title: Stability of operational taxonomic units: an important but neglected property for analyzing microbial diversity.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  Here, 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.
  
  
  CONCLUSIONS
  As 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.
authors.affiliation: State Key Laboratory of Organ Failure Research, Department of Environmental Health, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, 510515 China.
authors.forename: Yan
authors.initials: Y
authors.lastname: He
authors.affiliation: Department of Biological Sciences, Northern Arizona University, PO Box 5640, Flagstaff, AZ 86011-5640 USA ; Center for Microbial Genetics and Genomics, Northern Arizona University, PO Box 4073, Flagstaff, AZ 86011-4073 USA.
authors.forename: J Gregory
authors.initials: JG
authors.lastname: Caporaso
authors.affiliation: State Key Laboratory of Organ Failure Research, Department of Environmental Health, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, 510515 China.
authors.forename: Xiao-Tao
authors.initials: XT
authors.lastname: Jiang
authors.affiliation: State Key Laboratory of Organ Failure Research, Department of Environmental Health, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, 510515 China.
authors.forename: Hua-Fang
authors.initials: HF
authors.lastname: Sheng
authors.affiliation: Department of Pathology and Laboratory Science, Warren Alpert Medical School, Brown University, 70 Ship Street, Providence, RI 02912 USA.
authors.forename: Susan M
authors.initials: SM
authors.lastname: Huse
authors.affiliation: Center for Microbial Genetics and Genomics, Northern Arizona University, PO Box 4073, Flagstaff, AZ 86011-4073 USA.
authors.forename: Jai Ram
authors.initials: JR
authors.lastname: Rideout
authors.affiliation: ., Tiburon, CA 94920 USA.
authors.forename: Robert C
authors.initials: RC
authors.lastname: Edgar
authors.affiliation: Department of Pediatrics, University of California at San Diego, 9500 Gilman Drive MC0763, La Jolla, CA 92093-0763 USA.
authors.forename: Evguenia
authors.initials: E
authors.lastname: Kopylova
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, 526 Campus Road, Ithaca, NY 14853 USA.
authors.forename: William A
authors.initials: WA
authors.lastname: Walters
authors.affiliation: Department of Pediatrics, University of California at San Diego, 9500 Gilman Drive MC0763, La Jolla, CA 92093-0763 USA ; Department of Computer Science and Engineering, University of California at San Diego, 9500 Gilman Drive MC0763, La Jolla, CA 92093-0763 USA.
authors.forename: Rob
authors.initials: R
authors.lastname: Knight
authors.affiliation: State Key Laboratory of Organ Failure Research, Department of Environmental Health, Guangdong Provincial Key Laboratory of Tropical Disease Research, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, 510515 China.
authors.forename: Hong-Wei
authors.initials: HW
authors.lastname: Zhou