Average Nucleotide Identity Analysis for Diagnosis

M. Asaduzzaman Prodhan^*

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.

Contents

• ANI methods
  
• ANI tools
  
• How to run pyani
  
• Results
  
• References
  

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

• 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

• If you are working on HPC Cluster, load the required version of python

module load cray-python/3.10.10

• Create a conda environment with the compatible version of python, matplotlib and pyani

conda create -n pyani_env python=3.10 "matplotlib<=3.7" "pyani>=0.2.12" -c bioconda

• Activate the ani environment

conda activate pyani_env

• Alternatively, you can use my conda environment for pyani

• Download it HERE

• Then activate it as follows

conda env create -f pyani_env.yml

• Check it has been installed. Copy the following command and hit enter

average_nucleotide_identity.py --help

The above command will show the flags/options of the pyani program

• Install dos2unix for changing file format

conda install conda-forge::dos2unix

• Check it has been installed. Copy the following command and hit enter

dos2unix

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

<img src="https://github.com/asadprodhan/Average-Nucleotide-Identity-ANI-analysis/blob/main/classes.PNG"

Figure 1. Classes

<img src="https://github.com/asadprodhan/Average-Nucleotide-Identity-ANI-analysis/blob/main/labels.PNG"

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:

dos2unix *.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: https://github.com/widdowquinn/pyani/issues/56

Results

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

<img src="https://github.com/asadprodhan/Average-Nucleotide-Identity-ANI-analysis/blob/main/ANImpercentageidentity.png"

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