| Summary: | Due to the tremendous diversity and abundance of microbes in environmental and host-associated environments, adequate characterization of these samples remains a challenge for microbiologists. In order to increase the depth of sampling for diverse bacterial communities, this thesis research developed a novel method for sequencing and assembly of millions of paired-end reads from the 16S rRNA gene (spanning the V3 region; ~200 nucleotides), using Illumina-based next-generation sequencing. To confirm reproducibility and identify a suitable computational pipeline for data analysis, sequence libraries were prepared in duplicate for both a defined mixture of DNA from known cultured bacterial isolates (>1 million post-assembly sequences) and from an Arctic tundra soil sample (>6 million post-assembly sequences). These Illumina 16S rRNA gene libraries represent a substantial increase in number of sequences over all extant next-generation sequencing approaches (e.g. 454 pyrosequencing); the assembly of paired–end offers a methodological advantage by incorporating an initial quality control step for each 16S rRNA gene sequence. This method incorporates indexed primers to enable the characterization of multiple microbial communities in a single flow cell lane and may be readily modified to target other variable regions or genes. Soil pH is an important determinant of microbial community composition and diversity, yet few studies have characterized the specific effects of pH on individual bacterial taxa within bacterial communities, both abundant and rare. Composite soil samples were collected over two years from an experimentally maintained pH gradient ranging from 4.5 to 7.5 from the Craibstone Experimental Farm (Craibstone, Scotland). Extracted nucleic acids were characterized by bacterial and group-specific denaturing gradient gel electrophoresis (DGGE) and were sequenced using the Illumina sequencing method describe above. Both methods demonstrated comparable and reproducible shifts within higher taxonomic ...
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