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Cited by 5,748Genomics and Phylogenetic StudiesBacterial Identification and Susceptibility TestingMicrobial Community Ecology and Physiology

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High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries

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Jain, Chirag, Rodriguez-R, Luis M., Phillippy, Adam M., Konstantinidis, Konstantinos T., Aluru, Srinivas·2018·Nature Communications
DOI 10.1038/s41467-018-07641-9PMC PMC6269478

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Abstract

A fundamental question in microbiology is whether there is continuum of genetic diversity among genomes, or clear species boundaries prevail instead. Whole-genome similarity metrics such as Average Nucleotide Identity (ANI) help address this question by facilitating high resolution taxonomic analysis of thousands of genomes from diverse phylogenetic lineages. To scale to available genomes and beyond, we present FastANI, a new method to estimate ANI using alignment-free approximate sequence mapping. FastANI is accurate for both finished and draft genomes, and is up to three orders of magnitude faster compared to alignment-based approaches. We leverage FastANI to compute pairwise ANI values among all prokaryotic genomes available in the NCBI database. Our results reveal clear genetic discontinuity, with 99.8% of the total 8 billion genome pairs analyzed conforming to >95% intra-species and <83% inter-species ANI values. This discontinuity is manifested with or without the most frequently sequenced species, and is robust to historic additions in the genome databases.

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Figures

Fig. 1 Correlation of FastANI and Mash-based ANI output with ANIb values for datasets D1–D5. Because FastANI assumes a probabilistic identity cutoff that is set to 80% by default, it reports 76, 570, 4271, 464, and 130 genome matches for the individual queries in datasets D1–D5 respectively. To enable a direct quality comparison against FastANI, Mash is executed for only those pairs that are reported by FastANI. Notice that each dataset encompasses a different nucleotide identity range (x-axes). Gray line represents a straight line y = x plot for reference. Pearson correlation coefficients corresponding to these plots are listed separately in Table 2. Last plot shows error of these methods w.r.t. ANIb using all five datasets
Fig. 1 Correlation of FastANI and Mash-based ANI output with ANIb values for datasets D1–D5. Because FastANI assumes a probabilistic identity cutoff that is set to 80% by default, it reports 76, 570, 4271, 464, and 130 genome matches for the individual queries in datasets D1–D5 respectively. To enable a direct quality comparison against FastANI, Mash is executed for only those pairs that are reported by FastANI. Notice that each dataset encompasses a different nucleotide identity range (x-axes). Gray line represents a straight line y = x plot for reference. Pearson correlation coefficients corresponding to these plots are listed separately in Table 2. Last plot shows error of these methods w.r.t. ANIb using all five datasetsCC BY · PubMed Central
Fig. 2 Scaling up FastANI’s performance using multi-core parallel execution. We executed parallel FastANI processes on 40 physical cores, where each process was assigned an equally sized part of the reference D1–D5 databases. Left and right plots evaluate FastANI’s compute and indexing phase, respectively. FastANI achieves reasonable speedups on all datasets except the compute phase in D1 and D5, as their runtime on a single core is too small to begin with (Table 3)
Fig. 2 Scaling up FastANI’s performance using multi-core parallel execution. We executed parallel FastANI processes on 40 physical cores, where each process was assigned an equally sized part of the reference D1–D5 databases. Left and right plots evaluate FastANI’s compute and indexing phase, respectively. FastANI achieves reasonable speedups on all datasets except the compute phase in D1 and D5, as their runtime on a single core is too small to begin with (Table 3)CC BY · PubMed Central
Fig. 3 Genetic discontinuity observed using 90 K genomes. a Histogram plot showing the distribution of ANI values among the 90 K genomes. Only ANI values in the 76–100% range are shown. Out of total 8.01 billion pairwise genome comparisons, FastANI reported only 17M ANI values (0.21%) with ANI between 83 and 95% indicating a genetic discontinuum. Multiple colors are used to show how genomes from different genera are contributing to this distribution. Few peaks in the histogram arise from genera that have been extensively sequenced and dominate the database. b Density curves of ANI values in the ANI range 76–100%. Each curve shows the density curve corresponding to the database at a particular time period. Discontinuity in all four curves is observed consistently. c. Distribution of ANI values with each comparison labeled by the nomenclature of genomes being compared. All the comparisons between Escherichia coli and Shigella spp. have been labeled separately. The 95% ANI threshold on x-axis serves as a valid classifier for comparisons belonging to same and different species
Fig. 3 Genetic discontinuity observed using 90 K genomes. a Histogram plot showing the distribution of ANI values among the 90 K genomes. Only ANI values in the 76–100% range are shown. Out of total 8.01 billion pairwise genome comparisons, FastANI reported only 17M ANI values (0.21%) with ANI between 83 and 95% indicating a genetic discontinuum. Multiple colors are used to show how genomes from different genera are contributing to this distribution. Few peaks in the histogram arise from genera that have been extensively sequenced and dominate the database. b Density curves of ANI values in the ANI range 76–100%. Each curve shows the density curve corresponding to the database at a particular time period. Discontinuity in all four curves is observed consistently. c. Distribution of ANI values with each comparison labeled by the nomenclature of genomes being compared. All the comparisons between Escherichia coli and Shigella spp. have been labeled separately. The 95% ANI threshold on x-axis serves as a valid classifier for comparisons belonging to same and different speciesCC BY · PubMed Central
Fig. 4 FastANI algorithm explained using synthetic and real examples. a Illustration of FastANI’s work-flow for computing ANI between a query genome and a reference genome. Five mappings are obtained from three query fragments using Mashmap24. Mforward saves the maximum identity mapping for each query fragment. In this example, Mforward = {m2, m4, m5}. From this set, Mreciprocal picks m4 and m5 as the maximum identity mapping for each reference bin. Mapping identities of orthologous mappings, thus found in Mreciprocal, are finally averaged to compute ANI. b FastANI supports visualization of the orthologous mappings Mreciprocal that are used to estimate the ANI value using genoPlotR39. In this figure, ANI is computed between Bartonella quintana strain (NC_018533.1) as query and Bartonella henselae strain (NC_005956.1) as reference. Red line segments denote the orthologous mappings computed by FastANI for ANI estimation
Fig. 4 FastANI algorithm explained using synthetic and real examples. a Illustration of FastANI’s work-flow for computing ANI between a query genome and a reference genome. Five mappings are obtained from three query fragments using Mashmap24. Mforward saves the maximum identity mapping for each query fragment. In this example, Mforward = {m2, m4, m5}. From this set, Mreciprocal picks m4 and m5 as the maximum identity mapping for each reference bin. Mapping identities of orthologous mappings, thus found in Mreciprocal, are finally averaged to compute ANI. b FastANI supports visualization of the orthologous mappings Mreciprocal that are used to estimate the ANI value using genoPlotR39. In this figure, ANI is computed between Bartonella quintana strain (NC_018533.1) as query and Bartonella henselae strain (NC_005956.1) as reference. Red line segments denote the orthologous mappings computed by FastANI for ANI estimationCC BY · PubMed Central

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  1. Staley, J. The phylogenomic species concept. Microbiol. Today 36, 80–83 (2009).

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Metadata

Authors
Jain, C., Rodriguez-R, L. M., Phillippy, A. M., Konstantinidis, K. T., & Aluru, S.
Affiliations
  • National Institutes of Health (US)
  • Georgia Institute of Technology (US)
Corresponding author
  • Konstantinidis, Konstantinos T.
  • Aluru, Srinivas
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2018-11-30
Volume
9
Issue
1
Pages
5114-5114
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-018-07641-9
PMC
PMC6269478
OpenAlex
W2951254987
Cited by
5,748
References
40
Citations by year
2026: 1107 · 2025: 1110 · 2024: 977 · 2023: 789 · 2022: 695 · 2021: 617 · 2020: 305 · 2019: 128 · 2018: 16
FWCI
96.11
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
Funders
National Science Foundation (1416259, 1816027, 1356288)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
  • Health Sciences › Medicine › Microbiology (medical) › Bacterial Identification and Susceptibility Testing
  • Physical Sciences › Environmental Science › Ecology › Microbial Community Ecology and Physiology
Keywords
average nucleotide identity, prokaryotic genomes, species boundaries, taxonomic classification, draft genome sequence
MeSH terms
Phylogeny, Prokaryotic Cells, Genetic Variation, Databases, Factual, Genome, Bacterial, Sequence Analysis, DNA

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