diff --git a/PTL2/Scripts/guidance.py b/PTL2/Scripts/guidance.py index d4b6c39..3d68db2 100644 --- a/PTL2/Scripts/guidance.py +++ b/PTL2/Scripts/guidance.py @@ -1,10 +1,34 @@ +# Last updated Sept 2023 +# Authors: Auden Cote-L'Heureux and Mario Ceron-Romero + +# This script runs Guidance in an iterative fashion for more both MSA construction +# and more rigorous homology assessment than what is offered in PhyloToL 6 part 1. +# Guidance runs until the input number of iterations (--guidance_iters, default = 5) +# has been reached, or until there are no sequences below the sequence score cutoff. +# All sequences below the score cutoff (--seq_cutoff, default = 0.3) are removed at +# each iteration. By default, PhyloToL does not remove residues that fall below the +# given residue cutoff (--res_cutoff) and columns that fall below the given column +# cutoff (--col_cutoff, defaults are 0), though this can be turned on by adjusting +# these parameters. Outputs at this point are found in the “Guidance_NotGapTrimmed” +# output folder. We then run MSAs through TrimAl to remove all sites in the alignment +# that are at least 95% gaps (or --gap_trim_cutoff) generating files in the “Guidance” +# output folder. + +# This step is either intended to be run starting with --start = unaligned (but not raw) +# inputs, meaning one amino acid alignment per OG. It can also be run directly after the +# preguidance step. The run() function is called in two places: in phylotol.py generally, +# and in contamination.py if the contamination loop is using Guidance as the re-alignment +# method. + +#Dependencies import os, sys, re from Bio import SeqIO - +#Called in phylotol.py and contamination.py def run(params): if params.start == 'raw' or params.start == 'unaligned': + #Checking that pre-Guidance has been run or that unaligned files per OG are provided. if params.start == 'raw': preguidance_path = params.output + '/Output/Pre-Guidance' else: @@ -17,7 +41,8 @@ def run(params): if len([f for f in os.listdir(preguidance_path) if f.endswith('.fa') or f.endswith('.faa') or f.endswith('.fasta')]) == 0: print('\nERROR: No pre-Guidance (unaligned) files could be found at the path ' + preguidance_path + '. Make sure that the --start and --data parameters are correct, that the pre-Guidance step ran successfully, and that the unaligned files are formatted correctly (they must have the file extension .faa, .fa, or .fasta).\n') exit() - + + #Creating intermedate folders that will later be deleted unless running with --keep_temp os.mkdir(params.output + '/Output/Intermediate/Guidance') os.mkdir(params.output + '/Output/Intermediate/Guidance/Input') os.mkdir(params.output + '/Output/Intermediate/Guidance/Output') @@ -28,14 +53,17 @@ def run(params): guidance_removed_file = open(params.output + '/Output/GuidanceRemovedSeqs.txt', 'w') guidance_removed_file.write('Sequence\tScore\n') + #For each unaligned AA fasta file for file in [f for f in os.listdir(guidance_input) if f.endswith('.fa') or f.endswith('.faa') or f.endswith('.fasta')]: tax_guidance_outdir = params.output + '/Output/Intermediate/Guidance/Output/' + file.split('.')[0].split('_preguidance')[0] os.mkdir(tax_guidance_outdir) fail = False + #For each iteration for i in range(params.guidance_iters): n_recs = len([r for r in SeqIO.parse(guidance_input + '/' + file, 'fasta')]) + #Guidance can't handle inputs with fewer than 4 sequences if n_recs < 4: print('\nWARNING: Gene famiily ' + file.split('.')[0].split('_preguidance')[0] + ' contains fewer than 4 sequences after ' + str(i) + ' Guidance iterations, therefore no alignment will be produced for this gene family.\n') os.system('rm -rf ' + tax_guidance_outdir) @@ -43,44 +71,52 @@ def run(params): fail = True break + #Determining MAFFT algorithm based on the number of input sequences if n_recs < 200: mafft_alg = 'genafpair' else: mafft_alg = 'auto' + #Running Guidance (one per OG per iteration) os.system('Scripts/guidance.v2.02/www/Guidance/guidance.pl --seqFile ' + guidance_input + '/' + file + ' --msaProgram MAFFT --seqType aa --outDir ' + tax_guidance_outdir + ' --seqCutoff ' + str(params.seq_cutoff) + ' --colCutoff ' + str(params.col_cutoff) + " --outOrder as_input --bootstraps 10 --MSA_Param '\\--" + mafft_alg + " --maxiterate 1000 --thread " + str(params.guidance_threads) + " --bl 62 --anysymbol' > " + params.output + '/Output/Intermediate/Guidance/Output/' + file[:10] + '/log.txt') + #Checking for a sequence score file; if not available, Guidance failed. if os.path.isfile(tax_guidance_outdir + '/MSA.MAFFT.Guidance2_res_pair_seq.scr_with_Names'): + #All sequences below score cutoff seqs_below = len([line for line in open(tax_guidance_outdir + '/MSA.MAFFT.Guidance2_res_pair_seq.scr_with_Names').readlines()[1:-1] if float(line.split()[-1]) < params.seq_cutoff]) - + #If fewer than four were above the cutoff, this OG is done iterating. if n_recs - seqs_below < 4: print('\nWARNING: Gene famiily ' + file.split('.')[0].split('_preguidance')[0] + ' contains fewer than 4 sequences after ' + str(i + 1) + ' Guidance iterations, therefore no alignment will be produced for this gene family.\n') os.system('rm -rf ' + tax_guidance_outdir) break - + #If all sequences were above the cutoff, this OG is done iterating. if seqs_below == 0 or i == params.guidance_iters - 1: print('\nGuidance complete after ' + str(i + 1) + ' iterations for gene family ' + file.split('.')[0].split('_preguidance')[0] + '\n') break - + #Recording list of sequences removed by Guidance. for line in [line for line in open(tax_guidance_outdir + '/MSA.MAFFT.Guidance2_res_pair_seq.scr_with_Names').readlines()[1:-1] if float(line.split()[-1]) < params.seq_cutoff]: guidance_removed_file.write(line) - + #Copying over the old file with the new results os.system('cp ' + tax_guidance_outdir + '/Seqs.Orig.fas.FIXED.Without_low_SP_Seq.With_Names ' + guidance_input + '/' + file) - + #Cleaning up the intermediate files for the iteration. os.system('rm -r ' + tax_guidance_outdir + '/*') else: fail = True break + #After all iterations, THEN apply residue and column cutoffs if not fail: + #Getting a list of sequences to keep seqs2keep = [rec.description for rec in SeqIO.parse(tax_guidance_outdir + '/Seqs.Orig.fas.FIXED.Without_low_SP_Seq.With_Names', 'fasta')] orig_seqs = [rec.description for rec in SeqIO.parse(tax_guidance_outdir + '/MSA.MAFFT.aln.With_Names', 'fasta')] running_aln = { rec.description : str(rec.seq) for rec in SeqIO.parse(tax_guidance_outdir + '/MSA.MAFFT.aln.With_Names', 'fasta') if rec.description in seqs2keep } + #Residues that fall below the confidence cutoff (--res_cutoff) are replaced with 'X' for site in [(int(line.split()[1]), int(line.split()[0]) - 1) for line in open(tax_guidance_outdir + '/MSA.MAFFT.Guidance2_res_pair_seq.scr').readlines()[1:-1] if float(line.split(' ')[-1].strip()) < params.res_cutoff]: if(orig_seqs[site[0]] in seqs2keep): running_aln[orig_seqs[site[0]]][site[1]] = 'X' + #Removing columns below the --col_cutoff cols2remove = [int(line.split()[0]) - 1 for line in open(tax_guidance_outdir + '/MSA.MAFFT.Guidance2_res_pair_col.scr').readlines()[1:-1] if float(line.split(' ')[-1].strip()) < params.col_cutoff] for seq in running_aln: running_aln[seq] = ''.join([running_aln[seq][i] for i in range(len(running_aln[seq])) if i not in cols2remove]) @@ -89,15 +125,18 @@ def run(params): for seq in running_aln: o.write('>' + seq + '\n' + str(running_aln[seq]).replace('-', '') + '\n\n') + #Aligning one last time after removing the final set of sequences and applying the res and col cutoffs print('mafft ' + tax_guidance_outdir + '/postGuidance_preTrimAl_unaligned.fasta > ' + tax_guidance_outdir + '/' + file.split('.')[0].split('_preguidance')[0] + '_postGuidance_preTrimAl_aligned.fasta') os.system('mafft ' + tax_guidance_outdir + '/postGuidance_preTrimAl_unaligned.fasta > ' + tax_guidance_outdir + '/' + file.split('.')[0].split('_preguidance')[0] + '.postGuidance_preTrimAl_aligned.fasta') + #Gap trimming os.system('Scripts/trimal-trimAl/source/trimal -in ' + tax_guidance_outdir + '/' + file.split('.')[0].split('_preguidance')[0] + '.postGuidance_preTrimAl_aligned.fasta -out ' + tax_guidance_outdir + '/' + file.split('.')[0].split('_preguidance')[0] + '.95gapTrimmed.fasta -gapthreshold 0.05 -fasta') + #Copying over final aligments (pre and post gap trimming) into output folder. os.system('cp ' + tax_guidance_outdir + '/' + file.split('.')[0].split('_preguidance')[0] + '.95gapTrimmed.fasta ' + params.output + '/Output/Guidance/' + file.split('.')[0].split('_preguidance')[0] + '.95gapTrimmed.fasta') os.system('cp ' + tax_guidance_outdir + '/' + file.split('.')[0].split('_preguidance')[0] + '.postGuidance_preTrimAl_aligned.fasta ' + params.output + '/Output/NotGapTrimmed/' + file.split('.')[0].split('_preguidance')[0] + '.postGuidance_preTrimAl_aligned.fasta') - + #Removing intermediate files if not --keep_temp if not params.keep_temp: for gdir_file in os.listdir(tax_guidance_outdir): if gdir_file not in ('MSA.MAFFT.Guidance2_res_pair_seq.scr_with_Names', 'MSA.MAFFT.aln.With_Names', 'MSA.MAFFT.Guidance2_res_pair_col.scr', 'log', 'postGuidance_preTrimAl_unaligned.fasta'):