First, define some variables just to make the bash commands a bit cleaner. REF refers to the name of the Pfam hmm that we’re aligning against (Bac_rhodopsin.hmm in this case), while QUERY is the sequence file to be aligned (hop and bop gene products, plus a dinoflagellate rhodopsin as outgroup).

REF=Bac_rhodopsin
QUERY=uniprot_hop_bop_reviewed

Now, align and convert the alignment to fasta format (required by RAxML-ng).

hmmalign --amino -o $QUERY.sto $REF.hmm $QUERY.fasta
seqmagick convert $QUERY.sto $QUERY.align.fasta

Test which model is best for these data. Here we get LG+G4+F.

modeltest-ng -i $QUERY.align.fasta -d aa -p 8

Check your alignment!

raxml-ng --check --msa $QUERY.align.fasta --model LG+G4+F --prefix $QUERY

Oooh… I bet it failed. Exciting! In this case (using sequences from Uniprot) the long sequence descriptions are incompatible with RAxML-ng. Let’s do a little Python to clean that up.

from Bio import SeqIO

with open('uniprot_hop_bop_reviewed.align.clean.fasta', 'w') as clean_fasta:
    for record in SeqIO.parse('uniprot_hop_bop_reviewed.align.fasta', 'fasta'):
        record.description = ''
        SeqIO.write(record, clean_fasta, 'fasta')

Check again…

raxml-ng --check --msa $QUERY.align.clean.fasta --model LG+G4+F --prefix $QUERY

If everything is kosher go ahead and fire up your phylogenetic inference. Here I’ve limited bootstrapping to 100 trees. If you have the time/resources do more.

raxml-ng --all --msa $QUERY.align.clean.fasta --model LG+G4+F --prefix $QUERY --bs-trees 100

Superimpose the bootstrap support values on the best ML tree.

raxml-ng --support --tree $QUERY.raxml.bestTree --bs-trees $QUERY.raxml.bootstraps

And here’s our creation as rendered by Archaeopteryx. Some day I’ll create a tree that is visually appealing, but today is not that day. But you get the point.