Average Nucleotide Identity analysis

Asad Prodhan PhD

https://asadprodhan.github.io/

Average Nucleotide Identity (ANI) analysis calculates the percentage of nucleotide identity among the supplied nucleotide sequences. It produces a square matrix of the calculated values. This matrix allows for pairwise comparisons among the nucleotide sequences and helps determine their similarities.

ANI methods

There are several methods to calculate the ANI:

ANIb (based on BLAST algorithm)
ANIm (based on MUMmer algorithm)
TETRA (based on tetranucleotide signature occurrences)

ANI tools

There are several tools available for ANI analysis (Figueras et al., 2014). For example:

JSpecies (http://www.imedea.uib.es/jspecies) (multiple genome analysis)
Gegenees (http://www.gegenees.org/documentation.html) (Due to changes in NCBI Blast, Gegeneese may not function with Blast versions 2.10 and later. Blast version 2.9 should work OK)
EzGenome (http://www.ezbiocloud.net/ezgenome/ani) [online]
ANI calculator (http://enve-omics.ce.gatech.edu/ani/index) [online] (only two genomes per analysis)
Python3 package (pyani) (https://github.com/widdowquinn/pyani, https://pyani.readthedocs.io/en/latest/run_anim.html) based on (Richter and Rossello´-Mo´ra, 2009). Pyani uses MUMmer algorithm.

How to run pyani

Instal pyani as follows:

conda install -c bioconda pyani

Make two metadata files and name them as ‘classes.txt’ (Fig. 1) and ‘labels.txt’ (Fig. 2)

Figure 1. Classes

Figure 2. Labels

Note, the first column is the nucleotide sequences names

Second column is the label of the nucleotide sequences

Make a directory and name it as ‘ANI’ for example
Within the ‘ANI’ directory, make another directory and name it as ‘genomes’ for example
Keep all the nucleotide sequences, ‘classes.txt’ and ‘labels.txt’ in the ‘genomes’ directory
Check the line terminator of the ‘classes.txt’ and ‘labels.txt’ files as follows

file *.txt

If ‘classes.txt’ and ‘labels.txt’ have CRLF (Windows) format, then convert them into Unix format as follows:

dos2linux *.txt

Run the following command from the ‘ANI’ directory

average_nucleotide_identity.py -i genomes -o output_ANI --labels genomes/labels.txt --classes genomes/classes.txt -g --gmethod seaborn --gformat pdf,png -v -l ba_ANI.log

Note that you do not make the output directory beforehand. Otherwise, the command will exit with an ‘overwriting’ error
Command reference: widdowquinn/pyani#56

Results

The final output of the ANI analysis looks like this (Fig. 3):

Figure 3. Results

References

Figueras, M.J., Beaz-Hidalgo, R., Hossain, M.J., Liles, M.R., 2014. Taxonomic Affiliation of New Genomes Should Be Verified Using Average Nucleotide Identity and Multilocus Phylogenetic Analysis. Genome Announc 2, e00927-14. https://doi.org/10.1128/genomeA.00927-14

Richter, M., Rossello´-Mo´ra, R., 2009. Shifting the genomic gold standard for the prokaryotic species definition | Proceedings of the National Academy of Sciences. PNAS 106, 19126–19131. https://doi.org/10.1073/pnas.0906412106

asadprodhan / Average-Nucleotide-Identity-ANI-analysis