Molecular diversity of a North Carolina wastewater treatment plant as revealed by pyrosequencing

Abstract

We report the results of pyrosequencing of DNA collected from the activated sludge basin of a wastewater treatment plant in Charlotte, NC. Using the 454-FLX technology, we generated 378,601 sequences with an average read length of 250.4 bp. Running the 454 assembly algorithm over our sequences yielded very poor assembly, with only 0.3% of our sequences participating in assembly of significant contigs. Of the 117 contigs greater than 500 bp long that were assembled, the most common annotations were to transposases and hypothetical proteins. Comparing our sequences to known microbial genomes showed nonspecific recruitment, indicating that previously described taxa are only distantly related to the most abundant microbes in this treatment plant. A comparison of proteins generated by translating our sequence set to translations of other sequenced microbiomes shows a distinct metabolic profile for activated sludge with high counts for genes involved in metabolism of aromatic compounds and low counts for genes involved in photosynthesis. Taken together, these data document the substantial levels of microbial diversity within activated sludge and further establish the great utility of pyrosequencing for investigating diversity in complex ecosystems.

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.

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.

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.

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.

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.

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.

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).