Effects of Experimental Choices and Analysis Noise on Surveys of the “Rare Biosphere”

Abstract

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 variable 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. Pronounced differences between primer sets, however, occurred in the "rare biosphere" involving taxa that we observed in fewer than 11 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, we observed pronounced differences between these analysis schemes 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.For nearly 3 decades, investigations of the distribution of microbes in complex environments have focused on the use of rRNA genes (1,2,4,11,16,18,19,22,24). Because the full-length 16S rRNA sequence can be obtained with pairedend reads via traditional Sanger sequencing, until recently most studies of the 16S rRNA gene captured most or nearly most of the 16S sequence length. New pyrosequencing technologies, however, have recently been introduced that greatly reduce the per base cost of sequencing but with shorter read lengths than traditional Sanger sequencing (17). This new approach has proven powerful, yielding a previously unobtainable view of rare taxa (7,(12)(13)(14)25).The shorter reads produced by pyrosequencing require the choice of a particular region of the 16S rRNA gene to target for pyrosequencing as well as the choice of an algorithm to classify the taxonomy of the shorter reads. In their initial surveys of microbial diversity with pyrosequencing (12, 14, 25), Sogin and colleagues targeted the V6 variable region, in part because it is was small enough to be captured with the 100-bp reads of the pyrosequencing technology available at the time. Recently, the read length of 454 pyrosequencing machines has been increased to an average of ϳ250 bp. This allows for more flexibility in primer design and opens up the possibility of targeting regions of the 16S rRNA gene other than V6. In recent work, Huse et al. took advantage of this new capability to compare the classifications made for the human gut microbiome with the V6 and longer V3 regions (13). Plotti...

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