- Open the URLhttp://www.orthodb.org/.
- You can query the database for ortholog of your seed protein
using (1) the protein sequence, (2) the gene name, or (3) pro-
tein annotations. Select the species to display, i.e., the set of
taxa of interest, and click “Submit.” This will result in hierar-
chal orthologous groups at each major taxonomic level of the
selected species.
- Download all orthologous groups for PRKAA1 as FASTA files.
Filter for sequences from your target taxa and save into a new
file,AMPK_PRKAA1_Orthologs_Filtered.fa.
Optionalsteps 9– 18. Seesteps 19– 21 for an alternative approach.
You can use HaMStR [25] for extending precompiled ortho-
logous groups retrieved in the previous steps with sequences
from species not represented in the online ortholog collections
(seeNote 6). HaMStR lets you identify orthologs both in protein
sequence data and in transcript collections.
- Download and install HaMStR. We will refer in the following
to the HaMStR directory on your file systems as
pathToHaMStR.
- Follow the guidelines of the HaMStR package to set the stage
for your search. Per default, HaMStR comes with a ready-to-
use collection of protein sets from the Quest for Orthologs
Reference Species collection (http://www.ebi.ac.uk/refer
ence_proteomes). If your analysis requires protein sets from
further species, the HaMStR documentation provides the nec-
essary details for their addition.
- For every protein, create a directory insidepathToHaMStR/cor-
e_orthologs. For example, for the protein PRKAA1, create a
directory pathToHaMStR/core_orthologs/PRKAA1.Copythe
prepared ortholog fileAMPK_PRKAA1_Orthologs_Filtered.fa
(seestep 8in Subheading3.3 ) and rename the file asPRKAA1.
fa. Please note that the name of the file should be the same as the
name of the directory (PRKAA1 in this case). HaMStR is a bit
picky concerning the format of sequence headers. In brief, it
expects the following structure: proteinName|taxonName|
sequenceId. For example, if PRKAA1 has the sequence identifier
“Q13131” in the human gene set provided to HaMStR, the
sequence header should read as “>PRKAA1|HUMAN@9606|
Q13131” whereHUMAN@9606is the file name of the human
gene set (seeNote 7). Take into account that HaMStR assumes
that a sequence in a FASTA file is not interrupted by line breaks.
- Generate a multiple sequence alignment for ortholog
sequences in pathToHaMStR/core_orthologs/PRKAA1/
PRKAA1.fausing MUSCLE [32]:muscle -in PRKAA1.fa
-out PRKAA1.aln.
Tracing AMPK Evolution 121
