Aug 05, 2022

Public workspacePhylogenomic analysis of Xanthomonas

DOI
dx.doi.org/10.17504/protocols.io.261geny57g47/v1
  • 1University of Exeter
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DOI: dx.doi.org/10.17504/protocols.io.261geny57g47/v1
Protocol Citation: David J Studholme 2022. Phylogenomic analysis of Xanthomonas. protocols.io https://dx.doi.org/10.17504/protocols.io.261geny57g47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 03, 2022
Last Modified: August 05, 2022
Protocol Integer ID: 68154
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Abstract
This is a protocol for using PhaME to generate a phylogenomic tree from a set of Xanthomonas spp. genome sequences.
Create a directory for downloaded genome sequence data:

Command
Create directory (Ubuntu 22.04)
mkdir genomes

Enter the directory for downloaded genome sequence data:
Command
Enter directory (Ubuntu 22.04)
cd genomes

Ensure that NCBI Datasets command line tools are installed and executable (or a symbolic link to the executable) is in the current directory.
Software
NCBI Datasets command line tools
NAME
NCBI
DEVELOPER

Download the genome assemblies that will be included in the analysis:

Dataset
xanthomonas_assm_accs.txt
NAME
https://github.com/davidjstudholme/phylogenomics-Xanthomonas/blob/main/xanthomonas_assm_accs.txt
LINK

Command
Download assemblies from NCBI (Ubuntu 22.04 LTS)
./datasets download genome accession --inputfile xanthomonas_assm_accs.txt  --exclude-gff3 --exclude-protein --exclude-rna --exclude-genomic-cds --filename xanthomonas_genome_assemblies.zip



Expected result
You should receive a message something like:
"Downloading: xanthomonas_genome_assemblies.zip197MB done"

Command
Unzip the assemblies download (Ubuntu 22.04 LTS)
unzip xanthomonas_genome_assemblies.zip



Command
Make symbolic links to the downloaded assemblies (Ubuntu 22.04 LTS)
ln -s ncbi_dataset/data/GCA_*/GCA_*.fna .

Command
List the symbolic links to assembly sequence files (Ubuntu 22.04 LTS)
ls *.fna



Rename the symbolic links to more informative names. We will use the rename_files.pl script to effect this.
Software
rename_files.pl
NAME
https://github.com/davidjstudholme/phylogenomics-Xanthomonas/blob/main/rename_files.pl
SOURCE LINK

Dataset
genomes.list.all.txt
NAME
https://github.com/davidjstudholme/phylogenomics-Xanthomonas/blob/main/genomes.list.all.txt
LINK

Command
perl rename_files.pl  genomes.txt


Expected result
This should generate a set of .fasta files and .contig symbolic links with informative filenames.


Come back out of the directory for downloaded genome sequence data:

Command
Change to previous directory (Ubuntu LTS 22.04)
cd -

Set-up the reference genome sequence data
Command
Create reference directory (Ubuntu 22.04 LTS)
mkdir ref

Command
Enter the reference directory (Ubuntu 22.04 LTS)
cd ref

Command
Make symbolic link to reference genome assembly (Ubuntu 22.04 LTS)
ln -s ../genomes/X._campestris_pv._campestris_ATCC_33913_T.fasta .

Command
Change back to the root directory (Ubuntu 22.04 LTS)
cd -

Set-up the working directory.

Command
Create working directory (Ubuntu 20.04 LTS)
mkdir workdir

Command
cd workdir


Command
Make symbolic links to all the genome assemblies (Ubuntu 22.04 LTS)
ln -s ../genomes/*.contig .
Optionally, at this point, we can delete the symbolic links for any genomes that we want to exclude from the final analysis. It is also an option to add any genome assemblies as .contig files (inf FASTA format).
Command
Return to previous directory (Ubuntu 22.04 LTS)
cd -


Install PhaME software into a Conda environment called 'phame', following instructions on the software's GitHub page:
Software
PhaME
NAME
https://github.com/LANL-Bioinformatics/PhaME
SOURCE LINK
The PhaME software is described in this paper:
CITATION
Shakya M, Ahmed SA, Davenport KW, Flynn MC, Lo CC, Chain PSG (2020). Standardized phylogenetic and molecular evolutionary analysis applied to species across the microbial tree of life.. Scientific reports.
LINK
https://doi.org/10.1038/s41598-020-58356-1


Command
Activate Phame Conda environment (Ubuntu 22.04 LTS)
conda activate phame

Execute PhaME:
Command
Execute PhaME (Ubuntu 22.04 LTS)
phame ./phame.ctl

Expected result
This will generate output, including tree files, in directory:

./workdir/results/trees/

The tree file can now be visualised using any tree-viewing software, for example, iTOL.

CITATION
Letunic I, Bork P (2021). Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation.. Nucleic acids research.
LINK
https://doi.org/10.1093/nar/gkab301

Citations
Step 13
Letunic I, Bork P. Interactive Tree Of Life (iTOL) v5: an online tool for phylogenetic tree display and annotation.
https://doi.org/10.1093/nar/gkab301
Step 9
Shakya M, Ahmed SA, Davenport KW, Flynn MC, Lo CC, Chain PSG. Standardized phylogenetic and molecular evolutionary analysis applied to species across the microbial tree of life.
https://doi.org/10.1038/s41598-020-58356-1