditional approaches for bacterial identification include culturing isolates and molecular approaches involving Sanger and next generation sequencing (NGS) of the 16S ribosomal RNA gene (16S rRNA) but these are laborious and laboratory based. The ONT MinION sequencer can generate long read lengths that span the entire 16S rRNA gene and can accurately identify bacteria to the species level. This platform is also portable and allows for real-time analysis of the pathogen on-site, negating the need for transportation and laboratory analysis. However, it requires large quantities of pure high molecular weight (HMW) DNA compared to other sequencing platforms, thus, the bacterial DNA extraction method of choice is important for successful MinION sequencing. Most extraction methods are tailored to a priori knowledge of the taxonomic grouping, which is difficult to know for certain during an attack. Therefore, it is important to identify a universal extraction method that can isolate high-quality DNA for application to MinION sequencing.

Forensic plant science can be used for origin identification of seized illegal drugs derived from plants, such as marijuana, opium poppy, and kratom. P. somniferum, also known as the opium poppy, has been used for the opium stored in the flower pods. This opium contains morphine, codine, and thebaine, which have relaxing and pain-relieving properties. However, these alkaloids are highly addictive and are tightly controlled under the Controlled Substances Act (CSA). This control has led to the production of illegal heroin, which is derived from morphine. These substances have led to an epidemic that is responsible for many deaths due to overdoses.

Seized plant material and heroin are identified and sourced using morphological and chemical methods, which have been moderately successful but have major drawbacks. However, the use of the genome, especially the chloroplast genome (cpDNA), could aid in strengthening these methods. Two other species, P. setigerum and P. bracteatum, further complicate the analysis because they are morphologically similar and produce opium. However, these two species are not controlled and are used as a common defense in criminal proceedings. Therefore, methods must be developed that can individualize P. somniferum from its nearest neighbors and source the material to producers, suppliers, and distributors.

Mitragyna speciosa, also known as Kratom, is a ‘legal’ drug of abuse that is currently gaining popularity in the US. It has been banned in many countries, such as Thailand, but it is not federally controlled in the United States. Instead, each state has its own laws on sales. The effects are unique: in small quantities it acts like a stimulant, but in larger quantities it has opioid-like effects. There have been deaths documented from kratom use, many in polydrug users resulting in the safety of this product being questioned. The sale of this product is uncontrolled, and users often do not know what they are consuming, the total active alkaloid content, or if it has been adulterated.

The plant is very diverse morphologically, and seized components are usually powdered, making identification very difficult. Methods are needed to identify M. speciosa and help source and track the kratom coming into the US, especially in areas where it is illegal to possess and sell. This is confounded by the very limited literature and sequencing data available in databases.

The first aim of this study was to identify a universal extraction method that can be coupled with MinION sequencing for use in a forensic situation. It also evaluated the cloud-based data analysis software, EPI2ME, for accurate taxonomic identification. This study revealed that there was no true universal method, but the DNeasy PowerSoil kit and a Chelex-100 method described in the literature are compatible for rapid bacterial identification using MinION sequencing. However, the 16S Barcoding Kit 1-24 coupled with the EPI2ME 16S FASTQ data analysis workflow may not be appropriate for taxonomic identification of forensically relevant species. Furthermore, the intrinsic properties of the bacteria may play a greater role in sequencing accuracy rather than extraction methods.

The second aim of this study was to identify cpDNA barcoding markers that can be used as a comprehensive tool for the individualization of P. somniferum. Ten poppy seed samples (used as a proxy for heroin) were extracted, and the target regions of interest were sequenced using Sanger sequencing. Data were compared with previously published P. somniferum cpDNA genomes to assess intraspecies variability, as well as previously published genomes of P. setigerum and P. bracteatum for interspecies variation. There is a lack of sequencing data for the cpDNA genome of this species, and therefore, no hotspot regions were identified when comparing published genomes. Therefore, 11 previously described polymorphic barcode regions found in the literature were screened. Two regions, trnH-psbA and petA-psbJ, showed the most promise in their use for individualization of P. somniferum.

Lastly, the third aim of this study was to evaluate intraspecies variation of M. speciosa at recently published barcode markers, ITS, rbcL, matK, and trnH-psbA, for their utility in biogeographical source location. Commercially purchased kratom samples were extracted and sequenced using Sanger sequencing. The intraspecies variability was determined by aligning and comparing our data with the data available in public databases. A novel homopolymer T region was found in trnH-psbA, and two novel SNPs were found in the nuclear ITS region. However, our data appeared to group in two distinct haplotypes with no observable biogeographical trend.

These novel applications of sequencing to two distinct forensic fields can help aid investigations by providing intelligence, investigative leads, and for exclusionary purposes.The potential for bioterrorism or biocrime is not misplaced, and robust analytical methods for microbial forensics must be developed and tested. Drug trafficking has major economic and health impacts; therefore, identifying the source and trafficking routes is important to apprehending the individuals involved and stopping the import of illegal drugs. This work has also laid the foundation for future studies using sequencing data in microbial forensics and to aid in combating of drug trafficking.



Anthropology, Medical and Forensic, Biology, Microbiology, Biology, Botany