Abstract
The average nucleotide identity (ANI) determines if two genomes belong to the same species. Using ANI, we detected mislabeled genomes and recommend verifying with ANI and multilocus phylogenetic analysis the species affiliations of the announced genomes. The slightly different results obtained with different ANI calculation software can potentially mislead taxonomic inferences.
COMMENTARY
The average nucleotide identity (ANI) is a similarity index between a given pair of genomes that can be applicable to prokaryotic organisms independently of their G+C content, and a cutoff score of >95% indicates that they belong to the same species (1, 2). Despite the fact that some of the genomes published in Genome Announcements include ANI values with closely related genomes (3–6), these data are missing in many studies. The genome sequencing studies may use different software packages for ANI determination (3–6). The currently available software tools for ANI calculation include programs that have to be downloaded, like JSpecies (http://www.imedea.uib.es/jspecies) and Gegenees (http://www.gegenees.org/documentation.html), as well as online calculation tools, like the one at the EzGenome (http://www.ezbiocloud.net/ezgenome/ani) and the ANI calculator (http://enve-omics.ce.gatech.edu/ani/index). However, there is a lack of information indicating whether these tools provide consistent results.
In a recent study using ANI calculations and a multilocus phylogenetic analysis (MLPA), we discovered that 14 (36%) of the 39 Aeromonas genomes deposited in the GenBank genome database were taxonomically mislabeled and that ANI values can vary using different calculation tools (7). Of the different tools employed, the ANI calculator always provided higher values than the JSpecies and the EzGenome tools that provided very similar results, but the magnitude of the differences depended on the specific genomes that were compared. For instance, the ANI calculator provided results between the genomes Aeromonas hydrophila HZM (GenBank accession no. JEMQ01) and A. hydrophila ATCC 7966T (GenBank accession no. CP000462) of 89.0%, but when using JSpecies, the ANI was 86.6%. These results were <95% (the species ANI cutoff value) and therefore showed that the genome A. hydrophila HZM does not affiliate with A. hydrophila. This was also confirmed by the MLPA, because in the obtained tree, A. hydrophila HZM did not cluster with the type strain of A. hydrophila but with that of Aeromonas caviae instead. In fact, the ANI comparison between the genome of A. hydrophila HZM with that of A. caviae Ae398 (GenBank accession no. CACP01) showed results of >95% (98.4% with ANI calculator and 98.1% with JSpecies), clearly indicating that HZM belongs to the species A. caviae.
Typically, the differences between the ANI values obtained with the ANI calculator and JSpecies or EzGenome did not affect the species classification. The only exception might be a borderline ANI value (94.7%) obtained with the ANI calculator for the genomes of Aeromonas veronii AMC34 (GenBank accession no. AGWU01) and A. veronii B565 (GenBank accession no. CP002607) that might lead investigators to conclude that the two genomes belonged to the same species. However, the ANI values determined with JSpecies (93.7%) and EzGenome (93.5%), together with the MLPA results, confirmed that these two strains (AMC34 and B565) do not belong to the same species. Therefore, the use of other ANI determination methods in parallel with the ANI calculator is required to reinforce the correct interpretation of the results in borderline cases. Nevertheless, the ANI enabled us to easily detect wrongly labeled genomes.
The mistakes in the species names of deposited genomes are relevant because they may lead to incorrect conclusions in comparative genomic studies. Therefore, these data should motivate researchers to implement measures to prevent introducing taxonomical errors in public genome databases.
This commentary alerts authors about the ANI variations in relation to the calculation tool used and of the importance of using multiple approaches to confirm the taxonomic affiliations of announced genomes.
ACKNOWLEDGMENTS
This work was supported in part by the project with reference AGL2011-30461-C02-02 by the Ministerio de Ciencia e Innovación (Spain) and by funding from the European Union Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 311846.
The authors are solely responsible for the content of this publication. It does not represent the opinion of the European Commission. The European Commission is not responsible for any use that might be made of data appearing therein.
Footnotes
The views expressed in this Commentary do not necessarily reflect the views of the journal or of ASM.
REFERENCES
- 1. Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, Tiedje JM. 2007. DNA-DNA hybridization values and their relationship to whole-genome sequence similarities. Int. J. Syst. Evol. Microbiol. 57:81–91. 10.1099/ijs.0.64483-0. [DOI] [PubMed] [Google Scholar]
- 2. Richter M, Rosselló-Mora R. 2009. Shifting the genomic gold standard for the prokaryotic species definition. Proc. Natl. Acad. Sci. U. S. A. 106:19126–19131. 10.1073/pnas.0906412106. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Shagimardanova EI, Toymentseva AA, Balaban NP, Mardanova AM, Danilova YV, Gusev OA, Kostryukova E, Karpova I, Manolov A, Alexeev D, Sharipova MR. 2014. Draft genome sequence of Bacillus pumilus 7P, isolated from the soil of the Tatarstan Republic, Russia. Genome Announc. 2(3):e00599-14. 10.1128/genomeA.00599-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4. Komaki H, Ichikawa N, Hosoyama A, Fujita N, Igarashi Y. 2014. Draft genome sequence of marine-derived actinomycete Nocardiopsis sp. strain TP-A0876, a producer of polyketide pyrones. Genome Announc. 2(4):e00665-14. 10.1128/genomeA.00665-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5. Servín-Garcidueñas LE, Sánchez-Quinto A, Martínez-Romero E. 2014. Draft genome sequence of Commensalibacter papalotli MX01, a symbiont identified from the guts of overwintering monarch butterflies. Genome Announc. 2(2):e00128-14. 10.1128/genomeA.00128-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6. Kaden R, Agren J, Ferrari S, Lindberg M, Bäckman S, Wahab T. 2014. Whole-genome sequence of Brucella canis strain SVA13, isolated from an infected dog. Genome Announc. 2(4):e00700-14. 10.1128/genomeA.00700-14. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7. Beaz-Hidalgo R, Hossain MJ, Liles MR, Figueras MJ. 2014. Strategies to avoid wrongly labelled genomes using as example the detected wrong taxonomic affiliation for Aeromonas genomes in the GenBank database. PLOS One, in press. [DOI] [PMC free article] [PubMed] [Google Scholar]