Graduate Thesis Or Dissertation
 

Analysis of Bacteroidales 16S rRNA gene sequences from a geographically isolated human population

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  • Standard methods of measuring fecal pollution in water do not distinguish between human and non-human sources. Molecular technology enabled the development of host-specific markers that distinguish fecal sources. Human specific PCR primers, HF183F and HF134F, were designed based on phylogenetic analyses of partial 16S rRNA gene sequences from the Bacteroidales group of fecal anaerobes. Both primers amplify human fecal DNAs in the U.S., Europe, New Zealand, and Japan. However, they did not amplify human fecal DNAs from a geographically isolated population in Alaska, although amplification was possible with general Bacteroidales primers. We undertook phylogenetic analysis to compare Bacteroidales 16S rRNA genes from the isolated population to a non-isolated population. Our ultimate objective was to create new Bacteroidales human-specific primers from the full-length 16S rRNA gene to amplify fecal DNAs from both isolated and non-isolated geographic areas. Sequence libraries from the isolated Alaskan population and from Oregon were created by amplifying the full-length Bacteroidales 16S rRNA gene. Fragments were cloned and sequenced and 96 colonies from each geographic location were screened for inserts. Phylogenetic analysis and primer design used ARB software. Humans in the Alaskan isolated population did not have sequences from certain common human Bacteroidales groups, and also contained unique clades. The trees constructed showed that none of the Alaskan sequences grouped with sequences from which the current human primers were developed, explaining why fecal samples from this population did not amplify with those primers. A novel clade only contained human sequences from the Alaskan study and was one focus for primer design. Primers were designed from the sequences found in these two clades and from other human-specific clades. A promising primer in the latter half of the 16S rRNA gene targeted human-specific fecal bacteria in both Oregon and Alaskan populations. Humans in the isolated Alaskan population may differ in their fecal bacteria for reasons of geographic isolation, population history, or diet.
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