Next generation sequencing on soil samples from Etosha National Park: Diversity studies of an anthrax reservoir

• Main Author: Valseth, Karoline
• Format: Dissertation
• Language: English
• Subjects: anthracis; Bacillus; diversity; gun; metagenomic; microbial; sequencing; shot; soil

Nutrient availability is important for microbes in soil environments. When an animal dies and decomposes, nutrients are released into the soil. This may cause a shift in the diversity of microbes present, as well as lead to the introduction of new species into the already established microbial community. In the present study shot-gun metagenomic sequencing was used in order to investigate microbial changes in the soil before and after an influx of nutrients from two anthrax carcasses in Etosha National Park, Namibia. In addition, the study investigated temporal fluctuations in the microbial community after the introduction of nutrients and Bacillus anthracis (B. anthracis), the causative agent of anthrax. The final aspect of the study locked at the phylogeny of the two B. anthracis isolates from the anthrax carcasses and how they compared to published B. anthracis data. Soil samples were collected at six time-points during the first month after the death of two zebras, within the area of bloodspill from the carcasses. DNA was isolated and sequenced using Illumina MiSeq. The resulting metagenome sequences were cleaned, before taxonomical composition and abundance were determined using the programs metaxa2 and MetaAmp. A clear responsive community was found, were orders like Bacilliales, Fusobacteriales and Clostridiales increased after the zebras were introduced and then decreased during the sample period. The sequences that caused the increase of the Fusobacteriales order was from Fusobacteriales equinum, which is known to inhabit the mucosa of horses, indicating a species directly introduced by the zebra. Throughout the sampling period many orders were found to be non-responsive. These orders included the Frankiales, known to degrade organic matter, and Rhizobiales, known to fix nitrogen. These non-responsive orders likely had a stable level of nutrients available from the soil, and thus were unaffected by the carcass. From each carcass, a B. anthracis colony was grown, isolated and whole genome sequenced with Illumina MiSeq. The sequences were assembled into contigs using Spades and CLC. Various quality controls indicated that better assembly was achieved with the CLC assembly, and the resulting sequence strains were then sent for annotation to NCBI BioSample. Through phylogenetic analysis, the two CLC strains were found to belong to the A.Br.Aust94 branch of the B. anthracis phylogenetic tree. This is supported by several published studies where the A.B