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Related Articles The effects of experimental choices and analysis noise on surveys of the rare biosphere.
Appl Environ Microbiol. 2009 Mar 6;
Authors: Hamp TJ, Jones WJ, Fodor AA
When planning a survey of 16S rRNA genes from a complex environment, investigators face many choices including which primers to use and how to taxonomically classify sequences. In this study, we explored how these choices affected a survey of microbial diversity in a sample taken from the aerobic basin of the activated sludge of a North Carolina wastewater treatment plant. We performed pyrosequencing reactions on PCR products generated from primers targeting the V1-V2, V6 and V6-V7 regions of the 16S rRNA gene. We compared these sequences to 16S rRNA gene sequences found in a whole-genome shotgun pyrosequencing run performed on the same sample. We found that sequences generated from primers targeting the V1-V2 variable region had the best match to the whole-genome shotgun reaction across a range of taxonomic classifications from phylum to family. Most of the differences between primer sets, however, occurred in the "rare biosphere" involving taxa that we observed in fewer than 10 sequences. We also examined the results of analysis strategies comparing a classification scheme using a nearest neighbor approach to directly classifying sequences with a naïve Bayesian algorithm. Again, most of the differences between these analysis schemes occurred in infrequently observed taxa. We conclude that if a study is meant to probe the rare biosphere both the experimental conditions and analysis choices will have a profound impact on the observed results.
PMID: 19270149 [PubMed - as supplied by publisher]
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Related Articles The effects of experimental choices and analysis noise on surveys of the rare biosphere.
Appl Environ Microbiol. 2009 Mar 6;
Authors: Hamp TJ, Jones WJ, Fodor AA
When planning a survey of 16S rRNA genes from a complex environment, investigators face many choices including which primers to use and how to taxonomically classify sequences. In this study, we explored how these choices affected a survey of microbial diversity in a sample taken from the aerobic basin of the activated sludge of a North Carolina wastewater treatment plant. We performed pyrosequencing reactions on PCR products generated from primers targeting the V1-V2, V6 and V6-V7 regions of the 16S rRNA gene. We compared these sequences to 16S rRNA gene sequences found in a whole-genome shotgun pyrosequencing run performed on the same sample. We found that sequences generated from primers targeting the V1-V2 variable region had the best match to the whole-genome shotgun reaction across a range of taxonomic classifications from phylum to family. Most of the differences between primer sets, however, occurred in the "rare biosphere" involving taxa that we observed in fewer than 10 sequences. We also examined the results of analysis strategies comparing a classification scheme using a nearest neighbor approach to directly classifying sequences with a naïve Bayesian algorithm. Again, most of the differences between these analysis schemes occurred in infrequently observed taxa. We conclude that if a study is meant to probe the rare biosphere both the experimental conditions and analysis choices will have a profound impact on the observed results.
PMID: 19270149 [PubMed - as supplied by publisher]
More...