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Horses-Diseases, Gastrointestinal system-Diseases-Treatment, Helminthiasis-Treatment, Nucleotide sequence, Metagenomics, Biotic communities, Polymerase chain reaction, Bacterial diversity-Genetic aspects, Ribosomes, 16S ribosomal RNA, DNA extraction, Helminths, Massively parallel sequencing, Microbiome, Qiime, Miseq, Illumina
The advent of high-throughput genomic sequencing methods has become instrumental in the ability to study biological systems that are normally difficult to investigate using traditional culture-based techniques. Metagenomic analysis involves sequencing all DNA from a particular environment in order to reveal microbial diversity in a way that shotgun sequencing has not been able to do. The advantages of metagenomics are illustrated by this project that aimed to better characterize the equine gastrointestinal tract. Diseases affecting the gastrointestinal system are the main cause of mortality in horses and yet, our understanding of bacterial diversity and abundance is quite limited. To address these concerns, the project asks two questions: 1) which groups and species of bacteria are present in this environment before and after treatment with antihelminthics and in what abundance, and 2) does infection with parasitic helminths followed by antihelminthic treatment cause a shift in the composition of the equine GI tract microbiome? A metagenomic study was conducted in order to address the above two questions. Genomic DNA was extracted from equine fecal samples (n=6) and assessed for purity and quantity. PCR was performed using a modified protocol from Caporaso et al. (2012) to amplify the v4 variable region of the 16S ribosomal RNA gene, which has been widely used to classify bacterial representatives inhabiting different environmental niches (Wang and Qian, 2009). PCR amplification of DNA samples proved to be difficult due to residual inhibitors and as a result, amplicons were generated for only three of the four study horses. Each horse had two representative samples: August 25, 2011 and December 3, 2011, which correspond to pre-treatment and post-treatment samples, respectively. The 16S amplicons were sequenced by the Illumina MiSeq to assess bacterial diversity and abundance. Sequence data were analyzed using the software package, QIIME. The GreenGenes database was used as reference for classification of sequences into Operational Taxonomic Units. A total of 3,361,963 sequences representing read 1 were classified into 3,528 OTUs. Bar charts were generated for each of the samples to visualize the OTU taxonomical data at the phylum, class, order, family, and genus levels. The two most abundant phyla were Bacteriodetes and Firmicutes. In the "pre-treatment" samples, Bacteriodetes predominated (42.6%) followed by Firmicutes (27.1%) and Verrucomicrobia (12.7 %). Firmicutes was the most prevalent phylum among the "post-treatment" samples accounting for 34.6 % of sequences, followed by Bacteriodetes (31.5%) and Verrucomicrobia (21.7%). However, statistical tests must be performed to determine if abundances are significantly different between treatment groups and individual horses.
Sirois, Rachael Jean, "Comparison of the fecal microbiota of horses before and after treatment for parasitic helminths : massively parallel sequencing of the v4 region of the 16S ribosomal RNA gene" (2013). Masters Thesis, Smith College, Northampton, MA.
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