- Run the application and upload the customized phylogenetic
profile matrix (AMPK_1.matrix) as input file (seeNote 10).
Upload further theAMPK.domainsfile as “Additional annota-
tion.” Optionally, when hitting the “FASTA config” button,
you can provide a file,AMPK.extended.fa, with all ortholog
sequences for display within the application. - Set the taxonomic rank on which you want to group your
species. For a start, “species” is a good choice. - Choose your “taxon of interest”. Typically, one selects here the
seed species with which you started the phyletic profiling. In
the case of the AMPK pathway analysis, useHomo sapiens.
Note, you can change your choice at a later time point. - Upon clicking the “PLOT” button, the “Main profile” tab will
open, and the graphic representation of the phylogenetic pro-
file will be displayed. An example is shown in Fig.7a. - Adjust the display of the phylogenetic profile. In particular,
height and width of the plotting area and of the number of
genes displayed might require adjustment. - Take a look at the presence–absence pattern of orthologs to
your seed proteins and start exploring the data (seeNote 11). - The tab “Customized profile” allows you to sub-select genes
and species for display. Choose “PRKAA1” from the gene list
to see only the representation of orthologs for this gene. Mul-
tiple selections are possible (Fig.7b). - Click on individual dots in the phylogenetic profile matrix to
obtain detailed information, such as the FAS score, sequence
identifier of the ortholog, and, if provided, the sequence of the
ortholog and the graphical display of its feature architecture in
comparison to the seed (Fig.7c). - PhyloProfile offers you the option to dynamically explore the
phylogenetic profile. - Go back to the “Input & settings” tab, select “Phylum” as
taxonomic rank, and choose “Chordata” as “taxon of interest.”
After clicking the “PLOT” button, the updated profile will be
displayed. - In the “Main profile,” tab you can now apply a cutoff for the
minimal fraction of species in a phylum for which an ortholog
to a seed gene was detected (default is 0). This considers that
spurious orthology assignments will typically lead to only few
species in a systematic group displaying an ortholog candidate.
The more stringent the cutoff is, the sparser your phylogenetic
profile becomes. A reasonable cutoff helps reducing the impact
of spurious ortholog assignments.
128 Arpit Jain et al.