A comparison of methods for clustering 16S rRNA sequences into OTUs

PLoS One. 2013 Aug 13;8(8):e70837. doi: 10.1371/journal.pone.0070837. eCollection 2013.


Recent studies of 16S rRNA sequences through next-generation sequencing have revolutionized our understanding of the microbial community composition and structure. One common approach in using these data to explore the genetic diversity in a microbial community is to cluster the 16S rRNA sequences into Operational Taxonomic Units (OTUs) based on sequence similarities. The inferred OTUs can then be used to estimate species, diversity, composition, and richness. Although a number of methods have been developed and commonly used to cluster the sequences into OTUs, relatively little guidance is available on their relative performance and the choice of key parameters for each method. In this study, we conducted a comprehensive evaluation of ten existing OTU inference methods. We found that the appropriate dissimilarity value for defining distinct OTUs is not only related with a specific method but also related with the sample complexity. For data sets with low complexity, all the algorithms need a higher dissimilarity threshold to define OTUs. Some methods, such as, CROP and SLP, are more robust to the specific choice of the threshold than other methods, especially for shorter reads. For high-complexity data sets, hierarchical cluster methods need a more strict dissimilarity threshold to define OTUs because the commonly used dissimilarity threshold of 3% often leads to an under-estimation of the number of OTUs. In general, hierarchical clustering methods perform better at lower dissimilarity thresholds. Our results show that sequence abundance plays an important role in OTU inference. We conclude that care is needed to choose both a threshold for dissimilarity and abundance for OTU inference.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms
  • Cluster Analysis
  • Computational Biology / methods
  • Metagenomics / methods*
  • RNA, Ribosomal, 16S / genetics*
  • Reproducibility of Results
  • Time Factors


  • RNA, Ribosomal, 16S