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Int J Syst Evol Microbiol. 2016 Feb;66(2):1100-1103. doi: 10.1099/ijsem.0.000760. Epub 2015 Nov 9.

OrthoANI: An improved algorithm and software for calculating average nucleotide identity.

Lee I1,2, Ouk Kim Y2,3, Park SC2,3, Chun J2,1,3.

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1​School of Biological Sciences, Seoul National University, Seoul 151-742, Republic of Korea.
2​Institute of Molecular Biology & Genetics, Seoul National University, Seoul 151-742, Republic of Korea.
3​Interdisciplinary Program in Bioinformatics, Seoul National University, Seoul 151-742, Republic of Korea.


Species demarcation in Bacteria and Archaea is mainly based on overall genome relatedness, which serves a framework for modern microbiology. Current practice for obtaining these measures between two strains is shifting from experimentally determined similarity obtained by DNA-DNA hybridization (DDH) to genome-sequence-based similarity. Average nucleotide identity (ANI) is a simple algorithm that mimics DDH. Like DDH, ANI values between two genome sequences may be different from each other when reciprocal calculations are compared. We compared 63 690 pairs of genome sequences and found that the differences in reciprocal ANI values are significantly high, exceeding 1 % in some cases. To resolve this problem of not being symmetrical, a new algorithm, named OrthoANI, was developed to accommodate the concept of orthology for which both genome sequences were fragmented and only orthologous fragment pairs taken into consideration for calculating nucleotide identities. OrthoANI is highly correlated with ANI (using BLASTn) and the former showed approximately 0.1 % higher values than the latter. In conclusion, OrthoANI provides a more robust and faster means of calculating average nucleotide identity for taxonomic purposes. The standalone software tools are freely available at


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