pyani is a Python3 module and script 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 the prgram
pyani, which enables command-line based analysis of genomes.
The easiest way to install
pyani is to use
pip3 install pyani
From version 0.1.3.2 onwards, this should also install all the required Python package dependencies. Prior to this version (i.e. 0.1.3.1 and earlier), you can acquire these dependencies with
pip -r, and pointing at
requirements.txt from this repository:
pip3 install -r requirements.txt pip3 install -r requirements-pip.txt
With a working
anaconda installation, install the
conda config --add channels defaults conda config --add channels bioconda conda config --add channels conda-forge
conda install pyani
NOTE: The current
conda version is behind the development version and has a different command-line interface. Documentation for the
conda version (0.2.x) can be found at the link below:
The NCBI legacy
BLAST package is deprecated and not recommended. However, if you wish to use
pyani blastall or the
ANIblastall method with the legacy
pyani interface, you will require a locally-installed copy of this package. This is one of the packages linked in the
A guide to citing
pyani is included in the file
README.md file provides a quick overview and walkthrough for THE DEVELOPMENT VERSION (v0.3+) OF
pyani, and full documentation can be found at the link below:
If you are using an older version of
pyani (v0.2.x), then please note that the command-line API has changed, and documentation for this version can be found at the following page:
If wou would like to report a bug or problem with
pyani, or ask a question of the developer(s), please raise an issue at the link below:
If you would like to contribute code to the
pyani project (as a bug fix or new feature), please refer to the
CONTRIBUTING.md guide for more details.
The command-line interface to
pyani uses subcommands. These separate individual steps of an analysis into separate actions.
The steps are described in detail with examples, below.
The first step is to obtain genome data for analysis.
pyani expects to find each individual genome in its own FASTA file (that file can contain multiple sequences - chromosomes and plasmids; sequenced scaffolds, etc). All the FASTA files for an analysis are expected to be located in a single subdirectory (with optional
classes files). You can arrange your data manually, but
pyani provides a subcommand that downloads all genomes in a taxon subtree from NCBI, and organises them ready for use with
We’ll use the
pyani download subcommand to download all available genomes for Candidatus Blochmannia from NCBI. The taxon ID for this grouping is 203804.
pyani download C_blochmannia --email firstname.lastname@example.org -t 203804 -v -l C_blochmannia_dl.log
The first argument is the output directory into which the downloaded genomes will be written (
C_blochmannia). To download anything from NCBI we must provide an email address (
--email email@example.com), and to specify which taxon subtree we want to download we provide the taxon ID (
Here we have also requested verbose output (
-v), and write a log file for reproducible research/diagnosing bugs and errors (
This produces a new subdirectory (
C_blochmannia) with the following contents:
$ tree C_blochmannia C_blochmannia ├── GCF_000011745.1_ASM1174v1_genomic.fna ├── GCF_000011745.1_ASM1174v1_genomic.fna.gz ├── GCF_000011745.1_ASM1174v1_genomic.md5 ├── GCF_000011745.1_ASM1174v1_hashes.txt ├── GCF_000043285.1_ASM4328v1_genomic.fna ├── GCF_000043285.1_ASM4328v1_genomic.fna.gz ├── GCF_000043285.1_ASM4328v1_genomic.md5 ├── GCF_000043285.1_ASM4328v1_hashes.txt ├── GCF_000185985.2_ASM18598v2_genomic.fna ├── GCF_000185985.2_ASM18598v2_genomic.fna.gz ├── GCF_000185985.2_ASM18598v2_genomic.md5 ├── GCF_000185985.2_ASM18598v2_hashes.txt ├── GCF_000331065.1_ASM33106v1_genomic.fna ├── GCF_000331065.1_ASM33106v1_genomic.fna.gz ├── GCF_000331065.1_ASM33106v1_genomic.md5 ├── GCF_000331065.1_ASM33106v1_hashes.txt ├── GCF_000973505.1_ASM97350v1_genomic.fna ├── GCF_000973505.1_ASM97350v1_genomic.fna.gz ├── GCF_000973505.1_ASM97350v1_genomic.md5 ├── GCF_000973505.1_ASM97350v1_hashes.txt ├── GCF_000973545.1_ASM97354v1_genomic.fna ├── GCF_000973545.1_ASM97354v1_genomic.fna.gz ├── GCF_000973545.1_ASM97354v1_genomic.md5 ├── GCF_000973545.1_ASM97354v1_hashes.txt ├── classes.txt └── labels.txt
Seven genomes have been downloaded, and each is represented by four files:
_genomic.fna.gz: the compressed genome sequence
_genomic.fna: the uncompressed genome sequence
_genomic.md5: an MD5 hash/checksum of the (uncompressed) genome sequence; this was generated during the download
_hashes.txt: a list of MD5 hashes; this is provided by NCBI and is a reference to be sure that the download did not corrupt the genome sequence
There are two additional plain text files:
labels.txt, which provide alternative labels for use in the analysis. These files are generated during the download.
pyani uses a database to store genome data and analysis results. This is convenient for data sharing and developing custom analyses, but also makes it easier to extend an existing ANI analysis with new genomes, without having to repeat calculations that were already performed.
To create a new, clean, database in the default location (
.pyani/pyanidb) issue the command:
pyani createdb -v -l C_blochmannia_createdb.log
As above, the verbose (
-v) and log file (
-l C_blochmannia_createdb.log) options allow for reproducible work. The default database location is in the hidden directory (
$ tree .pyani .pyani └── pyanidb
pyani commands will assume this location for the database, but you can specify the location when creating a database, or using an existing database.
pyani provides four subcommands to run ANI analyses:
anib: ANIb, using BLAST+
aniblastall: ANIb, using legacy BLAST
In this walkthrough, we’ll run ANIm on the downloaded genomes, using the command:
pyani anim C_blochmannia C_blochmannia_ANIm -v -l C_blochmannia_ANIm.log \ --name "C. blochmannia run 1" \ --labels C_blochmannia/labels.txt --classes C_blochmannia/classes.txt
All four analysis commands operate in a similar way. The first two arguments are paths to directories: the first path is to a directory containing input genomes, and the second is the path to an output directory for storing intermediate results. The
-l arguments work as above, specifying verbose output and logging output to a file.
You will probably notice that the verbose output is very verbose, to enable informative identification of any problems. In particular, the verbose output (which is also written to the log file) writes out the command-lines used for the pairwise comparisons so, if something goes wrong, you can test whether a specific comparison can be run at the command-line at all, to aid diagnosis of any problems.
One reason for using a database backend for analysis results is so that, for very large analyses, we do not ever need to recalculate a pairwise genome comparison. All the analysis subcommands check whether input genomes have been used before (using the unique MD5 hash for each genome to identify whether it’s been used previously), and whether the comparison of two genomes has been run, with the particular analysis settings that were used. If either genome was not seen before, or if the analysis settings are different, the comparison is performed.
You can test this for yourself by running the analysis command again, as below. You will see a number of messages indicating that genomes have been seen before, and that analyses performed before were skipped:
$ pyani anim C_blochmannia C_blochmannia_ANIm -v -l C_blochmannia_ANIm.log \ --name "C. blochmannia run 2" \ --labels C_blochmannia/labels.txt --classes C_blochmannia/classes.txt INFO: command-line: pyani anim C_blochmannia C_blochmannia_ANIm -v -l C_blochmannia_ANIm.log INFO: Running ANIm analysis INFO: Adding analysis information to database .pyani/pyanidb INFO: Current analysis has ID 2 in this database INFO: Identifying input genome/hash files: […] INFO: Adding genome data to database... WARNING: Genome already in database with this hash and path! WARNING: Using existing genome from database, row 1 […] INFO: Complete pairwise comparison list: [(1, 2), (1, 3), (1, 4), (1, 5), (1, 6), (2, 3), (2, 4), (2, 5), (2, 6), (3, 4), (3, 5), (3, 6), (4, 5), (4, 6), (5, 6)] INFO: Excluding pre-calculated comparisons INFO: Comparisons still to be performed:  INFO: All comparison results already present in database (skipping comparisons) INFO: Completed. Time taken: 0.211
Once an analysis is run, the results are placed in a local
SQLite database, which can be queried for information about the analyses that have been run. You can request information about:
--runs: show all analysis runs with results stored in the database
--runs_genomes: show all the analysis runs with results in the database, and all the genomes analysed in each run
--genomes: show all the genomes used for any analysis in the database
--genomes_runs: for each genome in the database, also list the analysis results it participates in
--run_results: show all the pairwise comparison results for a named run (run IDs can be obtained with the
The report tables are written to a named directory (compulsory argument), and are written by default
.tab plain-text format, but HTML and Excel format can alos be requested with the
$ pyani report -v --runs C_blochmannia_ANIm/ --formats html,excel,stdout INFO: Processed arguments: Namespace(cmdline='./pyani report -v --runs C_blochmannia_ANIm/ --formats html,excel', dbpath='.pyani/pyanidb', formats='html,excel', func=<function subcmd_report at 0x10c674a60>, logfile=None, outdir='C_blochmannia_ANIm/', run_results=False, show_genomes=False, show_genomes_runs=False, show_runs=True, show_runs_genomes=False, verbose=True) INFO: command-line: ./pyani report -v --runs C_blochmannia_ANIm/ --formats html,excel INFO: Creating output in formats: ['excel', 'tab', 'html'] INFO: Using database: .pyani/pyanidb INFO: Writing table of pyani runs from the database to C_blochmannia_ANIm/runs.* INFO: Completed. Time taken: 0.937 $ tree -L 1 C_blochmannia_ANIm/ C_blochmannia_ANIm/ ├── nucmer_output ├── runs.html ├── runs.tab └── runs.xlsx
To see all the pairwise results for an individual run, the run ID must be provided. It is possible to get results for more than one run ID by providing a comma-separated list of run IDs (though each run’s results will be provided in a separate file):
$ pyani report -v --runs C_blochmannia_ANIm/ --formats html,excel --run_results 1,2,3,4 INFO: Processed arguments: Namespace(cmdline='./pyani report -v --runs C_blochmannia_ANIm/ --formats html,excel --run_results 1,2,3,4', dbpath='.pyani/pyanidb', formats='html,excel', func=<function subcmd_report at 0x108616a60>, logfile=None, outdir='C_blochmannia_ANIm/', run_results='1,2,3,4', show_genomes=False, show_genomes_runs=False, show_runs=True, show_runs_genomes=False, verbose=True) INFO: command-line: ./pyani report -v --runs C_blochmannia_ANIm/ --formats html,excel --run_results 1,2,3,4 INFO: Creating output in formats: ['tab', 'excel', 'html'] INFO: Using database: .pyani/pyanidb INFO: Writing table of pyani runs from the database to C_blochmannia_ANIm/runs.* INFO: Attempting to write results tables for runs: ['1', '2', '3', '4'] INFO: Collecting data for run with ID: 1 INFO: Collecting data for run with ID: 2 INFO: Collecting data for run with ID: 3 INFO: Collecting data for run with ID: 4 INFO: Completed. Time taken: 1.285
You can see a run’s results in the terminal by specifying the
stdout format. For example, to see the identity, coverage, and other output matrices, you would specify
--run_matrices <RUN> and
--formats=stdout as below:
$ pyani report C_blochmannia_ANIm --formats=stdout --run_matrices 1 TABLE: C_blochmannia_ANIm/matrix_identity_1 C. Blochmannia pennsylvanicus BPEN C. Blochmannia floridanus C. Blochmannia vafer BVAF C. Blochmannia chromaiodes 640 B. endosymbiont of Polyrhachis (Hedomyrma) turneri 675 B. endosymbiont of Camponotus (Colobopsis) obliquus 757 C. Blochmannia pennsylvanicus BPEN 1.000000 0.834866 0.836903 0.980244 0.843700 0.829509 C. Blochmannia floridanus 0.834866 1.000000 0.828733 0.834916 0.847060 0.857859 C. Blochmannia vafer BVAF 0.836903 0.828733 1.000000 0.837811 0.866015 0.844438 C. Blochmannia chromaiodes 640 0.980244 0.834916 0.837811 1.000000 0.849834 0.834769 B. endosymbiont of Polyrhachis (Hedomyrma) turn... 0.843700 0.847060 0.866015 0.849834 1.000000 0.844228 B. endosymbiont of Camponotus (Colobopsis) obli... 0.829509 0.857859 0.844438 0.834769 0.844228 1.000000 TABLE: C_blochmannia_ANIm/matrix_coverage_1 C. Blochmannia pennsylvanicus BPEN C. Blochmannia floridanus C. Blochmannia vafer BVAF C. Blochmannia chromaiodes 640 B. endosymbiont of Polyrhachis (Hedomyrma) turneri 675 B. endosymbiont of Camponotus (Colobopsis) obliquus 757 C. Blochmannia pennsylvanicus BPEN 1.000000 0.045736 0.041404 1.000306 0.017263 0.021027 C. Blochmannia floridanus 0.051317 1.000000 0.152609 0.054930 0.016366 0.010749 C. Blochmannia vafer BVAF 0.045362 0.149012 1.000000 0.046520 0.008356 0.014706 C. Blochmannia chromaiodes 640 1.000856 0.048983 0.042485 1.000000 0.014056 0.016140 B. endosymbiont of Polyrhachis (Hedomyrma) turn... 0.018238 0.015410 0.008058 0.014841 1.000000 0.020416 B. endosymbiont of Camponotus (Colobopsis) obli... 0.021508 0.009799 0.013730 0.016500 0.019766 1.000000 TABLE: C_blochmannia_ANIm/matrix_aln_lengths_1 C. Blochmannia pennsylvanicus BPEN C. Blochmannia floridanus C. Blochmannia vafer BVAF C. Blochmannia chromaiodes 640 B. endosymbiont of Polyrhachis (Hedomyrma) turneri 675 B. endosymbiont of Camponotus (Colobopsis) obliquus 757 C. Blochmannia pennsylvanicus BPEN 791654.0 36207.0 32778.0 791896.0 13666.0 16646.0 C. Blochmannia floridanus 36207.0 705557.0 107674.0 38756.0 11547.0 7584.0 C. Blochmannia vafer BVAF 32778.0 107674.0 722585.0 33615.0 6038.0 10626.0 C. Blochmannia chromaiodes 640 791896.0 38756.0 33615.0 791219.0 11121.0 12770.0 B. endosymbiont of Polyrhachis (Hedomyrma) turn... 13666.0 11547.0 6038.0 11121.0 749321.0 15298.0 B. endosymbiont of Camponotus (Colobopsis) obli... 16646.0 7584.0 10626.0 12770.0 15298.0 NaN TABLE: C_blochmannia_ANIm/matrix_sim_errors_1 C. Blochmannia pennsylvanicus BPEN C. Blochmannia floridanus C. Blochmannia vafer BVAF C. Blochmannia chromaiodes 640 B. endosymbiont of Polyrhachis (Hedomyrma) turneri 675 B. endosymbiont of Camponotus (Colobopsis) obliquus 757 C. Blochmannia pennsylvanicus BPEN 0.0 5979.0 5346.0 15645.0 2136.0 2838.0 C. Blochmannia floridanus 5979.0 0.0 18441.0 6398.0 1766.0 1078.0 C. Blochmannia vafer BVAF 5346.0 18441.0 0.0 5452.0 809.0 1653.0 C. Blochmannia chromaiodes 640 15645.0 6398.0 5452.0 0.0 1670.0 2110.0 B. endosymbiont of Polyrhachis (Hedomyrma) turn... 2136.0 1766.0 809.0 1670.0 0.0 2383.0 B. endosymbiont of Camponotus (Colobopsis) obli... 2838.0 1078.0 1653.0 2110.0 2383.0 0.0 TABLE: C_blochmannia_ANIm/matrix_hadamard_1 C. Blochmannia pennsylvanicus BPEN C. Blochmannia floridanus C. Blochmannia vafer BVAF C. Blochmannia chromaiodes 640 B. endosymbiont of Polyrhachis (Hedomyrma) turneri 675 B. endosymbiont of Camponotus (Colobopsis) obliquus 757 C. Blochmannia pennsylvanicus BPEN 1.000000 0.038183 0.034652 0.980543 0.014564 0.017442 C. Blochmannia floridanus 0.042843 1.000000 0.126472 0.045862 0.013863 0.009221 C. Blochmannia vafer BVAF 0.037964 0.123491 1.000000 0.038975 0.007237 0.012418 C. Blochmannia chromaiodes 640 0.981082 0.040896 0.035594 1.000000 0.011945 0.013473 B. endosymbiont of Polyrhachis (Hedomyrma) turn... 0.015387 0.013053 0.006978 0.012613 1.000000 0.017236 B. endosymbiont of Camponotus (Colobopsis) obli... 0.017841 0.008406 0.011594 0.013774 0.016687 1.000000
The output of a
pyani run can also be represented graphically, using the
plot subcommand. For example, the command:
pyani plot C_blochmannia_ANIm 1 -v --formats png,pdf
.png format output in the
C_blochmannia_ANIm output directory for the run wuth ID 1, generated above. Five heatmaps are generated:
The heatmaps also include dendrograms, clustering the rows and columns by overall similarity.
pyani includes tests that can be run with
nosetest (including coverage measurement using
coverage.py) with the following command, executed from the repository root directory:
nosetests --cover-erase --cover-package=pyani --cover-html --with-coverage
Coverage output will be placed (by default) in the
cover subdirectory, and can be loaded into the web browser.
average_nucleotide_identity.py script - installed as part of this package - enables straightforward ANI analysis at the command-line, and uses the
pyani module behind the scenes.
You can get a summary of available command-line options with
$ ./average_nucleotide_identity.py -h usage: average_nucleotide_identity.py [-h] [-o OUTDIRNAME] [-i INDIRNAME] [-v] [-f] [-s FRAGSIZE] [-l LOGFILE] [--skip_nucmer] [--skip_blastn] [--noclobber] [--nocompress] [-g] [--gformat GFORMAT] [--gmethod GMETHOD] [--labels LABELS] [--classes CLASSES] [-m METHOD] [--scheduler SCHEDULER] [--workers WORKERS] [--SGEgroupsize SGEGROUPSIZE] [--maxmatch] [--nucmer_exe NUCMER_EXE] [--blastn_exe BLASTN_EXE] [--makeblastdb_exe MAKEBLASTDB_EXE] [--blastall_exe BLASTALL_EXE] [--formatdb_exe FORMATDB_EXE] [--write_excel] [--subsample SUBSAMPLE] [--seed SEED] [--jobprefix JOBPREFIX] […]
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_011916 to the same species (C.crescentus), and the other two isolates to distinct species (
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,pdf,eps \ --classes tests/test_ani_data/classes.tab \ --labels tests/test_ani_data/labels.tab
genbank_get_genomes_by_taxon.py, installed by this package, enables download of genomes from NCBI, specified by taxon ID. The script will download all available assemblies for taxa at or below the specified node in the NCBI taxonomy tree.
Command-line options can be viewed using:
$ genbank_get_genomes_by_taxon.py -h usage: genbacnk_get_genomes_by_taxon.py [-h] [-o OUTDIRNAME] [-t TAXON] [-v] [-f] [--noclobber] [-l LOGFILE] [--format FORMAT] [--email EMAIL] [--retries RETRIES] [--batchsize BATCHSIZE] […]
For example, the NCBI taxonomy ID for Caulobacter is 75, so all publicly-available Caulobacter sequences can be obtained using the command-line:
$ genbank_get_genomes_by_taxon.py -o Caulobacter_downloads -v -t 75 -l Caulobacter_downloads.log --email firstname.lastname@example.org INFO: genbank_get_genomes_by_taxon.py: Mon Apr 18 17:22:54 2016 INFO: command-line: /Users/lpritc/Virtualenvs/pyani3/bin/genbank_get_genomes_by_taxon.py -o Caulobacter_downloads -v -t 75 -l Caulobacter_downloads.log --email email@example.com INFO: Namespace(batchsize=10000, firstname.lastname@example.org', force=False, format='gbk,fasta', logfile='Caulobacter_downloads.log', noclobber=False, outdirname='Caulobacter_downloads', retries=20, taxon='75', verbose=True) INFO: Set NCBI contact email to email@example.com INFO: Creating directory Caulobacter_downloads INFO: Output directory: Caulobacter_downloads INFO: Passed taxon IDs: 75 INFO: Entrez ESearch with query: txid75[Organism:exp] INFO: Entrez ESearch returns 29 assembly IDs INFO: Identified 29 unique assemblies INFO: Taxon 75: 29 assemblies […] INFO: Assembly 639581: 271 contigs INFO: Assembly 233261: 17 contigs INFO: Assembly 575291: 48 contigs INFO: Mon Apr 18 17:25:46 2016 INFO: Done.
NOTE: You must provide a valid email to identify yourself to NCBI for troubleshooting.
The number of attempted retries for each download, and the size of a batch download can be modified. By default, the script will attempt 20 download retries, and obtain sequences in batches of 10000.
Note that Python package dependencies should automatically be installed if you are using version 0.1.3.2 or greater, and installing with
pip install pyani.
For earlier versions, you can satisfy dependencies by using
pip install -r requirements.txt (using the
requirements.txt file in this repository).
$PATH, or available on the command line (required for ANIb analysis) ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/
$PATHor available on the command line (required for ANIblastall analysis) ftp://ftp.ncbi.nlm.nih.gov/blast/executables/release/LATEST/
This module calculates Average Nucleotide Identity (ANI) according to one of a number of alternative methods described in, e.g.
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:
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).
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.
If graphical output is chosen, the output directory will also contain PDF, PNG and EPS files representing the various output measures as a heatmap with row and column dendrograms. Other output formats (e.g. SVG) can be specified with the
Unless otherwise indicated, all code is subject to the following agreement:
(c) The James Hutton Institute 2014-2019 (c) The University of Strathclyde 2019 Author: Leighton Pritchard Contact: firstname.lastname@example.org Address: Leighton Pritchard, Strathclyde Institute of Pharmacy and Biomedical Sciences 161 Cathedral Street Glasgow G4 0RE, Scotland, UK The MIT License Copyright (c) 2014-2019 The James Hutton Institute Permission is hereby granted, free of charge, to any person obtaining a copy of this software and associated documentation files (the "Software"), to deal in the Software without restriction, including without limitation the rights to use, copy, modify, merge, publish, distribute, sublicense, and/or sell copies of the Software, and to permit persons to whom the Software is furnished to do so, subject to the following conditions: The above copyright notice and this permission notice shall be included in all copies or substantial portions of the Software. THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM, OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE SOFTWARE.