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Developing population genetics microsatellite markers from metagenomic shotgun next generation sequencing data

Barton, XavierORCID: 0000-0002-5783-4941 (2021) Developing population genetics microsatellite markers from metagenomic shotgun next generation sequencing data. Honours thesis, Murdoch University.

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Population genetics allows the measure of genetic variation between individuals within a population. Population genetics relies upon the use of genetic markers, which include Amplified fragment length polymorphism, Random amplified polymorphic DNA, Single nucleotide polymorphisms, and Short tandem repeats (microsatellites). Microsatellite markers are an excellent tool for measuring genetic variation due to their high mutation rate and ease of experimentation using polymerase chain reactions. However, the generation of microsatellite markers is costly and complicated. The workflow presented in this thesis aimed at creating an effective microsatellite marker design process. A mixed-species shotgun sequencing sample of an Ixodes holocyclus tick was used to create a workflow, with the objective of creating microsatellite population genetic markers. However, as ticks are hematophagous arthropods, the generated shotgun data would also include vertebrate host, microbiome and tick DNA. Therefore, a pipeline was developed, which included read mapping, read classification and metagenomic assembly, to isolate tick genomic content for downstream microsatellite retrieval. Four additional next generation sequencing datasets were obtained from NCBI in order to validate this workflow. In total, read mapping identified 441 microsatellite primer sets, metagenomic assembly identified 2,792, and read classification identified 617 with the un-isolated reads recovering 578 microsatellite primer sets. The results obtained from this study show that performing a Kraken2 custom database classification, read mapping to a close target reference genome (using Bowtie2) and metagenomic assembly (using MEGAHIT) all aid in isolating DNA of a target organism. Overall, the pipeline developed in this study was able to isolate target organism sequences, from which microsatellites were discovered and primer sets generated. The pipeline outlined in this study will provide future researchers a more streamlined approach to creating microsatellite markers for their target organism using standard shotgun next generation sequencing data.

Item Type: Thesis (Honours)
Murdoch Affiliation(s): Medical, Molecular and Forensic Sciences
Supervisor(s): Oskam, Charlotte and Tobe, Shane
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