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1. Kennedy, Katherine Margaret. Molecular methods for evaluating the human microbiome.

Degree: 2014, University of Waterloo

In human microbiome analysis, sequencing of bacterial 16S rRNA genes has revealed a role for the gut microbiota in maintaining health and contributing to various pathologies. Novel community analysis techniques must be evaluated in terms of bias, sensitivity, and reproducibility and compared to existing techniques to be effectively implemented. Next- generation sequencing technologies offer many advantages over traditional fingerprinting methods, but this extensive evaluation required for the most efficacious use of data has not been performed previously. Illumina libraries were generated from the V3 region of the 16S rRNA gene of samples taken from 12 unique sites within the gastrointestinal tract for each of 4 individuals. Fingerprint data were generated from these samples and prominent bands were sequenced. Sequenced bands were matched with OTUs within their respective libraries. The results demonstrate that denaturing gradient gel electrophoresis (DGGE) represents relatively abundant bacterial taxa (>0.1%) beta-diversity of all samples was compared using Principal Coordinates Analysis (PCoA) of UniFrac distances and Multi-Response Permutation Procedure (MRPP) was applied to measure sample cluster strength and significance; indicator species analysis of fingerprint bands and Illumina OTUs were also compared. The results demonstrate overall similarities between community profiling methods but also indicate that sequence data were not subject to the same limitations observed with the DGGE method (i.e., only abundant taxa bands are resolved, unable to distinguish disparate samples). In addition, the effect of stochastic fluctuations in ␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣␣ differ for DGGE and next-generation sequencing. I compared pooled and individual reactions for samples of high and low template concentration for both Illumina and DGGE using the combined V3-V4 region of the 16S rRNA gene, and demonstrated that template concentration has a greater impact on reproducibility than pooling. This research shows congruity between two disparate molecular methods, identifies sources of bias, and establishes new guidelines for minimizing bias in microbial community analyses.

Subjects/Keywords: microbiome; human microbiome; DGGE; denaturing gradient gel electrophoresis; Illumina; gut microbiome; NGS; next generation sequencing; MRPP; NMS; CM2BL; 16S; PCoA; PCR drft

…Immunoglobulin IL-10 IV kCal MRPP NAFLD ng NMS OTU PCoA PCR QIIME rRNA SCFA SOLiD T TLR Treg UPGMA V… …When characterizing microbial communities, marker genes are targeted for amplification by PCR… …x28;PCR) is useful for effective analysis of the human microbiome and other microbial… …in a visual representation of a sample’s community structure. DGGE uses a modified PCR to… …PCR amplicons from a single molecule become spatially clustered, allowing for a signal… 

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Kennedy, K. M. (2014). Molecular methods for evaluating the human microbiome. (Thesis). University of Waterloo. Retrieved from http://hdl.handle.net/10012/8230

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16th Edition):

Kennedy, Katherine Margaret. “Molecular methods for evaluating the human microbiome.” 2014. Thesis, University of Waterloo. Accessed October 30, 2020. http://hdl.handle.net/10012/8230.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7th Edition):

Kennedy, Katherine Margaret. “Molecular methods for evaluating the human microbiome.” 2014. Web. 30 Oct 2020.

Vancouver:

Kennedy KM. Molecular methods for evaluating the human microbiome. [Internet] [Thesis]. University of Waterloo; 2014. [cited 2020 Oct 30]. Available from: http://hdl.handle.net/10012/8230.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Kennedy KM. Molecular methods for evaluating the human microbiome. [Thesis]. University of Waterloo; 2014. Available from: http://hdl.handle.net/10012/8230

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

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