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Updated: Sep 15, 2025

Novel Sequence Discovery by Subtractive Genomics
Published on: January 25, 2019
PyOrthoANI, PyFastANI, and Pyskani: a suite of Python libraries for computation of average nucleotide identity
Martin Larralde1,2, Georg Zeller1,2,3, Laura M Carroll4,5,6,7
1Structural and Computational Biology Unit, EMBL, 69117 Heidelberg, Germany.
Abstract:
The average nucleotide identity (ANI) metric has become the gold standard for prokaryotic species delineation in the genomics era. The most popular ANI algorithms are available as command-line tools and/or web applications, making it inconvenient to incorporate them into bioinformatic workflows, which utilize the popular Python programming language. Here, we present PyOrthoANI, PyFastANI, and Pyskani, Python libraries for three popular ANI computation methods. ANI values produced by PyOrthoANI, PyFastANI, and Pyskani are virtually identical to those produced by OrthoANI, FastANI, and skani, respectively (adjusted R 2 >0.999). Compared to OrthoANI, PyOrthoANI is, on average, 3× faster per genome, while PyFastANI has multithreading support for single queries. All three libraries integrate seamlessly with BioPython, making it easy and convenient to use, compare, and benchmark popular ANI algorithms within Python-based bioinformatic workflows, software programs, and notebooks. Each library is available as part of the Python Package Index repository under the open-source MIT license, with source code available via GitHub (PyOrthoANI, https://github.com/althonos/orthoani; PyFastANI, https://github.com/althonos/pyfastani; Pyskani, https://github.com/althonos/pyskani).
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