Begin by collecting the sequences of interest, in this case all animal homeobox proteins:
./fastaCollection.pl --terms "Homeobox,Homeodomain,ANTP,PRD,LIM,POU,HNF,SINE,TALE,CUT,PROS,ZF,CERS" --fastafile "<file>.csv" --occfile "<file>.fasta"
Next the fastas should be sorted and sepearted into individual HG fasta files:
./sortFasta.pl <file>.fasta
Next for the classification step:
Download database of homeodb homeoboxes: http://homeodb.zoo.ox.ac.uk/download.get - Selecting the appropriate parameters.
cat Fastas/*.fasta > All.fasta
makeblastdb -dbtype 'prot' -in homeodb.fasta
blastp -db 'homeodb' -query 'All.fasta' -evalue 10e-6 -out All.blast
And run InterProScan:
./interproscan.sh -appl 'Panther,Pfam' -i </path/to/All.fasta> -b </path/to/results.interpro>
Run the domain extraction program:
./domainPuller - This one is hardcoded so may need adjustments to fit user directories etc. For future use, this may remain hardcoded for a single command pipeline with full automation.
Finally to actually classify:
./classifyWrapper.pl or to individually classify each $hg.fasta file:
./homeoClassifier.pl --fastafile path/to/$hg.fasta --blastfile All.blast --interprofile new-interpro.tsv
./classifyChecker.pl Quick checks for formatting as well as classifications
Then run the trees:
treebuilder.pl <FastaDir>
SortFastaMore.pl <Intermediate File Directory with alignments> To remove duplicate domains after trimming ahead of IQ-TREE. (Not necessary in most cases).
Final classification step:
./homeoTreeParser.pl <IntermediateFiles> <classificationTable.tsv> With directory containing all inferred trees in Newick format, and classificationTable.tsv as current classification log as verbose output from classifyWrapper.pl/homeoClassifier.pl.
Additional Extra:
sh speciesReplace.sh <speciesList> This script replaces any 4-letter species code as used in the pipeline with the species name for publication. Takes a speciesList.tsv as input.
./allhbxcounttable.pl TreeFiles > hbxCount.csv To produce a tablet of occupancy for each species and homeobox family.
./homeocountconvert.pl hbxCount.csv > hbxCountMelt.csv To produce an easily parseable file for the rest of the display results, such as the R scripts and the following analyses.
./hbxOrigins.pl hbxCountMelt.csv > hbxOrigins.csv To produce a list of homeobox families and the last shared ancestor within animals or before first splitting animals.
./lossGainhbx.pl hbxCountMelt.csv > hbxLossGain.csv To produce a list of homeobox reduction and expansion for each animal clade/node.
./hbxResultsTable.pl <classifiedTable.tsv> Takes produced classification log table from homeoTreeParser.pl to produce a table of gene evidence for each species and homeobox family.