treesak 1.51.2__py3-none-any.whl

TreeSAK/AssessMarkerDeltaLL.py

@@ -0,0 +1,257 @@
+import os
+import glob
+import argparse
+from Bio import SeqIO
+from Bio import AlignIO
+
+
+AssessMarkerDeltaLL_usage = '''
+============================= AssessMarkerDeltaLL example commands =============================
+
+Dependencies: iqtree
+
+# example commands
+TreeSAK AssessMarkerDeltaLL -deltall DeltaLL_stdout.txt -o op_dir -c 25-50-75-100 -mmn 20 -aln trimmed_aln_dir -jst 6 -qsub -f 
+
+================================================================================================
+'''
+
+
+def sep_path_basename_ext(file_in):
+
+    # separate path and file name
+    file_path, file_name = os.path.split(file_in)
+    if file_path == '':
+        file_path = '.'
+
+    # separate file basename and extension
+    file_basename, file_extension = os.path.splitext(file_name)
+
+    return file_path, file_basename, file_extension
+
+
+def catfasta2phy(msa_dir, msa_ext, concatenated_msa_phy, partition_file):
+
+    concatenated_msa_fasta = '%s.fasta' % concatenated_msa_phy
+    msa_file_re            = '%s/*.%s'  % (msa_dir, msa_ext)
+    msa_file_list          = [os.path.basename(file_name) for file_name in glob.glob(msa_file_re)]
+    msa_file_list_sorted   = sorted(msa_file_list)
+
+    complete_gnm_set = set()
+    for each_msa_file in msa_file_list:
+        pwd_msa = '%s/%s' % (msa_dir, each_msa_file)
+        for each_seq in SeqIO.parse(pwd_msa, 'fasta'):
+            complete_gnm_set.add(each_seq.id)
+
+    complete_gnm_list_sorted = sorted([i for i in complete_gnm_set])
+
+    # initialize concatenated msa dict
+    gnm_to_seq_dict = {i: '' for i in complete_gnm_list_sorted}
+    msa_len_dict = dict()
+    for each_msa_file in msa_file_list_sorted:
+        gene_id = each_msa_file.split('.' + msa_ext)[0]
+
+        # read in msa
+        current_msa_len = 0
+        current_msa_len_set = set()
+        pwd_current_msa = '%s/%s' % (msa_dir, each_msa_file)
+        current_msa_seq_dict = dict()
+        for each_seq in SeqIO.parse(pwd_current_msa, 'fasta'):
+            complete_gnm_set.add(each_seq.id)
+            current_msa_seq_dict[each_seq.id] = str(each_seq.seq)
+            current_msa_len_set.add(len(each_seq.seq))
+            current_msa_len = len(each_seq.seq)
+
+        if len(current_msa_len_set) != 1:
+            print('Sequences with different length were found in %s, program exited!' % each_msa_file)
+            exit()
+
+        msa_len_dict[gene_id] = current_msa_len
+
+        # add sequence to concatenated msa dict
+        for each_gnm in complete_gnm_list_sorted:
+            msa_seq = current_msa_seq_dict.get(each_gnm, current_msa_len*'-')
+            gnm_to_seq_dict[each_gnm] += msa_seq
+
+    # write out concatenated msa
+    concatenated_msa_handle = open(concatenated_msa_fasta, 'w')
+    for each_gnm in complete_gnm_list_sorted:
+        concatenated_msa_handle.write('>%s\n' % each_gnm)
+        concatenated_msa_handle.write('%s\n' % gnm_to_seq_dict[each_gnm])
+    concatenated_msa_handle.close()
+
+    # write out partition file
+    end_pos = 0
+    partition_file_handle = open(partition_file, 'w')
+    for each_m in msa_file_list_sorted:
+        gene_id = each_m.split('.' + msa_ext)[0]
+        current_m_len = msa_len_dict[gene_id]
+        partition_file_handle.write('%s = %s-%s\n' % (each_m, (end_pos + 1), (end_pos + current_m_len)))
+        end_pos += current_m_len
+    partition_file_handle.close()
+
+    # convert msa in fasta to phy
+    AlignIO.convert(concatenated_msa_fasta, 'fasta', concatenated_msa_phy, 'phylip-relaxed')
+
+
+def submit_js(js):
+    current_wd = os.getcwd()
+    js_path, js_basename, js_ext = sep_path_basename_ext(js)
+    os.chdir(js_path)
+    os.system('qsub %s%s' % (js_basename, js_ext))
+    os.chdir(current_wd)
+
+
+def AssessMarkerDeltaLL(args):
+
+    deltall_stdout_txt      = args['deltall']
+    op_dir                  = args['o']
+    deltall_keep_pct_str    = args['c']
+    min_marker_num          = args['mmn']
+    trimmed_aln_dir         = args['aln']
+    js_cpu_num              = args['jst']
+    submit_job              = args['qsub']
+    force_overwrite         = args['f']
+
+    deltall_keep_pct_list = [int(i) for i in deltall_keep_pct_str.split('-')]
+
+    deltall_stdout_path, deltall_stdout_basename, deltall_stdout_ext = sep_path_basename_ext(deltall_stdout_txt)
+
+    # create dir
+    if os.path.isdir(op_dir) is True:
+        if force_overwrite is True:
+            os.system('rm -r %s' % op_dir)
+        else:
+            print('output folder detected, program exited!')
+            exit()
+    os.system('mkdir %s' % op_dir)
+
+    # define file name
+    deltall_stdout_summary_txt = '%s/%s_summary.txt' % (op_dir, deltall_stdout_basename)
+
+    # read in deltall_stdout_txt
+    deltall_op_dict = dict()
+    for each_line in open(deltall_stdout_txt):
+        if not ((each_line.startswith('WARNING:')) or (each_line.startswith('awk:'))):
+            each_line_split = each_line.strip().split('\t')
+            marker_id = each_line_split[0]
+            value = float(each_line_split[1])
+            if marker_id not in deltall_op_dict:
+                deltall_op_dict[marker_id] = [value]
+            else:
+                deltall_op_dict[marker_id].append(value)
+
+    # assigned score to marker
+    metric_1_dict = dict()
+    metric_2_dict = dict()
+    for each_marker in deltall_op_dict:
+        metric_1_value = float("{0:.2f}".format(deltall_op_dict[each_marker][0]))
+        metric_2_value = float("{0:.2f}".format(deltall_op_dict[each_marker][1]))
+        metric_1_dict[each_marker] = metric_1_value
+        metric_2_dict[each_marker] = metric_2_value
+
+    metric_1_dict_sorted = {k: v for k, v in sorted(metric_1_dict.items(), key=lambda item: item[1])[::-1]}
+    metric_2_dict_sorted = {k: v for k, v in sorted(metric_2_dict.items(), key=lambda item: item[1])}
+
+    metric_1_score_dict = dict()
+    metric_1_score = 1
+    for each_marker_1 in metric_1_dict_sorted:
+        metric_1_score_dict[each_marker_1] = metric_1_score
+        metric_1_score += 1
+
+    metric_2_score_dict = dict()
+    metric_2_score = 1
+    for each_marker_2 in metric_2_dict_sorted:
+        metric_2_score_dict[each_marker_2] = metric_2_score
+        metric_2_score += 1
+
+    overall_score_dict = dict()
+    for each_marker in deltall_op_dict:
+        metric_score_1 = metric_1_score_dict[each_marker]
+        metric_score_2 = metric_2_score_dict[each_marker]
+        metric_score_overall = metric_score_1 + metric_score_2
+        overall_score_dict[each_marker] = metric_score_overall
+
+    overall_score_dict_sorted     = {k: v for k, v in sorted(overall_score_dict.items(), key=lambda item: item[1])}
+    marker_list_sorted_by_deltall = [k for k, v in sorted(overall_score_dict.items(), key=lambda item: item[1])]
+
+    # write out summary_txt
+    summary_txt_handle = open(deltall_stdout_summary_txt, 'w')
+    summary_txt_handle.write('Marker\tmetric1\tmetric1_score\tmetric2\tmetric2_score\toverall_score\n')
+    for each_marker in overall_score_dict_sorted:
+        metric_value_1 = metric_1_dict[each_marker]
+        metric_value_2 = metric_2_dict[each_marker]
+        metric_score_1 = metric_1_score_dict[each_marker]
+        metric_score_2 = metric_2_score_dict[each_marker]
+        metric_score_overall = overall_score_dict_sorted[each_marker]
+        summary_txt_handle.write('%s\t%s\t%s\t%s\t%s\t%s\n' % (each_marker, metric_value_1, metric_score_1, metric_value_2, metric_score_2, metric_score_overall))
+    summary_txt_handle.close()
+
+    # get qualified marker list
+    for each_keep_pct in deltall_keep_pct_list:
+        marker_num_to_keep = round(len(marker_list_sorted_by_deltall)*each_keep_pct/100)
+        markers_to_keep_id_list = marker_list_sorted_by_deltall[:marker_num_to_keep]
+
+        if marker_num_to_keep < min_marker_num:
+            print('Ignored DeltaLL cutoff at %s , the number of qualified markers (%s) less than %s' % (each_keep_pct, marker_num_to_keep, min_marker_num))
+        else:
+            pwd_aln_dir                     = '%s/%s_DeltaLL_%s_trimmed_aln'                % (op_dir, deltall_stdout_basename.split('_DeltaLL_stdout')[0], each_keep_pct)
+            pwd_aln_concatenated            = '%s/%s_DeltaLL_%s_concatenated.phy'           % (op_dir, deltall_stdout_basename.split('_DeltaLL_stdout')[0], each_keep_pct)
+            pwd_aln_concatenated_partitions = '%s/%s_DeltaLL_%s_concatenated_partition.txt' % (op_dir, deltall_stdout_basename.split('_DeltaLL_stdout')[0], each_keep_pct)
+            pwd_iqtree_guide_tree_wd        = '%s/%s_DeltaLL_%s_iqtree_guide_tree'          % (op_dir, deltall_stdout_basename.split('_DeltaLL_stdout')[0], each_keep_pct)
+            pwd_iqtree_c60_pmsf_wd          = '%s/%s_DeltaLL_%s_iqtree_C60_PMSF'            % (op_dir, deltall_stdout_basename.split('_DeltaLL_stdout')[0], each_keep_pct)
+            pwd_iqtree_js                   = '%s/js_%s_DeltaLL_%s_iqtree.sh'               % (op_dir, deltall_stdout_basename.split('_DeltaLL_stdout')[0], each_keep_pct)
+
+            # create dir
+            os.mkdir(pwd_aln_dir)
+
+            # copy msa of qualified markers
+            for each_marker in markers_to_keep_id_list:
+                pwd_marker_aln = '%s/%s.aln' % (trimmed_aln_dir, each_marker)
+                cp_cmd = 'cp %s %s/' % (pwd_marker_aln, pwd_aln_dir)
+                os.system(cp_cmd)
+
+            # concatenate msa
+            catfasta2phy(pwd_aln_dir, 'aln', pwd_aln_concatenated, pwd_aln_concatenated_partitions)
+
+            # create dir
+            os.mkdir(pwd_iqtree_guide_tree_wd)
+            os.mkdir(pwd_iqtree_c60_pmsf_wd)
+
+            # run iqtree
+            get_guide_tree_cmd = 'iqtree -s ../%s_DeltaLL_%s_concatenated.phy --prefix guide_tree --seqtype AA -m LG -T %s -B 1000 --alrt 1000 --quiet'                                                                      % (deltall_stdout_basename.split('_DeltaLL_stdout')[0], each_keep_pct, js_cpu_num)
+            get_c60_tree_cmd   = 'iqtree -s ../%s_DeltaLL_%s_concatenated.phy --prefix concatenated --seqtype AA -m LG+G+F+C60 -T %s -B 1000 --alrt 1000 --quiet -ft ../%s_DeltaLL_%s_iqtree_guide_tree/guide_tree.treefile' % (deltall_stdout_basename.split('_DeltaLL_stdout')[0], each_keep_pct, js_cpu_num, deltall_stdout_basename.split('_DeltaLL_stdout')[0], each_keep_pct)
+
+            # write job script
+            with open(pwd_iqtree_js, 'w') as pwd_iqtree_js_handle:
+                pwd_iqtree_js_handle.write('#!/bin/bash\n#SBATCH --ntasks 1\n#SBATCH --cpus-per-task %s\n\n' % js_cpu_num)
+                pwd_iqtree_js_handle.write('cd %s/%s\n' % (os.getcwd(), pwd_iqtree_guide_tree_wd))
+                pwd_iqtree_js_handle.write(get_guide_tree_cmd + '\n\n')
+                pwd_iqtree_js_handle.write('cd %s/%s\n' % (os.getcwd(), pwd_iqtree_c60_pmsf_wd))
+                pwd_iqtree_js_handle.write(get_c60_tree_cmd + '\n')
+
+            if submit_job is True:
+                print(pwd_iqtree_js)
+                submit_js(pwd_iqtree_js)
+            else:
+                print('Job script for running iqtree exported to %s' % pwd_iqtree_js)
+
+    # prepare files for performing dating
+    print('Preparing files for performing dating')
+
+
+
+if __name__ == '__main__':
+
+    # initialize the options parser
+    parser = argparse.ArgumentParser()
+    parser.add_argument('-deltall', required=True,                          help='DeltaLL stdout')
+    parser.add_argument('-o',       required=True,                          help='output dir')
+    parser.add_argument('-c',       required=False, default='25-50-75-100', help='cutoffs, default: 25-50-75-100')
+    parser.add_argument('-mmn',     required=False, default=20, type=int,   help='minimal marker number, default: 20')
+    parser.add_argument('-aln',     required=True,                          help='faa file dir')
+    parser.add_argument('-jst',     required=False, default='6',            help='threads to request in job script, for running iqtree')
+    parser.add_argument('-qsub',    required=False, action="store_true",    help='submit job scripts')
+    parser.add_argument('-f',       required=False, action="store_true",    help='force overwrite')
+    args = vars(parser.parse_args())
+    AssessMarkerDeltaLL(args)

TreeSAK/AssessMarkerPA.py

@@ -0,0 +1,317 @@
+import os
+import glob
+import argparse
+from Bio import SeqIO
+from Bio import AlignIO
+
+
+AssessMarkerPA_usage = '''
+=========================== AssessMarkerPA example commands ===========================
+
+BioSAK AssessMarkerPA -ta trimmed_aln -tax aln -aa faa_files -aax faa -g gnm_group.txt -c 25-50-75-100 -o s10_assess_marker_PA
+
+Note
+1. Extra genomes in gnm_metadata.txt won't affect assessment results.
+2. Genomes can not be found in gnm_metadata.txt will trigger an error.
+3. Alignments in {trimmed_aln_dir} need to be trimmed before assessment
+4. Sequences in MSAs need to be named by genome id.
+
+=======================================================================================
+'''
+
+
+def sep_path_basename_ext(file_in):
+    file_path, file_name = os.path.split(file_in)
+    if file_path == '':
+        file_path = '.'
+    file_basename, file_extension = os.path.splitext(file_name)
+    return file_path, file_basename, file_extension
+
+
+def catfasta2phy(msa_dir, msa_ext, concatenated_msa_phy, partition_file):
+
+    concatenated_msa_fasta = '%s.fasta' % concatenated_msa_phy
+    msa_file_re            = '%s/*.%s'  % (msa_dir, msa_ext)
+    msa_file_list          = [os.path.basename(file_name) for file_name in glob.glob(msa_file_re)]
+    msa_file_list_sorted   = sorted(msa_file_list)
+
+    complete_gnm_set = set()
+    for each_msa_file in msa_file_list:
+        pwd_msa = '%s/%s' % (msa_dir, each_msa_file)
+        for each_seq in SeqIO.parse(pwd_msa, 'fasta'):
+            complete_gnm_set.add(each_seq.id)
+
+    complete_gnm_list_sorted = sorted([i for i in complete_gnm_set])
+
+    # initialize concatenated msa dict
+    gnm_to_seq_dict = {i: '' for i in complete_gnm_list_sorted}
+    msa_len_dict = dict()
+    for each_msa_file in msa_file_list_sorted:
+        gene_id = each_msa_file.split('.' + msa_ext)[0]
+
+        # read in msa
+        current_msa_len = 0
+        current_msa_len_set = set()
+        pwd_current_msa = '%s/%s' % (msa_dir, each_msa_file)
+        current_msa_seq_dict = dict()
+        for each_seq in SeqIO.parse(pwd_current_msa, 'fasta'):
+            complete_gnm_set.add(each_seq.id)
+            current_msa_seq_dict[each_seq.id] = str(each_seq.seq)
+            current_msa_len_set.add(len(each_seq.seq))
+            current_msa_len = len(each_seq.seq)
+
+        if len(current_msa_len_set) != 1:
+            print('Sequences with different length were found in %s, program exited!' % each_msa_file)
+            exit()
+
+        msa_len_dict[gene_id] = current_msa_len
+
+        # add sequence to concatenated msa dict
+        for each_gnm in complete_gnm_list_sorted:
+            msa_seq = current_msa_seq_dict.get(each_gnm, current_msa_len*'-')
+            gnm_to_seq_dict[each_gnm] += msa_seq
+
+    # write out concatenated msa
+    concatenated_msa_handle = open(concatenated_msa_fasta, 'w')
+    for each_gnm in complete_gnm_list_sorted:
+        concatenated_msa_handle.write('>%s\n' % each_gnm)
+        concatenated_msa_handle.write('%s\n' % gnm_to_seq_dict[each_gnm])
+    concatenated_msa_handle.close()
+
+    # write out partition file
+    end_pos = 0
+    partition_file_handle = open(partition_file, 'w')
+    for each_m in msa_file_list_sorted:
+        gene_id = each_m.split('.' + msa_ext)[0]
+        current_m_len = msa_len_dict[gene_id]
+        partition_file_handle.write('%s = %s-%s\n' % (each_m, (end_pos + 1), (end_pos + current_m_len)))
+        end_pos += current_m_len
+    partition_file_handle.close()
+
+    # convert msa in fasta to phy
+    AlignIO.convert(concatenated_msa_fasta, 'fasta', concatenated_msa_phy, 'phylip-relaxed')
+
+
+def AssessMarkerPA(args):
+
+    trimmed_aln_dir   = args['ta']
+    trimmed_aln_ext   = args['tax']
+    faa_file_dir      = args['aa']
+    faa_file_ext      = args['aax']
+    gnm_group_txt     = args['g']
+    cutoff_str        = args['c']
+    op_dir            = args['o']
+    force_overwriting = args['f']
+    #catfasta2phyml_pl = args['pl']
+    js_cpu_num        = args['jst']
+
+    # get gnm id list
+    faa_file_re   = '%s/*.%s' % (faa_file_dir, faa_file_ext)
+    faa_file_list = [os.path.basename(file_name) for file_name in glob.glob(faa_file_re)]
+    gnm_set = set()
+    for each_faa_file in faa_file_list:
+        faa_path, faa_basename, faa_ext = sep_path_basename_ext(each_faa_file)
+        gnm_set.add(faa_basename)
+
+    # read in genome metadata
+    group_to_gnm_dict       = dict()
+    group_to_gnm_num_dict   = dict()
+    gnm_to_group_dict       = dict()
+    for each_gnm in open(gnm_group_txt):
+        each_gnm_split = each_gnm.strip().split('\t')
+        gnm_id = each_gnm_split[0]
+        domain_name = each_gnm_split[1]
+
+        if gnm_id in gnm_set:
+            gnm_to_group_dict[gnm_id] = domain_name
+
+            if domain_name not in group_to_gnm_num_dict:
+                group_to_gnm_num_dict[domain_name] = 1
+            else:
+                group_to_gnm_num_dict[domain_name] += 1
+
+            if domain_name not in group_to_gnm_dict:
+                group_to_gnm_dict[domain_name] = {gnm_id}
+            else:
+                group_to_gnm_dict[domain_name].add(gnm_id)
+
+    group_id_list_sorted = sorted(list(group_to_gnm_dict.keys()))
+
+    # exit program if group information is missing
+    gnms_without_group_info = set()
+    for gnm in gnm_set:
+        if gnm not in gnm_to_group_dict:
+            gnms_without_group_info.add(gnm)
+
+    if len(gnms_without_group_info) > 0:
+        print('Group information for the following genomes are missing from %s, program exited!' % gnm_group_txt)
+        print(','.join(gnms_without_group_info))
+        print('Group information for the above genomes are missing from %s, program exited!' % gnm_group_txt)
+        exit()
+
+    # create folder
+    if force_overwriting is True:
+        if os.path.isdir(op_dir) is True:
+            os.system('rm -r %s' % op_dir)
+    else:
+        if os.path.isdir(op_dir) is True:
+            print('Output folder already exist, program exited!')
+            exit()
+    os.system('mkdir %s' % op_dir)
+
+    # read in provided cutoffs
+    present_pct_cutoff_list = [int(i) for i in cutoff_str.split('-')]
+    assess_summary_1_txt    = '%s/assessment_PA.txt'                % op_dir
+    assess_summary_2_txt    = '%s/assessment_PA_summary.txt'        % op_dir
+    itol_binary_txt         = '%s/assessment_PA_iTOL_binary.txt'    % op_dir
+
+    trimmed_aln_file_re = '%s/*.%s' % (trimmed_aln_dir, trimmed_aln_ext)
+    trimmed_aln_file_list = [os.path.basename(file_name) for file_name in glob.glob(trimmed_aln_file_re)]
+
+    assess_summary_1_txt_handle = open(assess_summary_1_txt, 'w')
+    assess_summary_1_txt_handle.write('Marker\t%s\n' % '\t'.join([str(i) for i in group_id_list_sorted]))
+    assess_summary_2_txt_handle = open(assess_summary_2_txt, 'w')
+    assess_summary_2_txt_handle.write('Marker\t%s\n' % '\t'.join([str(i) for i in present_pct_cutoff_list]))
+    cutoff_to_qualified_marker_dict = dict()
+    gnm_to_identified_marker_dict = dict()
+    marker_id_list = []
+    for each_aln in trimmed_aln_file_list:
+
+        marker_id = each_aln.split(('.%s' % trimmed_aln_ext))[0]
+        marker_id_list.append(marker_id)
+        pwd_aln = '%s/%s' % (trimmed_aln_dir, each_aln)
+
+        current_marker_num_by_group_dict = dict()
+        for each_seq in SeqIO.parse(pwd_aln, 'fasta'):
+            gnm_id = each_seq.id
+
+            # get genome to marker dist
+            if gnm_id not in gnm_to_identified_marker_dict:
+                gnm_to_identified_marker_dict[gnm_id] = {marker_id}
+            else:
+                gnm_to_identified_marker_dict[gnm_id].add(marker_id)
+
+            if gnm_id in gnm_to_group_dict:
+                gnm_group = gnm_to_group_dict[gnm_id]
+                if gnm_group not in current_marker_num_by_group_dict:
+                    current_marker_num_by_group_dict[gnm_group] = 1
+                else:
+                    current_marker_num_by_group_dict[gnm_group] += 1
+            else:
+                print('Not all genomes used to generate the MSA being found in -aa, program exited!')
+                exit()
+
+        # write out assess_summary_1_txt
+        pct_list = []
+        for each_grp in group_id_list_sorted:
+            grp_pct = current_marker_num_by_group_dict.get(each_grp, 0)*100/group_to_gnm_num_dict[each_grp]
+            grp_pct = float("{0:.2f}".format(grp_pct))
+            pct_list.append(grp_pct)
+        assess_summary_1_txt_handle.write('%s\t%s\n' % (marker_id, '\t'.join([str(i) for i in pct_list])))
+
+        # write out assess_summary_2_txt
+        assess_list = []
+        for each_cutoff in present_pct_cutoff_list:
+
+            good_marker = True
+            for each_pct in pct_list:
+                if each_pct < each_cutoff:
+                    good_marker = False
+
+            if each_cutoff not in cutoff_to_qualified_marker_dict:
+                cutoff_to_qualified_marker_dict[each_cutoff] = {marker_id}
+
+            if good_marker is True:
+                assess_list.append('1')
+                cutoff_to_qualified_marker_dict[each_cutoff].add(marker_id)
+            else:
+                assess_list.append('0')
+        assess_summary_2_txt_handle.write('%s\t%s\n' % (marker_id, '\t'.join(assess_list)))
+
+    # write out total in assess_summary_2_txt
+    total_stats_list = [str(len(cutoff_to_qualified_marker_dict[each_c])) for each_c in present_pct_cutoff_list]
+    assess_summary_2_txt_handle.write('Total\t%s\n' % ('\t'.join(total_stats_list)))
+    assess_summary_1_txt_handle.close()
+    assess_summary_2_txt_handle.close()
+
+    # copy alignments of qualified marker to corresponding folders
+    for each_cutoff in cutoff_to_qualified_marker_dict:
+        qualified_marker_set = cutoff_to_qualified_marker_dict[each_cutoff]
+
+        qualified_marker_phy           = 'qualified_marker_PA_%s_concatenated.phy'               % each_cutoff
+        pwd_qualified_marker_dir       = '%s/qualified_marker_PA_%s'                             % (op_dir, each_cutoff)
+        pwd_qualified_marker_id_txt    = '%s/qualified_marker_PA_%s_id.txt'                      % (op_dir, each_cutoff)
+        pwd_qualified_marker_phy       = '%s/qualified_marker_PA_%s_concatenated.phy'            % (op_dir, each_cutoff)
+        pwd_qualified_marker_partition = '%s/qualified_marker_PA_%s_concatenated_partition.txt'  % (op_dir, each_cutoff)
+        pwd_js_iqtree                  = '%s/js_iqtree_PA_%s.sh'                                 % (op_dir, each_cutoff)
+
+        os.system('mkdir %s' % pwd_qualified_marker_dir)
+        for each_marker in qualified_marker_set:
+            pwd_marker_aln = '%s/%s.%s' % (trimmed_aln_dir, each_marker, trimmed_aln_ext)
+            cp_cmd = 'cp %s %s/' % (pwd_marker_aln, pwd_qualified_marker_dir)
+            os.system(cp_cmd)
+
+        # write out id
+        with open(pwd_qualified_marker_id_txt, 'w') as pwd_qualified_marker_id_txt_handle:
+            pwd_qualified_marker_id_txt_handle.write('%s\n' % '\n'.join(qualified_marker_set))
+
+        # concatenate qualified alignments
+        #catfasta2phyml_cmd = 'perl %s --sequential --concatenate %s/*.aln > %s 2> %s' % (catfasta2phyml_pl, pwd_qualified_marker_dir, pwd_qualified_marker_phy, pwd_qualified_marker_partition)
+        #print('running: ' + catfasta2phyml_cmd)
+        #os.system(catfasta2phyml_cmd)
+        catfasta2phy(pwd_qualified_marker_dir, 'aln', pwd_qualified_marker_phy, pwd_qualified_marker_partition)
+
+        # write out iqtree js
+        guide_tree_dir          = 'qualified_marker_PA_%s_guide_tree'       % each_cutoff
+        iqtree_C60_PMSF_dir     = 'qualified_marker_PA_%s_iqtree_C60_PMSF'  % each_cutoff
+        pwd_guide_tree_dir      = '%s/%s'                                   % (op_dir, guide_tree_dir)
+        pwd_iqtree_C60_PMSF_dir = '%s/%s'                                   % (op_dir, iqtree_C60_PMSF_dir)
+
+        with open(pwd_js_iqtree, 'w') as pwd_js_iqtree_handle:
+            pwd_js_iqtree_handle.write('#!/bin/bash\n#SBATCH --ntasks 1\n#SBATCH --cpus-per-task %s\n' % js_cpu_num)
+            pwd_js_iqtree_handle.write('mkdir %s/%s\n' % (os.getcwd(), pwd_guide_tree_dir))
+            pwd_js_iqtree_handle.write('cd %s/%s\n'    % (os.getcwd(), pwd_guide_tree_dir))
+            pwd_js_iqtree_handle.write('iqtree -s ../%s --prefix guide_tree --seqtype AA -m LG -T %s -B 1000 --alrt 1000\n' % (qualified_marker_phy, js_cpu_num))
+            pwd_js_iqtree_handle.write('mkdir %s/%s\n' % (os.getcwd(), pwd_iqtree_C60_PMSF_dir))
+            pwd_js_iqtree_handle.write('cd %s/%s\n'    % (os.getcwd(), pwd_iqtree_C60_PMSF_dir))
+            pwd_js_iqtree_handle.write('iqtree -s ../%s --prefix concatenated --seqtype AA -m LG+G+F+C60 -T %s -B 1000 --alrt 1000 -ft ../%s/guide_tree.treefile\n' % (qualified_marker_phy, js_cpu_num, guide_tree_dir))
+
+    # write out iTOL file
+    itol_binary_txt_handle = open(itol_binary_txt, 'w')
+    itol_binary_txt_handle.write('DATASET_BINARY\n\nSEPARATOR TAB\nDATASET_LABEL\tlabel1\nCOLOR\t#85C1E9\n')
+    itol_binary_txt_handle.write('SHOW_LABELS\t1\nLABEL_ROTATION\t45\nLABEL_SHIFT\t5\n')
+    itol_binary_txt_handle.write('FIELD_LABELS\t%s\n' % '\t'.join(sorted(marker_id_list)))
+    itol_binary_txt_handle.write('FIELD_SHAPES\t%s\n' % '\t'.join(['1']*len(marker_id_list)))
+    itol_binary_txt_handle.write('\nDATA\n')
+    for each_g in gnm_to_identified_marker_dict:
+        g_identified_marker_set = gnm_to_identified_marker_dict[each_g]
+
+        pa_list = []
+        for each_m in sorted(marker_id_list):
+            if each_m in g_identified_marker_set:
+                pa_list.append('1')
+            else:
+                pa_list.append('-1')
+        itol_binary_txt_handle.write('%s\t%s\n' % (each_g, '\t'.join(pa_list)))
+    itol_binary_txt_handle.close()
+
+    print('Assessment results exported to:\n%s\n%s' % (assess_summary_1_txt, assess_summary_2_txt))
+    print('Done!')
+
+
+if __name__ == '__main__':
+
+    parser = argparse.ArgumentParser()
+    parser.add_argument('-ta',         required=True,                               help='trimmed alignments')
+    parser.add_argument('-tax',        required=True,                               help='extension of trimmed alignments')
+    parser.add_argument('-aa',         required=True,                               help='faa file dir')
+    parser.add_argument('-aax',        required=True,                               help='faa file ext')
+    parser.add_argument('-g',          required=True,                               help='genome group')
+    parser.add_argument('-c',          required=False, default='50-75-100',         help='cutoffs, default: 50-75-100')
+    parser.add_argument('-o',          required=True,                               help='output dir')
+    parser.add_argument('-f',          required=False, action="store_true",         help='force overwrite existing output folder')
+    #parser.add_argument('-pl',         required=True,                               help='path to catfasta2phyml.pl')
+    parser.add_argument('-jst',        required=False, default='3',                 help='threads to request in job script')
+    parser.add_argument('-qsub',       required=False, action="store_true",         help='submit job script')
+    args = vars(parser.parse_args())
+    AssessMarkerPA(args)