PyamilySeq 0.4.0__py3-none-any.whl → 0.5.0__py3-none-any.whl

PyamilySeq/PyamilySeq_Genus.py

@@ -0,0 +1,659 @@
+#from line_profiler_pycharm import profile
+
+from collections import OrderedDict,defaultdict
+import copy
+import math
+import sys
+from tempfile import NamedTemporaryFile
+
+
+
+try:
+    from .Constants import *
+    from .utils import *
+except (ModuleNotFoundError, ImportError, NameError, TypeError) as error:
+    from Constants import *
+    from utils import *
+
+
+def custom_sort_key(k, dict1, dict2):
+    return (len(dict1[k]), len(dict2[k]))
+
+def sort_keys_by_values(dict1, dict2):
+    sorted_keys = sorted(dict1.keys(), key=lambda k: custom_sort_key(k, dict1, dict2), reverse=True)
+    return sorted_keys
+
+def select_longest_gene(sequences):
+    """Select the longest sequence for each genome."""
+    longest_sequences = {}
+    for seq_id, sequence in sequences.items():
+        genome = seq_id.split('|')[0]  # Assuming genome name can be derived from the sequence ID
+        if genome not in longest_sequences or len(sequence) > len(longest_sequences[genome][1]):
+            longest_sequences[genome] = (seq_id, sequence)
+    return longest_sequences
+
+
+def run_mafft_on_sequences(options, sequences, output_file):
+    print("Conducting MAFFT alignment.")
+    """Run mafft on the given sequences and write to output file."""
+    # Create a temporary input file for mafft
+    with NamedTemporaryFile('w', delete=False) as temp_input_file:
+        for header, sequence in sequences.items():
+            temp_input_file.write(f">{header}\n{sequence}\n")
+        temp_input_file_path = temp_input_file.name
+
+    # Run mafft
+    try:
+        with open(output_file, 'w') as output_f:
+            if options.verbose == True:
+                subprocess.run(
+                    ['mafft', '--auto', temp_input_file_path],
+                    stdout=output_f,
+                    stderr=sys.stderr,
+                    check=True
+                )
+            else:
+                subprocess.run(
+                    ['mafft', '--auto', temp_input_file_path],
+                    stdout=output_f,
+                    stderr=subprocess.DEVNULL,  # Suppress stderr
+                    check=True
+                )
+    finally:
+        os.remove(temp_input_file_path)  # Clean up the temporary file
+
+
+def process_gene_families(options, directory, output_file):
+    """Process each gene family file to select the longest sequence per genome and concatenate aligned sequences."""
+    concatenated_sequences = {}
+    output_file = directory.replace('Gene_Families_Output',output_file)
+
+    # Iterate over each gene family file
+    for gene_file in os.listdir(directory):
+        if gene_file.endswith('.fasta'):
+            gene_path = os.path.join(directory, gene_file)
+
+            # Read sequences from the gene family file
+            sequences = read_fasta(gene_path)
+
+            # Select the longest sequence for each genome
+            longest_sequences = select_longest_gene(sequences)
+
+            # Run mafft on the longest sequences
+            aligned_file = f"{gene_file}_aligned.fasta"
+            run_mafft_on_sequences(options, {seq_id: seq for seq_id, seq in longest_sequences.values()}, aligned_file)
+
+            # Read aligned sequences and concatenate them
+            aligned_sequences = read_fasta(aligned_file)
+            for genome, aligned_seq in aligned_sequences.items():
+                genome_name = genome.split('|')[0]
+                if genome_name not in concatenated_sequences:
+                    concatenated_sequences[genome_name] = ""
+                concatenated_sequences[genome_name] += aligned_seq
+
+            # Clean up aligned file
+            os.remove(aligned_file)
+
+    # Write the concatenated sequences to the output file
+    with open(output_file, 'w') as out:
+        for genome, sequence in concatenated_sequences.items():
+            out.write(f">{genome}\n")
+            wrapped_sequence = wrap_sequence(sequence, 60)
+            out.write(f"{wrapped_sequence}\n")
+
+def gene_presence_absence_output(options, genome_dict, pangenome_clusters_First_sorted, pangenome_clusters_First_sequences_sorted):
+    print("Outputting gene_presence_absence file")
+    in_name = options.clusters.split('.')[0]
+    gpa_outfile = open(in_name+'_gene_presence_absence.csv','w')
+    gpa_outfile.write('"Gene","Non-unique Gene name","Annotation","No. isolates","No. sequences","Avg sequences per isolate","Genome Fragment","Order within Fragment","'
+                     '"Accessory Fragment","Accessory Order with Fragment","QC","Min group size nuc","Max group size nuc","Avg group size nuc","')
+    gpa_outfile.write('","'.join(genome_dict.keys()))
+    gpa_outfile.write('"\n')
+    for cluster, sequences in pangenome_clusters_First_sequences_sorted.items():
+        average_sequences_per_genome = len(sequences) / len(pangenome_clusters_First_sorted[cluster])
+        gpa_outfile.write('"group_'+str(cluster)+'","","'+str(len(pangenome_clusters_First_sorted[cluster]))+'","'+str(len(sequences))+'","'+str(average_sequences_per_genome)+
+                         '","","","","","","","","",""')
+
+        full_out = ''
+        for genome in genome_dict.keys():
+            tmp_list = []
+            for value in sequences:
+                if value.split('|')[0] == genome:
+                    tmp_list.append(value)
+            if tmp_list:
+                full_out += ',"'+''.join(tmp_list)+'"'
+            gpa_outfile.write(full_out)
+        gpa_outfile.write('\n')
+
+### Below is some unfinished code
+    # edge_list_outfile = open(in_name+'_edge_list.csv','w')
+    # for cluster, sequences in pangenome_clusters_First_sequences_sorted.items():
+    #     output = []
+    #     for entry in sequences:
+    #         # Split each entry at '|'
+    #         genome, gene = entry.split('|')
+    #         # Format the result as "gene  genome"
+    #         output.append(f"{gene}\t{genome}")
+    #     for line in output:
+    #         edge_list_outfile.write(line + '\n')
+
+
+def wrap_sequence(sequence, width=60):
+    wrapped_sequence = []
+    for i in range(0, len(sequence), width):
+        wrapped_sequence.append(sequence[i:i + width])
+    return "\n".join(wrapped_sequence)
+
+
+def read_fasta(fasta_file):
+    sequences = {}
+    current_sequence = None
+    with open(fasta_file, 'r') as file:
+        for line in file:
+            line = line.strip()
+            if not line:
+                continue  # Skip empty lines
+            if line.startswith('>'):
+                current_sequence = line[1:]  # Remove '>' character
+                sequences[current_sequence] = ''
+            else:
+                sequences[current_sequence] += line
+    return sequences
+
+
+def reorder_dict_by_keys(original_dict, sorted_keys):
+    return {k: original_dict[k] for k in sorted_keys}
+
+def get_cores(options,genome_dict):
+    ##Calculate core groups
+    groups = OrderedDict()
+    cores = OrderedDict()
+    prev_top = len(genome_dict)
+    first = True
+    for group in options.core_groups.split(','):
+        calculated_floor = math.floor(int(group) / 100 * len(genome_dict))
+        if first == False:
+            # Ensure no overlap
+            # if calculated_floor <= prev_top:
+            #     calculated_floor = prev_top - 1
+
+            groups[group] = (calculated_floor,prev_top)
+        else:
+            groups[group] = (calculated_floor, prev_top)
+            first = False
+        prev_top = calculated_floor
+        first_core_group = 'first_core_' + group
+        cores[first_core_group] = []
+        if options.reclustered != None:
+            extended_core_group = 'extended_core_' + group
+            cores[extended_core_group] = []
+            combined_core_group = 'combined_core_' + group
+            cores[combined_core_group] = []
+            second_core_group = 'second_core_' + group
+            cores[second_core_group] = []
+            only_second_core_group = 'only_second_core_' + group
+            cores[only_second_core_group] = []
+    return cores, groups
+
+#@profile
+def calc_First_only_core(cluster, pep_num, groups, cores):
+    groups_as_list = list(groups.values())
+    for idx in (idx for idx, (sec, fir) in enumerate(groups_as_list) if sec <= pep_num <= fir):
+        res = idx
+    family_group = list(groups)[res]
+    cores['first_core_'+family_group].append(cluster)
+
+#@profile
+def calc_single_First_extended_Second_only_core(pep_num, groups, cores, second_num): # Count gene families extended with StORFs
+    groups_as_list = list(groups.values())
+    for idx in (idx for idx, (sec, fir) in enumerate(groups_as_list) if sec <= pep_num+second_num <= fir):
+        res = idx
+    family_group = list(groups)[res]
+    cores['extended_core_' + family_group].append(pep_num)
+
+
+#@profile
+def calc_multi_First_extended_Second_only_core(pep_num, groups, cores, second_num): # Count seperately those gene families extended with StORF_Reporter but combined >1 PEP
+    groups_as_list = list(groups.values())
+    for idx in (idx for idx, (sec, fir) in enumerate(groups_as_list) if sec <= pep_num+second_num <= fir):
+        res = idx
+    family_group = list(groups)[res]
+    cores['combined_core_' + family_group] += 1
+
+
+#@profile
+def calc_Second_only_core(groups, cores, second_num):
+    groups_as_list = list(groups.values())
+    for idx in (idx for idx, (sec, fir) in enumerate(groups_as_list) if sec <= second_num <= fir):
+        res = idx
+    family_group = list(groups)[res]
+    cores['second_core_' + family_group] += 1
+
+#@profile
+def calc_only_Second_only_core(groups, cores, second_num): # only count the true storf onlies
+    groups_as_list = list(groups.values())
+    for idx in (idx for idx, (sec, fir) in enumerate(groups_as_list) if sec <= second_num <= fir):
+        res = idx
+    family_group = list(groups)[res]
+    cores['only_second_core_' + family_group] += 1
+
+
+
+
+
+#@profile
+def combined_clustering_counting(options, pangenome_clusters_First, reps, combined_pangenome_clusters_First_Second_clustered):
+    num_clustered_First = defaultdict(list)
+    pangenome_clusters_Type = copy.deepcopy(pangenome_clusters_First)
+    list_of_reps = list(reps.keys())
+    for cluster, pep_genomes in pangenome_clusters_First.items():
+        rep = list_of_reps[int(cluster)]  # get the rep of the current pep cluster
+        Com_PEP_Genomes = 0
+        Seconds = 0
+        seen_Seconds = []
+        added_Second_genomes = 0
+        try:  # get the cluster from the storf clusters which contains this rep
+            clustered_combined = combined_pangenome_clusters_First_Second_clustered[rep]  # Not true clusters - I put a PEP as key myself
+            seen_clust_Genomes = []
+            num_clustered_First[cluster].append(rep + '_' + str(len(pep_genomes)))
+            for clust in clustered_combined:
+                if options.sequence_tag not in clust:  # Not good enough at the moment
+                    clust_Genome = clust.split('|')[0]
+                    if clust_Genome not in seen_clust_Genomes:
+                        seen_clust_Genomes.append(clust_Genome)
+                        if clust_Genome not in pep_genomes:
+                            Com_PEP_Genomes += 1
+                    num_clustered_First[cluster].append(clust + '_' + str(reps[clust][1]))
+                elif options.sequence_tag in clust:
+                    Seconds += 1
+                    clust_Genome = clust.split('|')[0]
+                    if clust_Genome not in seen_Seconds:
+                        seen_Seconds.append(clust_Genome)
+                    if clust_Genome not in seen_clust_Genomes:
+                        seen_clust_Genomes.append(clust_Genome)
+                        if clust_Genome not in pep_genomes:
+                            added_Second_genomes += 1
+                else:
+                    sys.exit("Error: looking for sequence_tag")
+
+            size_of_pep_clusters = []
+            peps = num_clustered_First[cluster]
+            for pep in peps:
+                pep = pep.rsplit('_', 1)
+                size_of_pep_clusters.append(int(pep[1]))
+            pangenome_clusters_Type[cluster] = [len(num_clustered_First[cluster]), sum(size_of_pep_clusters),
+                                                size_of_pep_clusters, added_Second_genomes, Seconds, len(seen_Seconds)]
+
+        except KeyError:
+            ###Singleton
+            num_pep_genomes = [len(pep_genomes)]
+            pangenome_clusters_Type[cluster] = [1, len(pep_genomes), num_pep_genomes, added_Second_genomes, Seconds,
+                                                len(seen_Seconds)]
+
+    return pangenome_clusters_Type
+
+#@profile
+def single_clustering_counting(options, pangenome_clusters_First, reps):
+    num_clustered_First = defaultdict(list)
+    recorded_First = []
+    pangenome_clusters_Type = copy.deepcopy(pangenome_clusters_First)
+    list_of_reps = list(reps.keys())
+    for cluster, First_genomes in pangenome_clusters_First.items():
+        rep = list_of_reps[int(cluster)]  # get the rep of the current pep cluster
+
+        try:  # get the cluster from the storf clusters which contains this rep
+            num_clustered_First[cluster].append(rep + '_' + str(len(First_genomes)))
+            size_of_First_clusters = []
+            Firsts = num_clustered_First[cluster]
+            for First in Firsts:
+                First = First.rsplit('_', 1)
+                size_of_First_clusters.append(int(First[1]))
+                recorded_First.append(First[0])
+            pangenome_clusters_Type[cluster] = [len(num_clustered_First[cluster]), sum(size_of_First_clusters),
+                                                size_of_First_clusters, 0, 0, 0]
+
+        except KeyError:
+            ###Singleton
+            num_pep_genomes = [len(First_genomes)]
+            pangenome_clusters_Type[cluster] = [1, len(First_genomes), num_pep_genomes, 0, 0, 0]
+
+    return pangenome_clusters_Type
+
+
+
+#@profile
+def combined_clustering_CDHIT(options, genome_dict):
+    unique_genomes = []
+    Second_in = open(options.reclustered, 'r')
+    combined_pangenome_clusters_First = OrderedDict()
+    combined_pangenome_clusters_First_sequences = OrderedDict()
+    combined_pangenome_clusters_Second = OrderedDict()
+    combined_pangenome_clusters_Second_sequences = OrderedDict()
+    combined_pangenome_clusters_First_Second_clustered = OrderedDict()
+
+    not_Second_only_cluster_ids = []
+    already_seen_PEP = []
+    Combined_clusters = OrderedDict()
+    Combined_reps = OrderedDict()
+    first = True
+    for line in Second_in:
+        if line.startswith('>'):
+            if first == False:
+                cluster_size = len(Combined_clusters[cluster_id])
+                Combined_reps.update({rep: cluster_size})
+                for pep in combined_pangenome_clusters_First_sequences[cluster_id]:
+                    if pep != []:
+                        if pep in already_seen_PEP:
+                            continue
+                        else:
+                            already_seen_PEP.append(pep)
+                if len(combined_pangenome_clusters_Second_sequences[cluster_id]) > 0 and len(combined_pangenome_clusters_First_sequences[cluster_id]) > 0:
+                    if len(combined_pangenome_clusters_First_sequences[cluster_id]) > 1:  # If we have clustered >1 PEP family, we need to record 1 as key and all others are val
+                        all_but_first = combined_pangenome_clusters_First_sequences[cluster_id][1:]
+                        storfs_clustered = combined_pangenome_clusters_Second_sequences[cluster_id]
+                        VALUE = all_but_first + storfs_clustered
+                    else:
+                        VALUE = combined_pangenome_clusters_Second_sequences[cluster_id]
+                    KEY = combined_pangenome_clusters_First_sequences[cluster_id][0]
+                    combined_pangenome_clusters_First_Second_clustered.update({KEY: VALUE})
+            cluster_id = line.strip('>')
+            cluster_id = cluster_id.strip('\n')
+            cluster_id = cluster_id.split(' ')[1]
+            Combined_clusters.update({cluster_id: []})
+            combined_pangenome_clusters_First.update({cluster_id: []})
+            combined_pangenome_clusters_First_sequences.update({cluster_id: []})
+            combined_pangenome_clusters_Second.update({cluster_id: []})
+            combined_pangenome_clusters_Second_sequences.update({cluster_id: []})
+
+            first = False
+        else:
+            clustered = line.split('\t')[1]
+            clustered = clustered.split('>')[1]
+            clustered = clustered.split('...')[0]
+            genome = clustered.split('|')[0]
+            genome_dict[genome] += 1
+            if '*' in line:
+                rep = clustered
+                Combined_reps.update({rep: 0})
+            if first == False:
+                Combined_clusters[cluster_id].append(clustered)
+                clustered_genome = clustered.split('|')[0]
+                if options.sequence_tag in line:
+                    if clustered_genome not in combined_pangenome_clusters_Second[cluster_id]:
+                        combined_pangenome_clusters_Second[cluster_id].append(clustered_genome)
+                    combined_pangenome_clusters_Second_sequences[cluster_id].append(clustered)
+                else:
+                    if cluster_id not in not_Second_only_cluster_ids:
+                        not_Second_only_cluster_ids.append(cluster_id)  # Tell us which StORF_Reporter clustered are unmatched to a PEP
+                    if clustered_genome not in combined_pangenome_clusters_First[cluster_id]:
+                        combined_pangenome_clusters_First[cluster_id].append(clustered_genome)
+                    combined_pangenome_clusters_First_sequences[cluster_id].append(clustered)
+
+
+    return combined_pangenome_clusters_First_Second_clustered,not_Second_only_cluster_ids, combined_pangenome_clusters_Second, unique_genomes
+
+def combined_clustering_Edge_List(options, genome_dict):
+    if options.cluster_format == 'TSV':
+        separator = '\t'
+    elif options.cluster_format == 'CSV':
+        separator = ','
+    unique_genomes = []
+    cluster_id = 0
+    last_rep = ''
+    Second_in = open(options.reclustered, 'r')
+    combined_pangenome_clusters_First = OrderedDict()
+    combined_pangenome_clusters_First_sequences = OrderedDict()
+    combined_pangenome_clusters_Second = OrderedDict()
+    combined_pangenome_clusters_Second_sequences = OrderedDict()
+    combined_pangenome_clusters_First_Second_clustered = OrderedDict()
+
+    not_Second_only_cluster_ids = []
+    already_seen_PEP = []
+    Combined_clusters = OrderedDict()
+    Combined_reps = OrderedDict()
+    first = True
+    for line in Second_in:
+        rep, child = line.strip().split(separator)
+        child_genome = child.split('|')[0]  # Extracting the genome identifier from the child sequence
+
+        if first == True:
+            Combined_clusters.update({cluster_id: []})
+            combined_pangenome_clusters_First.update({cluster_id: []})
+            combined_pangenome_clusters_First_sequences.update({cluster_id: []})
+            combined_pangenome_clusters_Second.update({cluster_id: []})
+            combined_pangenome_clusters_Second_sequences.update({cluster_id: []})
+            Combined_reps.update({rep: 0})
+            first = False
+
+        if first == False:
+            if rep != last_rep and last_rep != '':
+                cluster_size = len(Combined_clusters[cluster_id])
+                Combined_reps.update({rep: cluster_size})
+                for pep in combined_pangenome_clusters_First_sequences[cluster_id]:
+                    if pep != []:
+                        if pep in already_seen_PEP:
+                            continue
+                        else:
+                            already_seen_PEP.append(pep)
+                if len(combined_pangenome_clusters_Second_sequences[cluster_id]) > 0 and len(combined_pangenome_clusters_First_sequences[cluster_id]) > 0:
+                    if len(combined_pangenome_clusters_First_sequences[cluster_id]) > 1:  # If we have clustered >1 PEP family, we need to record 1 as key and all others are val
+                        all_but_first = combined_pangenome_clusters_First_sequences[cluster_id][1:]
+                        storfs_clustered = combined_pangenome_clusters_Second_sequences[cluster_id]
+                        VALUE = all_but_first + storfs_clustered
+                    else:
+                        VALUE = combined_pangenome_clusters_Second_sequences[cluster_id]
+                    KEY = combined_pangenome_clusters_First_sequences[cluster_id][0]
+                    combined_pangenome_clusters_First_Second_clustered.update({KEY: VALUE})
+
+                cluster_id += 1
+                Combined_clusters.update({cluster_id: []})
+                combined_pangenome_clusters_First.update({cluster_id: []})
+                combined_pangenome_clusters_First_sequences.update({cluster_id: []})
+                combined_pangenome_clusters_Second.update({cluster_id: []})
+                combined_pangenome_clusters_Second_sequences.update({cluster_id: []})
+                Combined_reps.update({rep: 0})
+
+
+        Combined_clusters[cluster_id].append(child)
+        if options.sequence_tag in line:
+            if child_genome not in combined_pangenome_clusters_Second[cluster_id]:
+                combined_pangenome_clusters_Second[cluster_id].append(child_genome)
+            combined_pangenome_clusters_Second_sequences[cluster_id].append(child)
+        else:
+            if cluster_id not in not_Second_only_cluster_ids:
+                not_Second_only_cluster_ids.append(cluster_id)  # Tell us which StORF_Reporter clustered are unmatched to a PEP
+            if child_genome not in combined_pangenome_clusters_First[cluster_id]:
+                combined_pangenome_clusters_First[cluster_id].append(child_genome)
+            combined_pangenome_clusters_First_sequences[cluster_id].append(child)
+
+        last_rep = rep
+
+    return combined_pangenome_clusters_First_Second_clustered,not_Second_only_cluster_ids, combined_pangenome_clusters_Second, unique_genomes
+
+
+def cluster_EdgeList(options):
+    if options.cluster_format == 'TSV':
+        separator = '\t'
+    elif options.cluster_format == 'CSV':
+        separator = ','
+    cluster_id = 0
+    last_rep = ''
+    first = True
+    First_in = open(options.clusters, 'r')
+    pangenome_clusters_First = OrderedDict()
+    pangenome_clusters_First_sequences = OrderedDict()
+    genome_dict = defaultdict(int)
+    reps = OrderedDict()
+    for line in First_in:
+        rep, child = line.strip().split(separator)
+        child_genome = child.split('|')[0]  # Extracting the genome identifier from the child sequence
+        # Counting occurrences of genomes
+        genome_dict[child_genome] += 1
+        if first == True:
+            pangenome_clusters_First[0] = []
+            pangenome_clusters_First_sequences[0] = []
+            first = False
+
+        if rep != last_rep and last_rep != '':
+            cluster_id +=1
+            pangenome_clusters_First[cluster_id] = []
+            pangenome_clusters_First_sequences[cluster_id] = []
+            cluster_size = len(pangenome_clusters_First_sequences[cluster_id-1])
+            reps.update({last_rep: [cluster_size, len(pangenome_clusters_First[cluster_id-1])]})
+            pangenome_clusters_First[cluster_id] = []
+            pangenome_clusters_First_sequences[cluster_id] = []
+        if child_genome not in pangenome_clusters_First[cluster_id]:
+            pangenome_clusters_First[cluster_id].append(child_genome)
+
+        pangenome_clusters_First_sequences[cluster_id].append(child)
+        last_rep = rep
+        cluster_size = len(pangenome_clusters_First_sequences[cluster_id])
+        reps.update({rep: [cluster_size, len(pangenome_clusters_First[cluster_id])]})
+
+
+    return genome_dict, pangenome_clusters_First, pangenome_clusters_First_sequences, reps
+
+
+
+def cluster_CDHIT(options):
+    First_in = open(options.clusters, 'r')
+    clusters = OrderedDict()
+    pangenome_clusters_First = OrderedDict()
+    pangenome_clusters_First_sequences = OrderedDict()
+    first = True
+    genome_dict = defaultdict(int)
+    reps = OrderedDict()
+    ## Load in all data for easier reuse later
+    for line in First_in:
+        if line.startswith('>'):
+            if first == False:
+                cluster_size = len(clusters[cluster_id])
+                reps.update({rep: [cluster_size, len(pangenome_clusters_First[cluster_id])]})
+            cluster_id = line.strip('>')
+            cluster_id = cluster_id.strip('\n')
+            cluster_id = cluster_id.split(' ')[1]
+            clusters.update({cluster_id: []})
+            pangenome_clusters_First.update({cluster_id: []})
+            pangenome_clusters_First_sequences.update({cluster_id: []})
+
+            first = False
+        else:
+            clustered = line.split('\t')[1]
+            clustered = clustered.split('>')[1]
+            clustered = clustered.split('...')[0]
+            genome = clustered.split('|')[0]
+            genome_dict[genome] += 1
+            if '*' in line:
+                rep = clustered
+                reps.update({rep: [0, 0]})
+            if first == False:
+                clusters[cluster_id].append(clustered)
+                clustered_genome = clustered.split('|')[0]
+                if clustered_genome not in pangenome_clusters_First[cluster_id]:
+                    pangenome_clusters_First[cluster_id].append(clustered_genome)
+                pangenome_clusters_First_sequences[cluster_id].append(clustered)
+    return genome_dict, pangenome_clusters_First, pangenome_clusters_First_sequences, reps
+
+#@profile
+def cluster(options):
+
+    if options.cluster_format == 'CD-HIT':
+        genome_dict, pangenome_clusters_First, pangenome_clusters_First_sequences, reps = cluster_CDHIT(options)
+    elif options.cluster_format in ['TSV','CSV']:
+        genome_dict, pangenome_clusters_First, pangenome_clusters_First_sequences, reps = cluster_EdgeList(options)
+
+    ######################################
+    cores, groups = get_cores(options, genome_dict)
+    ###
+
+    if options.reclustered != None:
+        if options.cluster_format == 'CD-HIT':
+            combined_pangenome_clusters_First_Second_clustered,not_Second_only_cluster_ids,combined_pangenome_clusters_Second,\
+                unique_genomes = combined_clustering_CDHIT(options, genome_dict)
+        if options.cluster_format == 'TSV':
+            combined_pangenome_clusters_First_Second_clustered,not_Second_only_cluster_ids,combined_pangenome_clusters_Second,\
+                unique_genomes = combined_clustering_Edge_List(options, genome_dict)
+        pangenome_clusters_Type = combined_clustering_counting(options, pangenome_clusters_First, reps, combined_pangenome_clusters_First_Second_clustered)
+    else:
+        pangenome_clusters_Type = single_clustering_counting(options, pangenome_clusters_First, reps)
+
+
+
+    Number_Of_StORF_Extending_But_Same_Genomes = 0
+
+    sorted_first_keys = sort_keys_by_values(pangenome_clusters_First, pangenome_clusters_First_sequences)
+    pangenome_clusters_First_sorted = reorder_dict_by_keys(pangenome_clusters_First, sorted_first_keys)
+    pangenome_clusters_First_sequences_sorted = reorder_dict_by_keys(pangenome_clusters_First_sequences, sorted_first_keys)
+    pangenome_clusters_Type_sorted = reorder_dict_by_keys(pangenome_clusters_Type, sorted_first_keys)
+
+    print("Calculating Groups")
+    for cluster, numbers in pangenome_clusters_Type_sorted.items():
+    ############################### Calculate First only
+        #if numbers[0] == 1 and numbers[1] >=2:
+        calc_First_only_core(cluster, numbers[1],groups,cores)
+
+        # elif numbers[0] >1 and numbers[1] >=2:
+        #     calc_First_only_core(cluster, numbers[2][0],groups,cores)
+
+
+    if options.reclustered != None:
+        ############################# Calculate First and Reclustered-Second
+        if numbers[0] == 1 and numbers[3] >= 1:  # If Seconds did not combine First reps
+            calc_single_First_extended_Second_only_core(numbers[1], groups, cores, numbers[3])
+        elif numbers[0] > 1 and numbers[3] >= 1:  # If unique Secondss combined multiple Firsts
+            calc_multi_First_extended_Second_only_core(numbers[1], groups, cores, numbers[3])
+        elif numbers[4] >= 1:
+            Number_Of_StORF_Extending_But_Same_Genomes += 1
+        combined_pangenome_clusters_ONLY_Second_Type = defaultdict(list)
+        combined_pangenome_clusters_Second_Type = defaultdict(list)
+        for cluster, genomes in combined_pangenome_clusters_Second.items():
+            if cluster in not_Second_only_cluster_ids:
+                combined_pangenome_clusters_Second_Type[cluster] = [cluster, len(genomes)]
+            else:
+                combined_pangenome_clusters_ONLY_Second_Type[cluster] = [cluster, len(genomes)]
+        for cluster, data in combined_pangenome_clusters_Second_Type.items():
+            calc_Second_only_core(groups, cores, data[1])
+        for cluster, data in combined_pangenome_clusters_ONLY_Second_Type.items():
+            if data[1] >= 2:
+                calc_only_Second_only_core(groups, cores, data[1])
+    ###########################
+    key_order = ['first_core_', 'extended_core_', 'combined_core_', 'second_core_','only_second_core_']
+    print("Gene Groups:")
+    for key_prefix in key_order:
+        for key, value in cores.items():
+            if key.startswith(key_prefix):
+                print(f"{key}: {len(value)}")
+    print("Total Number of Gene Groups (Including Singletons): " + str(len(pangenome_clusters_First_sequences_sorted)))
+
+    if options.gene_presence_absence_out != None:
+        gene_presence_absence_output(options,genome_dict, pangenome_clusters_First_sorted, pangenome_clusters_First_sequences_sorted)
+
+    if options.write_families != None and options.fasta != None:
+        sequences = read_fasta(options.fasta)
+        input_dir = os.path.dirname(os.path.abspath(options.clusters))
+        output_dir = os.path.join(input_dir, 'Gene_Families_Output')
+
+        # Create output directory if it doesn't exist
+        if not os.path.exists(output_dir):
+            os.makedirs(output_dir)
+        for key_prefix in key_order:
+            for key, values in cores.items():
+                if any(part in options.write_families.split(',') for part in key.split('_')):
+                    if key.startswith(key_prefix):
+                        for value in values:
+                            output_filename = f"{key}_{value}.fasta"
+                            sequences_to_write = pangenome_clusters_First_sequences_sorted[value]
+                            # Write sequences to output file that are in the sequences dictionary
+                            with open(os.path.join(output_dir, output_filename), 'w') as outfile:
+                                for header in sequences_to_write:
+                                    if header in sequences:
+                                        outfile.write(f">{header}\n")
+                                        wrapped_sequence = wrap_sequence(sequences[header])
+                                        outfile.write(f"{wrapped_sequence}\n")
+
+    if options.con_core != None and options.fasta != None and options.write_families != None:
+        process_gene_families(options, os.path.join(input_dir, 'Gene_Families_Output'), 'concatonated_genes_aligned.fasta')
+
+
+
+