>Magister Scientiae - MSc / Aims DNA metabarcoding has recently emerged as a valuable supplementary tool to ensure food authenticity within the global food market. However, it is widely known that highly processed food samples are one of DNA metabarcoding’s greatest shortfalls due to high DNA degradation, presence of PCR inhibitors and the incomplete removal of several undesirable compounds (such as polysaccharides) that makes the amplification of desired DNA challenging. This project has two main aims, the first of which was to determine and develop a cost and time effective DNA metabarcoding system that could successfully describe to species level the ingredient composition of highly processed vegetarian food products. The DNA metabarcoding system was thoroughly evaluated and tested by combining well-researched primers with varying concentrations into a multiplex reaction. The combination of plant and animal primers selected that yielded the best results were used to determine the species composition in the samples. The second aim is to determine the possible presence of meat contaminants within the highly processed vegetarian food samples. Numerous studies have shown that food adulteration is a wide-spread phenomenon throughout the world due to the economic gains it can provide. Animal primers were introduced into the multiplex reaction to aid in the identification of any meat products that could have been inserted into the vegetarian products to lower the overall cost to company. Methodology Thirty-two highly processed vegetarian food samples were collected in the Cape Town area from local and franchised supermarkets. DNA was extracted using the Chloroform/Isoamyl alcohol method best suited for plant-based samples followed by amplification of the following mini-barcoding regions: the mitochondrial 16S ribosomal rRNA, cytochrome B, tRNALeu – trnL – UAA intron and the ribosomal internal transcribed spacer region – ITS2 for plant and fungi identification. The PCR products were purified using the Qiaquick kit and library preparation and building was conducted using the TruSeq DNA PCR-free Library kit. Final purification was completed using AMPure XP kit and the pooled libraries were sequenced on an Illumina Miseq using 300bp paired-end run. Statistical and bioinformatic analysis on the NGS raw sequence reads was performed in R version 3.6.3. Results The results of the data analysis showed that the cytochrome B primer couldn’t detect any animal DNA in the vegetarian samples, however animal-derived sequences were detected in the positives present, validating the efficacy of the multiplex reaction. Mitochondrial 16S ribosomal rRNA was only able to detect plant-based DNA due to the structural homology between chloroplast and mitochondrial DNA. The fungal ribosomal internal transcribed spacer region – ITS2 detected sequences deriving from “Viridiplantae”. This result could have been due to the fungal and plant ribosomal internal transcribed spacer region – ITS2 sharing a reverse primer during amplification. The trnL region was able to detect the presence of undeclared coriander, mustard and wheat in 8 (29%), 6 (21%) and 5 (18%) samples respectively. Additionally, trnL was able to detect the presence of tobacco in 11 (35%) samples. This could have been due to cross-contamination between samples being co-extracted and amplified at the same time for separate studies. The PITS2 region was able to detect the presence of undeclared barley, mustard and wheat in 8 (25%), 4 (14%) and 4 (14%) samples respectively. Our results show the possibility of DNA metabarcoding for the authentication of a wide range of species present in highly processed vegetarian samples using a single assay. However, further optimization of the technique for the identification of both plant and animal species within vegetarian samples needs to be performed before the wide-spread implementation of this technology would be both feasible and viable. Eliminating primer biases, decreasing the risk of homology between different primers in the same assay as well as preventing the amplification of sequencing of undesirable DNA need to be further explored and ultimately mitigated before DNA metabarcoding can be widely seen as an effective and cost-effective method for authentication and food control.