Get started

The data for this guide is in the example directory in the repo. A quick overview of the program is given in the Introduction. The installation process is described here

Step 0. Prepare a database

The first step after cloning the repo (with wget <repo-link>) is to go to the rRNADif/datasets/computed folder and to extract the archive in the same folder. The archive holds a folder with all fasta files with pre-computed 16S rRNA and a csv with pre-computed mean/median branch length between those sequences.

Step 1. Prepare input files

The inputs are:

  1. A csv file, downloaded from a "Browse by organism" tab in NCBI Genome database. This csv file holds genomes to filter from pre-computed database.
  2. Genome sequence in fasta format.

The optional input is a set of 16S rRNAs (with provided --step 2 flag in a script. See sh rrnadif -h for more help). Then the barrnap step will be omitted. More about inputs can be read here.

Step 2. Run the script with minimal tweaks

To run a script with provided genome sequence and csv file you can enter:

sh rrnadif -d <csv.file> -i <genome.fasta> -p <project-name>

The project name will affect the result folder name. The results then will be printed into the terminal.

caution

rRNADif was only tested when run under the repo directory. Please try to navigate and run the sh rrnadif.sh from repo directory to avoid any errors (:

To run rRNADif with the same project name, --redo flag should be used!

Step 3. Compute phylogeny

The phylogenetic tree can be computed as such

sh rrnadif -d <csv.file> -i <genome.fasta> -p <project-name> --tree
note

Default programs for phylogenetic tree computation are mafft and fasttree. They should be installed and be accessible from a terminal.

The rRNADif will compute the tree from all strains/genomes. used in the analysis. Also the script is capable of computing only species phylogenetic tree, discarding all the strains:

sh rrnadif -d <csv.file> -i <genome.fasta> -p <project-name> --tree --only_species
note

Variability comparison will be done on a whole set of sequences. They only be discarded for final tree computation.

tip

You can choose the tree computation algorithms via -m and -t flags. The chosen algorithm can be used for whole analysis, or only for final tree computation (--only_phylo flag). More parameters see at sh rrnadif.sh -h

Step 4. Get the results

The results of an analysis are under project-name/results directory in the rRNADif folder, named Results_...csv.

  • Results_all.csv contains the main result info. Each row represents every used genome. There are three columns - name (strain name), mean (mean branch length value for 16S sequences for this strain), median (median values - same logic as for mean). The values for analysed sequences are included as well.
  • Results_mean_outliers.csv - contains genomes, which mean value was an outlier within the analyzed dataset. The structure is the same as in Results_all.csv
  • Results_median_outliers.csv - contains genomes, which median values are an outlier within the analyzed dataset. Can be identical to Results_mean_outliers.csv. The structure is the same as in Results_all.csv
  • Results_no_outliers.csv - contains genomes, which values are not within outliers. The structure is the same as in Results_all.csv.

The tree results are in "phylogeny" folder. The folder itself contains 3 files:

  1. FINAL.fasta - the sequences used in phylogeny computation in fasta format.
  2. FINAL.mafft - the MSA with the chosen algorithm in fasta format. The extension will not change with a change of MSA program - used for script stability.
  3. FINAL.nwk - the phylogenetic tree in newick format.

For more extensive results description see this tutorial

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