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Microbial Ecology

Molecular Diversity of a North Carolina Wastewater Treatment Plant as Revealed by Pyrosequencing

Nina Sanapareddy, Timothy J. Hamp, Luis C. Gonzalez, Helene A. Hilger, Anthony A. Fodor, Sandra M. Clinton
Nina Sanapareddy
1Bioinformatics Research Center
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Timothy J. Hamp
1Bioinformatics Research Center
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Luis C. Gonzalez
1Bioinformatics Research Center
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Helene A. Hilger
2Department of Civil and Environmental Engineering
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Anthony A. Fodor
1Bioinformatics Research Center
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  • For correspondence: anthony.fodor@gmail.com
Sandra M. Clinton
3Department of Biology, University of North Carolina at Charlotte, 9201 University City Blvd., Charlotte, North Carolina 28223
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DOI: 10.1128/AEM.01210-08
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  • FIG. 1.
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    FIG. 1.

    Pie charts showing taxonomic assignments for 148 16S rRNA sequences in our data set that could be classified to the phylum level with RDP confidence scores of ≥80. At the phylum level, the Simpsons diversity index is 0.48.

  • FIG. 2.
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    FIG. 2.

    Results obtained with the RDP classification algorithm for 148 16S rRNA sequences that can be assigned at the phylum level with a confidence score of ≥80. The x axis of each graph shows the confidence in assignments as reported by the RDP classification algorithm. The y axis of each graph shows the level of identity (expressed as a percentage) between our query sequence and the best Blastn hit in the RDP database (version 9.52). The horizontal and vertical lines indicate 95% sequence identity and an RDP confidence score of 80, respectively.

  • FIG. 3.
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    FIG. 3.

    Locations (as determined by manual annotation) and E-values of sequences from the 648-member pyrosequencing data set that matched the RDP 16S rRNA database at an E-value cutoff of 0.01.

  • FIG. 4.
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    FIG. 4.

    Fraction covered as a function of the size of each assembled sequence for each of the 1,442 assembled plasmids and chromosomes in the NCBI datadase. The fraction covered is defined as the number of nucleotides in the assembled sequence that match at least one of our wastewater sequences divided by the total number of nucleotides in the assembled sequence.

  • FIG. 5.
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    FIG. 5.

    Nonspecific recruitment against the Acidovorax sp. strain JS42 genome. BLAST hits with alignment lengths less than 75 nucleotides (for the 20 March run) or 250 nucleotides (for the environmental sequence database) were removed. Protein annotations are derived from the full NCBI core nucleotide report for the Acidovorax sp. strain JS42 genome (http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=121592436 ).

  • FIG. 6.
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    FIG. 6.

    Region involving a transposase from the JS42 genome that shows an exception to the pattern of nonspecific recruitment. For visualization, a small amount of random noise was added to the y axis (as otherwise most of the hits to the transposase region would be superimposed). The red sequences matching the transposase region are from the GOS (28).

  • FIG. 7.
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    FIG. 7.

    Functional categories provided for our data set by the SEED server (http://www.theseed.org ). The data for microbial genomes are averages for sequences gathered from multiple biomes (10).

Tables

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  • TABLE 1.

    Top 10 assembled microbial genomes as sorted by the number of hits recruited from our wastewater metagenomea

    No. of hitsFraction of genome coveredAnnotation
    181100.26gi 121592436 ref NC_008782.1 Acidovorax sp. JS42
    173410.20gi 120608714 ref NC_008752.1 Acidovorax avenae subsp. citrulli AAC00-1
    171000.16gi 160895450 ref NC_010002.1 Delftia acidovorans SPH-1
    168000.20gi 171056692 ref NC_010524.1 Leptothrix cholodnii SP-6
    157520.23gi 124265193 ref NC_008825.1 Methylibium petroleiphilum PM1
    156950.22gi 121602919 ref NC_008781.1 Polaromonas naphthalenivorans
    154680.18gi 91785913 ref NC_007948.1 Polaromonas sp. JS666
    147350.16gi 121607004 ref NC_008786.1 Verminephrobacter eiseniae EF01-2
    135900.18gi 89898822 ref NC_007908.1 Rhodoferax ferrireducens T118
    115950.15gi 119896292 ref NC_008702.1 Azoarcus sp. BH72
    • ↵ a A complete list of all assembled microbial genomes is shown in File S6 in the supplemental material.

Additional Files

  • Figures
  • Tables
  • Supplemental material

    Files in this Data Supplement:

    • Supplemental file 1 - Schematic diagram of the wastewater treatment plant from which the samples in this study were taken (Fig. S1).
      Zipped PDF, 319K.
    • Supplemental file 2 - Summary of the bioinformatics analyses used in the study (Fig. S2).
      Zipped PDF file, 173K.
    • Supplemental file 3 - Distribution of sequence lengths of the 454-FLX pyrosequence reads from this study (Fig. S3).
      Zipped PDF, 168K.
    • Supplemental file 4 - Large contigs produced by the 454 assembler (File S1).
      Zipped FNA file, 32K.
    • Supplemental file 5 - Functional assignments for assembled contigs (File S2).
      Zipped TXT file, 7K.
    • Supplemental file 6 - Putative 16S rRNA gene sequences within the sequences in our data set as identified by BLAST analysis (File S3).
      Zipped FNA file, 61K.
    • Supplemental file 7 - RDP assignments of putative 16S rRNA genes (File S4).
      Zipped TXT file, 19K.
    • Supplemental file 8 - The locations of 16S rRNA sequences of samples as determined by manual inspection of GenBank records (File S5).
      Zipped TXT file, 73K.
    • Supplemental file 9 - The fraction of each assembled genome at NCBI covered by reads recruited from our sequences (File S6).
      Zipped TXT file, 80K.
    • Supplemental file 10 - Figure and file legends.
      MS Word document, 25K.
    • Supplemental file 11 - Supplemental bioinformatics methods.
      Zipped MS Word document, 74K.
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Molecular Diversity of a North Carolina Wastewater Treatment Plant as Revealed by Pyrosequencing
Nina Sanapareddy, Timothy J. Hamp, Luis C. Gonzalez, Helene A. Hilger, Anthony A. Fodor, Sandra M. Clinton
Applied and Environmental Microbiology Mar 2009, 75 (6) 1688-1696; DOI: 10.1128/AEM.01210-08

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Molecular Diversity of a North Carolina Wastewater Treatment Plant as Revealed by Pyrosequencing
Nina Sanapareddy, Timothy J. Hamp, Luis C. Gonzalez, Helene A. Hilger, Anthony A. Fodor, Sandra M. Clinton
Applied and Environmental Microbiology Mar 2009, 75 (6) 1688-1696; DOI: 10.1128/AEM.01210-08
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KEYWORDS

biodiversity
sewage
Water Microbiology

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