Create plots and phylogenetic tree

Compute density plot

One of analysis options is a density plot of analyzed database subset values. The plot is drawn for non-outliers only. Example: plot

Just use the --plot flag with no options. (Boolean one)

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

You should install r packages in the environment you are running rRNADif from to draw the plot. Please refer to the installation guide for more details.

Compute final phylogenetic tree

The main advantage of rRNADif is fast and flexible computation of 16S rRNA based species phylogenetic trees from the comprehensive database. Therefore, no misclassified and/or partly sequenced 16S are included, leading to better species/genus-level classification of genomes based on their rRNAs. As was stated in get started guide, final phylogenetic tree can be computed with --tree flag.

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

The defaults are mafft and fasttree. Also, all sequences are used for outliers and 1 sequence (genome-representative) for non-outlier for giver subset of the database.

However, the algorithm for Multiple Sequence alignment or program for phylogeny can be changed with -m and -t flags respectively. For now, rRNADif supports clustalo, muscle and mafft for MSA, and fasttree, iqtree and raxml for phylogeny.

sh rrnadif -d <csv.file> -i <genome.fasta> -p <project-name> --tree -m muscle -t iqtree
caution

By default, branch length for annotated 16S in the input genome will also be computed using provided options, not default ones. Hence, the provided pre-computed database is generated with mafft and fasttree, we suggest to use --only_phylo flag. Therefore, only the final tree will be computed with desired algorithms.

The computed phylogenetic tree will contain every strain and therefore can be simply unreadable. To use only species information and compute species-level phylogenetic tree use --only_species flag.

In the example below, default options will be used to measure intragenomic variability (construct individual phylogenetic tree) (--only_phylo flag), the muscle for MSA and iqtree for phylogeny (-m and -t flags) will be used for final phylogenetic tree computation. The data for the final phylogenetic tree will contain only species (--only species). This would drastically reduce tree size and respective computation time for it, also making tree human readable.

sh rrnadif -d <csv.file> -i <genome.fasta> -p <project-name> --tree -m muscle -t iqtree --only_species --only_phylo
note

Species, when using --only_species flag, will be chosen randomly from the database subset (if multiple strains are available). By default, the first in alphabetical order is kept. Prioritized are genomes with multiple rrnas which are non-outliers.

warning

If --only_species flag is used, intragenomic variability of input 16S will be compared to the whole subset. Strains, that are the same species will be dropped only for the final tree computation.

The output of a run is organized under <project-name>_results/results/phylogeny directory. Three files should be there:

  1. FINAL.fasta - sequences used for constructing of a phylogenetic tree
  2. FINAL.mafft - MSA in fasta format. The extension .mafft is used for the sake of consistency in outputs from different MSA programs. This is NOT an indicator of the used algorithm.
  3. FINAL.nwk - phylogenetic tree in newick format. Ready to view in almost every phylogenetic tree viewer.
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