pyani is a Python module that provides support for calculating average nucleotide identity (ANI) and related measures for whole genome comparisons, and rendering relevant graphical summary output. Where available, it takes advantage of multicore systems, and can integrate with SGE/OGE-type job schedulers for the sequence comparisons.
pyani also installs a script: average_nucleotide_identity.py that enables command-line ANI analysis.
Script: average_nucleotide_identity.py
The average_nucleotide_identity.py script - part of this module - enables ANI analysis at the command-line, and uses the pyani module behind the scenes.
average_nucleotide_identity.py [-h] [-o OUTDIRNAME] [-i INDIRNAME] [-v]
[-f] [-s] [-l LOGFILE] [--skip_nucmer]
[--skip_blastn] [--noclobber] [-g]
[--gformat GFORMAT] [--gmethod GMETHOD]
[--labels LABELS] [--classes CLASSES]
[-m METHOD] [--scheduler SCHEDULER]
[--maxmatch] [--nucmer_exe NUCMER_EXE]
[--blastn_exe BLASTN_EXE]
[--makeblastdb_exe MAKEBLASTDB_EXE]
[--blastall_exe BLASTALL_EXE]
[--formatdb_exe FORMATDB_EXE]
optional arguments:
-h, --help show this help message and exit
-o OUTDIRNAME, --outdir OUTDIRNAME
Output directory
-i INDIRNAME, --indir INDIRNAME
Input directory name
-v, --verbose Give verbose output
-f, --force Force file overwriting
-s, --fragsize Sequence fragment size for ANIb
-l LOGFILE, --logfile LOGFILE
Logfile location
--skip_nucmer Skip NUCmer runs, for testing (e.g. if output already
present)
--skip_blastn Skip BLASTN runs, for testing (e.g. if output already
present)
--noclobber Don't nuke existing files
-g, --graphics Generate heatmap of ANI
--gformat GFORMAT Graphics output format [pdf|png|jpg|svg]
--gmethod GMETHOD Graphics output method [mpl|R]
--labels LABELS Path to file containing sequence labels
--classes CLASSES Path to file containing sequence classes
-m METHOD, --method METHOD
ANI method [ANIm|ANIb|ANIblastall|TETRA]
--scheduler SCHEDULER
Job scheduler [multiprocessing|SGE]
--maxmatch Override MUMmer to allow all NUCmer matches
--nucmer_exe NUCMER_EXE
Path to NUCmer executable
--blastn_exe BLASTN_EXE
Path to BLASTN+ executable
--makeblastdb_exe MAKEBLASTDB_EXE
Path to BLAST+ makeblastdb executable
--blastall_exe BLASTALL_EXE
Path to BLASTALL executable
--formatdb_exe FORMATDB_EXE
Path to BLAST formatdb executable
Example data and output can be found in the directory test_ani_data. The data are chromosomes of four isolates of Caulobacter. Basic analyses can be performed with the command lines:
$ ./average_nucleotide_identity.py -i tests/test_ani_data/ -o tests/test_ANIm_output -m ANIm -g
$ ./average_nucleotide_identity.py -i tests/test_ani_data/ -o tests/test_ANIb_output -m ANIb -g
$ ./average_nucleotide_identity.py -i tests/test_ani_data/ -o tests/test_ANIblastall_output -m ANIblastall -g
$ ./average_nucleotide_identity.py -i tests/test_ani_data/ -o tests/test_TETRA_output -m TETRA -g
The graphical output below, supporting assignment of NC_002696 and NC_011916 to the same species (C.crescentus), and the other two isolates to distinct species (NC_014100:C.segnis; NC_010338:C. sp K31), was generated with the command-line:
./average_nucleotide_identity.py -v -i tests/test_ani_data/ \
-o tests/test_ANIm_output/ -g --gformat png \
--classes tests/test_ani_data/classes.tab \
--labels tests/test_ani_data/labels.tab
-
Biopython http://www.biopython.org
-
NumPy http://www.numpy.org/
-
pandas http://pandas.pydata.org/
-
SciPy http://www.scipy.org/
-
BLAST+ executable in the
$PATH, or available on the command line (required for ANIb analysis) ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/ -
legacy BLAST executable in the
$PATHor available on the command line (required for ANIblastall analysis) ftp://ftp.ncbi.nlm.nih.gov/blast/executables/release/LATEST/ -
MUMmer executables in the $PATH, or available on the command line (required for ANIm analysis) http://mummer.sourceforge.net/
- matplotlib http://matplotlib.org/
and/or
- R with shared libraries installed on the system http://cran.r-project.org/
- Rpy2 http://rpy.sourceforge.net/rpy2.html
If you have downloaded v0.1.0 or greater, and the dependencies above are satisfied, then installation should be as simple as downloading the latest release and uncompressing it, or cloning the repository as below:
$ git clone https://github.com/widdowquinn/pyani
then changing to the appropriate directory:
$ cd pyani
then issuing:
$ python setup.py install
(or whatever variant you wish, e.g. for a home directory-local installation) from the top directory in the repository (with root permissions, if necessary).
This module calculates Average Nucleotide Identity (ANI) according to one of a number of alternative methods described in, e.g.
- Richter M, Rossello-Mora R (2009) Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci USA 106: 19126-19131. doi:10.1073/pnas.0906412106. (ANI1020, ANIm, ANIb)
- Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, et al. (2007) DNA-DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Micr 57: 81-91. doi:10.1099/ijs.0.64483-0.
ANI is proposed to be the appropriate in silico substitute for DNA-DNA hybridisation (DDH), and so useful for delineating species boundaries. A typical percentage threshold for species boundary in the literature is 95% ANI (e.g. Richter et al. 2009).
All ANI methods follow the basic algorithm:
- Align the genome of organism 1 against that of organism 2, and identify the matching regions
- Calculate the percentage nucleotide identity of the matching regions, as an average for all matching regions
Methods differ on: (1) what alignment algorithm is used, and the choice of parameters (this affects the aligned region boundaries); (2) what the input is for alignment (typically either fragments of fixed size, or the most complete assembly available).
- ANIm: uses MUMmer (NUCmer) to align the input sequences.
- ANIb: uses BLASTN+ to align 1020nt fragments of the input sequences
- ANIblastall: uses legacy BLASTN to align 1020nt fragments of the input sequences
- TETRA: calculates tetranucleotide frequencies of each input sequence
The algorithms takes as input correctly-formatted FASTA multiple sequence files. All sequences for a single organism should be contained in only one sequence file. Although it is possible to provide new labels for each input genome, for rendering graphical output, the names of these files are used for identification so it is best to name them sensibly.
Output is written to a named directory. The output files differ depending on the chosen ANI method.
- ANIm: MUMmer/NUCmer .delta files, describing each pairwise sequence alignment. Output as tab-separated plain text and Excel format tables describing: alignment coverage; total alignment lengths; similarity errors; and percentage identity (ANIm).
- ANIb and ANIblastall: FASTA sequences describing 1020nt fragments of each input sequence; BLAST nucleotide databases - one for each set of fragments; and BLASTN output files (tab-separated tabular format plain text) - one for each pairwise comparison of input sequences. Output as tab-separated plain text and Excel format tables describing: alignment coverage; total alignment lengths; similarity errors; and percentage identity (ANIb or ANIblastall).
- TETRA: Tab-separated plain text and Excel format files describing the Pearson correlations between Z-score distributions for each tetranucleotide in each input sequence (TETRA).
If graphical output is chosen, the output directory will also contain PDF files representing the similarity between sequences as a heatmap with row and column dendrograms.
Unless otherwise indicated, all code is subject to the following agreement:
(c) The James Hutton Institute 2014, 2015
Author: Leighton Pritchard
Contact: leighton.pritchard@hutton.ac.uk
Address:
Leighton Pritchard,
Information and Computational Sciences,
James Hutton Institute,
Errol Road,
Invergowrie,
Dundee,
DD6 9LH,
Scotland,
UK
The MIT License
Copyright (c) 2014-2015 The James Hutton Institute
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