Abstract
BackgroundThe operational taxonomic unit (OTU) is widely used in microbial ecology. Reproducibility in microbial ecology research depends on the reliability of OTU-based 16S ribosomal subunit RNA (rRNA) analyses.ResultsHere, we report that many hierarchical and greedy clustering methods produce unstable OTUs, with membership that depends on the number of sequences clustered. If OTUs are regenerated with additional sequences or samples, sequences originally assigned to a given OTU can be split into different OTUs. Alternatively, sequences assigned to different OTUs can be merged into a single OTU. This OTU instability affects alpha-diversity analyses such as rarefaction curves, beta-diversity analyses such as distance-based ordination (for example, Principal Coordinate Analysis (PCoA)), and the identification of differentially represented OTUs. Our results show that the proportion of unstable OTUs varies for different clustering methods. We found that the closed-reference method is the only one that produces completely stable OTUs, with the caveat that sequences that do not match a pre-existing reference sequence collection are discarded.ConclusionsAs a compromise to the factors listed above, we propose using an open-reference method to enhance OTU stability. This type of method clusters sequences against a database and includes unmatched sequences by clustering them via a relatively stable de novo clustering method. OTU stability is an important consideration when analyzing microbial diversity and is a feature that should be taken into account during the development of novel OTU clustering methods.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-015-0081-x) contains supplementary material, which is available to authorized users.
Highlights
The operational taxonomic unit (OTU) is widely used in microbial ecology
Due to an artifact of many OTU clustering methods, the equivalent situation is common in microbial ecology studies that use taxonomic-independent methods
This study demonstrated for the first time that the problem of depth-dependent rarefaction curves that is inherent in most of the de novo clustering algorithms arises from unstable OTUs and not from sequencing errors
Summary
The operational taxonomic unit (OTU) is widely used in microbial ecology. Reproducibility in microbial ecology research depends on the reliability of OTU-based 16S ribosomal subunit RNA (rRNA) analyses. Rapid advances in DNA sequencing technologies over the past decade have allowed us to study communities of microorganisms in far greater depth than was previously possible Many of these studies involve PCR amplification and sequencing of marker genes (often the 16S small ribosomal subunit RNA (rRNA)) from complex communities of organisms, which can be compared to databases of known sequences to identify the taxa present in the microbial community. These methods have led to the discovery of new organisms at a much. Sequences assigned to different OTUs can be assigned to a single OTU
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