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Adapting Polony Technology to Oligonucleotide Fingerprinting of Ribosomal rRNA Genes for Microbial Community Analysis

  • Author(s): Ruegger, Paul
  • Advisor(s): Borneman, James
  • et al.
Abstract

Bacteria are present in nearly all terrestrial environments and play varied and important roles. Understanding their impacts on the environments and hosts where they reside is greatly aided by an accurate estimation of the number and types present. We have adapted polony technology to Oligonucleotide Fingerprinting of Ribosomal rRNA Genes (OFRG), a hybridization-based method for clustering similar 16S rDNA sequences. We present a new OFRG probe set design method that utilizes the available taxonomic information of training sequences to improve the clustering of fingerprints into biologically meaningful groups. A software tool is presented that quickly and accurately identifies randomly placed polonies in microarray images. The polony OFRG method is applied to DNA from a mock bacterial community created from a clone library, as well as to PCR amplicons made from the same mock community to examine PCR bias. We also examine several natural bacterial communities, making polonies starting directly from genomic DNA templates. The method successfully clusters the known bacterial community and reveals the presence of artifacts in template from the mock community PCR. Natural bacterial communities are differentiated using a weighted UniFrac analysis. Due to the initial spatial separation of sample DNA strands, polonies are essentially free of the PCR bias and chimeric sequence formation that occurs in mixed-template PCR reactions. An additional benefit of the polony format is that sequences of near full-length rDNA can be obtained when desired - a feature not possible with current high-throughput sequencing methods. We anticipate polony OFRG may be an invaluable tool for microbial population studies where these two characteristics are required.

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