view scripts/ReMatCh/modules/rematch_module.py @ 0:c6bab5103a14 draft

"planemo upload commit 6abf3e299d82d07e6c3cf8642bdea80e96df64c3-dirty"

import os.path
import multiprocessing
import functools
import sys
import pickle

# https://chrisyeh96.github.io/2017/08/08/definitive-guide-python-imports.html#case-2-syspath-could-change
sys.path.insert(0, os.path.dirname(os.path.realpath(__file__)))
import utils


def index_fasta_samtools(fasta, region_none, region_outfile_none, print_comand_true):
    command = ['samtools', 'faidx', fasta, '', '', '']
    shell_true = False
    if region_none is not None:
        command[3] = region_none
    if region_outfile_none is not None:
        command[4] = '>'
        command[5] = region_outfile_none
        shell_true = True
    run_successfully, stdout, stderr = utils.run_command_popen_communicate(command, shell_true, None, print_comand_true)
    return run_successfully, stdout


# Indexing reference file using Bowtie2
def index_sequence_bowtie2(reference_file, threads):
    if os.path.isfile(str(reference_file + '.1.bt2')):
        run_successfully = True
    else:
        command = ['bowtie2-build', '--threads', str(threads), reference_file, reference_file]
        run_successfully, stdout, stderr = utils.run_command_popen_communicate(command, False, None, True)
    return run_successfully


# Mapping with Bowtie2
def mapping_bowtie2(fastq_files, reference_file, threads, outdir, num_map_loc,
                    bowtie_algorithm='--very-sensitive-local', bowtie_opt=None):
    sam_file = os.path.join(outdir, str('alignment.sam'))

    # Index reference file
    run_successfully = index_sequence_bowtie2(reference_file, threads)

    if run_successfully:
        command = ['bowtie2', '', '', '-q', bowtie_algorithm, '--threads', str(threads), '-x',
                   reference_file, '', '--no-unal', '', '-S', sam_file]

        if num_map_loc is not None and num_map_loc > 1:
            command[1] = '-k'
            command[2] = str(num_map_loc)

        if len(fastq_files) == 1:
            command[9] = '-U ' + fastq_files[0]
        elif len(fastq_files) == 2:
            command[9] = '-1 ' + fastq_files[0] + ' -2 ' + fastq_files[1]
        else:
            return False, None

        if bowtie_opt is not None:
            command[11] = bowtie_opt

        run_successfully, stdout, stderr = utils.run_command_popen_communicate(command, False, None, True)

    if not run_successfully:
        sam_file = None

    return run_successfully, sam_file


def split_cigar(cigar):
    cigars = ['M', 'I', 'D', 'N', 'S', 'H', 'P', '=', 'X']

    splited_cigars = []
    numbers = ''
    for char in cigar:
        if char not in cigars:
            numbers += char
        else:
            splited_cigars.append([int(numbers), char])
            numbers = ''

    return splited_cigars


def recode_cigar_based_on_base_quality(cigar, bases_quality, soft_clip_base_quality, mapping_position,
                                       direct_strand_true, soft_clip_cigar_flag_recode):
    cigar = split_cigar(cigar)
    soft_left = []
    soft_right = []
    cigar_flags_for_reads_length = ('M', 'I', 'S', '=', 'X')
    read_length_without_right_s = sum([cigar_part[0] for cigar_part in cigar if
                                       cigar_part[1] in cigar_flags_for_reads_length]) - \
                                  (cigar[len(cigar) - 1][0] if cigar[len(cigar) - 1][1] == 'S' else 0)
    for x, base in enumerate(bases_quality):
        if ord(base) - 33 >= soft_clip_base_quality:
            if x <= cigar[0][0] - 1:
                if cigar[0][1] == 'S':
                    soft_left.append(x)
            elif x > read_length_without_right_s - 1:
                if cigar[len(cigar) - 1][1] == 'S':
                    soft_right.append(x)

    left_changed = (False, 0)
    if len(soft_left) > 0:
        soft_left = min(soft_left) + 1
        if soft_left == 1:
            cigar = [[cigar[0][0], soft_clip_cigar_flag_recode]] + cigar[1:]
            left_changed = (True, cigar[0][0])
        elif cigar[0][0] - soft_left > 0:
            cigar = [[soft_left, 'S']] + [[cigar[0][0] - soft_left, soft_clip_cigar_flag_recode]] + cigar[1:]
            left_changed = (True, cigar[0][0] - soft_left)

    right_changed = (False, 0)
    if len(soft_right) > 0:
        soft_right = max(soft_right) + 1
        cigar = cigar[:-1]
        if soft_right - read_length_without_right_s > 0:
            cigar.append([soft_right - read_length_without_right_s, soft_clip_cigar_flag_recode])
            right_changed = (True, soft_right - read_length_without_right_s)
        if len(bases_quality) - soft_right > 0:
            cigar.append([len(bases_quality) - soft_right, 'S'])

    if left_changed[0]:
        # if direct_strand_true:
        mapping_position = mapping_position - left_changed[1]
    # if right_changed[0]:
    #     if not direct_strand_true:
    #         mapping_position = mapping_position + right_changed[1]

    return ''.join([str(cigar_part[0]) + cigar_part[1] for cigar_part in cigar]), str(mapping_position)


def verify_is_forward_read(sam_flag_bit):
    # 64 = 1000000
    forward_read = False
    bit = format(sam_flag_bit, 'b').zfill(7)
    if bit[-7] == '1':
        forward_read = True
    return forward_read


def verify_mapped_direct_strand(sam_flag_bit):
    # 16 = 10000 -> mapped in reverse strand
    direct_strand = False
    bit = format(sam_flag_bit, 'b').zfill(5)
    if bit[-5] == '0':
        direct_strand = True
    return direct_strand


def verify_mapped_tip(reference_length, mapping_position, cigar):
    tip = False
    if 'S' in cigar:
        cigar = split_cigar(cigar)
        if cigar[0][1] == 'S':
            if mapping_position - cigar[0][0] < 0:
                tip = True
        if cigar[len(cigar) - 1][1] == 'S':
            if mapping_position + cigar[len(cigar) - 1][0] > reference_length:
                tip = True
    return tip


def change_sam_flag_bit_mapped_reverse_strand_2_direct_strand(sam_flag_bit):
    bit = list(format(sam_flag_bit, 'b').zfill(5))
    bit[-5] = '0'
    return int(''.join(bit), 2)


def change_sam_flag_bit_mate_reverse_strand_2_direct_strand(sam_flag_bit):
    bit = list(format(sam_flag_bit, 'b').zfill(6))
    bit[-6] = '0'
    return int(''.join(bit), 2)


def move_read_mapped_reverse_strand_2_direct_strand(seq, bases_quality, sam_flag_bit, cigar):
    seq = utils.reverse_complement(seq)
    bases_quality = ''.join(reversed(list(bases_quality)))
    sam_flag_bit = change_sam_flag_bit_mapped_reverse_strand_2_direct_strand(sam_flag_bit)
    cigar = ''.join([str(cigar_part[0]) + cigar_part[1] for cigar_part in reversed(split_cigar(cigar))])
    return seq, bases_quality, str(sam_flag_bit), cigar


@utils.trace_unhandled_exceptions
def parallelized_recode_soft_clipping(line_collection, pickle_file, soft_clip_base_quality, sequences_length,
                                      soft_clip_cigar_flag_recode):
    lines_sam = []
    for line in line_collection:
        line = line.rstrip('\r\n')
        if len(line) > 0:
            if line.startswith('@'):
                lines_sam.append(line)
            else:
                line = line.split('\t')
                if not verify_mapped_tip(sequences_length[line[2]], int(line[3]), line[5]):
                    line[5], line[3] = recode_cigar_based_on_base_quality(line[5], line[10], soft_clip_base_quality,
                                                                          int(line[3]),
                                                                          verify_mapped_direct_strand(int(line[1])),
                                                                          soft_clip_cigar_flag_recode)
                lines_sam.append('\t'.join(line))
    with open(pickle_file, 'wb') as writer:
        pickle.dump(lines_sam, writer)


def recode_soft_clipping_from_sam(sam_file, outdir, threads, soft_clip_base_quality, reference_dict,
                                  soft_clip_cigar_flag_recode):
    pickle_files = []
    sequences_length = {}
    for x, seq_info in list(reference_dict.items()):
        sequences_length[seq_info['header']] = seq_info['length']

    with open(sam_file, 'rtU') as reader:
        pool = multiprocessing.Pool(processes=threads)
        line_collection = []
        x = 0
        for x, line in enumerate(reader):
            line_collection.append(line)
            if x % 10000 == 0:
                pickle_file = os.path.join(outdir, 'remove_soft_clipping.' + str(x) + '.pkl')
                pickle_files.append(pickle_file)
                pool.apply_async(parallelized_recode_soft_clipping, args=(line_collection, pickle_file,
                                                                          soft_clip_base_quality, sequences_length,
                                                                          soft_clip_cigar_flag_recode,))
                line_collection = []
        if len(line_collection) > 0:
            pickle_file = os.path.join(outdir, 'remove_soft_clipping.' + str(x) + '.pkl')
            pickle_files.append(pickle_file)
            pool.apply_async(parallelized_recode_soft_clipping, args=(line_collection, pickle_file,
                                                                      soft_clip_base_quality, sequences_length,
                                                                      soft_clip_cigar_flag_recode,))
        pool.close()
        pool.join()

    os.remove(sam_file)

    new_sam_file = os.path.join(outdir, 'alignment_with_soft_clipping_recoded.sam')
    with open(new_sam_file, 'wt') as writer:
        for pickle_file in pickle_files:
            if os.path.isfile(pickle_file):
                lines_sam = None
                with open(pickle_file, 'rb') as reader:
                    lines_sam = pickle.load(reader)
                if lines_sam is not None:
                    for line in lines_sam:
                        writer.write(line + '\n')
                os.remove(pickle_file)

    return new_sam_file


# Sort alignment file
def sort_alignment(alignment_file, output_file, sort_by_name_true, threads):
    out_format_string = os.path.splitext(output_file)[1][1:].lower()
    command = ['samtools', 'sort', '-o', output_file, '-O', out_format_string, '', '-@', str(threads), alignment_file]
    if sort_by_name_true:
        command[6] = '-n'
    run_successfully, stdout, stderr = utils.run_command_popen_communicate(command, False, None, True)
    if not run_successfully:
        output_file = None
    return run_successfully, output_file


# Index alignment file
def index_alignment(alignment_file):
    command = ['samtools', 'index', alignment_file]
    run_successfully, stdout, stderr = utils.run_command_popen_communicate(command, False, None, True)
    return run_successfully


def mapping_reads(fastq_files, reference_file, threads, outdir, num_map_loc, rematch_run,
                  soft_clip_base_quality, soft_clip_recode_run, reference_dict, soft_clip_cigar_flag_recode,
                  bowtie_algorithm, bowtie_opt, clean_run=True):
    # Create a symbolic link to the reference_file
    if clean_run:
        reference_link = os.path.join(outdir, os.path.basename(reference_file))
        if os.path.islink(reference_link):
            os.unlink(reference_link)
        os.symlink(reference_file, reference_link)
        reference_file = reference_link

    bam_file = None
    # Mapping reads using Bowtie2
    run_successfully, sam_file = mapping_bowtie2(fastq_files=fastq_files, reference_file=reference_file,
                                                 threads=threads, outdir=outdir, num_map_loc=num_map_loc,
                                                 bowtie_algorithm=bowtie_algorithm, bowtie_opt=bowtie_opt)

    if run_successfully:
        # Remove soft clipping
        if rematch_run == soft_clip_recode_run or soft_clip_recode_run == 'both':
            print('Recoding soft clipped regions')
            sam_file = recode_soft_clipping_from_sam(sam_file, outdir, threads, soft_clip_base_quality, reference_dict,
                                                     soft_clip_cigar_flag_recode)

        # Convert sam to bam and sort bam
        run_successfully, bam_file = sort_alignment(sam_file, str(os.path.splitext(sam_file)[0] + '.bam'), False,
                                                    threads)

        if run_successfully:
            os.remove(sam_file)
            # Index bam
            run_successfully = index_alignment(bam_file)

    return run_successfully, bam_file, reference_file


def create_vcf(bam_file, sequence_to_analyse, outdir, counter, reference_file):
    gene_vcf = os.path.join(outdir, 'samtools_mpileup.sequence_' + str(counter) + '.vcf')

    command = ['samtools', 'mpileup', '--count-orphans', '--no-BAQ', '--min-BQ', '0', '--min-MQ', str(7), '--fasta-ref',
               reference_file, '--region', sequence_to_analyse, '--output', gene_vcf, '--VCF', '--uncompressed',
               '--output-tags', 'INFO/AD,AD,DP', bam_file]

    run_successfully, stdout, stderr = utils.run_command_popen_communicate(command, False, None, False)
    if not run_successfully:
        gene_vcf = None
    return run_successfully, gene_vcf


# Read vcf file
class Vcf:
    def __init__(self, vcf_file, encoding=None, newline=None):
        try:
            self.vcf = open(vcf_file, 'rt', encoding=encoding, newline=newline)
        except TypeError:
            self.vcf = open(vcf_file, 'rt')
        self.line_read = self.vcf.readline()
        self.contigs_info_dict = {}
        while self.line_read.startswith('#'):
            if self.line_read.startswith('##contig=<ID='):
                seq = self.line_read.split('=')[2].split(',')[0]
                seq_len = self.line_read.split('=')[3].split('>')[0]
                self.contigs_info_dict[seq] = int(seq_len)
            self.line_read = self.vcf.readline()
        self.line = self.line_read

    def readline(self):
        line_stored = self.line
        self.line = self.vcf.readline()
        return line_stored

    def close(self):
        self.vcf.close()

    def get_contig_legth(self, contig):
        return self.contigs_info_dict[contig]


def get_variants(gene_vcf, seq_name, encoding=None, newline=None):
    variants = {}

    vfc_file = Vcf(vcf_file=gene_vcf, encoding=encoding, newline=newline)
    line = vfc_file.readline()
    counter = 1
    while len(line) > 0:
        fields = line.rstrip('\r\n').split('\t')
        if len(fields) > 0:
            fields[1] = int(fields[1])

            info_field = {}
            try:
                for i in fields[7].split(';'):
                    i = i.split('=')
                    if len(i) > 1:
                        info_field[i[0]] = i[1]
                    else:
                        info_field[i[0]] = None
            except IndexError:
                if counter > vfc_file.get_contig_legth(contig=seq_name):
                    break
                else:
                    raise IndexError

            format_field = {}
            format_field_name = fields[8].split(':')
            format_data = fields[9].split(':')

            for i in range(0, len(format_data)):
                format_field[format_field_name[i]] = format_data[i].split(',')

            fields_to_store = {'REF': fields[3], 'ALT': fields[4].split(','), 'info': info_field,
                               'format': format_field}
            if fields[1] in variants:
                variants[fields[1]][len(variants[fields[1]])] = fields_to_store
            else:
                variants[fields[1]] = {0: fields_to_store}

        try:
            line = vfc_file.readline()
        except UnicodeDecodeError:
            if counter + 1 > vfc_file.get_contig_legth(contig=seq_name):
                break
            else:
                raise UnicodeDecodeError

        counter += 1
    vfc_file.close()

    return variants


def indel_entry(variant_position):
    entry_with_indel = []
    entry_with_snp = None
    for i in variant_position:
        keys = list(variant_position[i]['info'].keys())
        if 'INDEL' in keys:
            entry_with_indel.append(i)
        else:
            entry_with_snp = i

    return entry_with_indel, entry_with_snp


def get_alt_no_matter(variant_position, indel_true):
    dp = sum(map(int, variant_position['format']['AD']))
    index_alleles_sorted_position = sorted(zip(list(map(int, variant_position['format']['AD'])),
                                               list(range(0, len(variant_position['format']['AD'])))),
                                           reverse=True)
    index_dominant_allele = None
    if not indel_true:
        ad_idv = index_alleles_sorted_position[0][0]

        if len([x for x in index_alleles_sorted_position if x[0] == ad_idv]) > 1:
            index_alleles_sorted_position = sorted([x for x in index_alleles_sorted_position if x[0] == ad_idv])

        index_dominant_allele = index_alleles_sorted_position[0][1]
        if index_dominant_allele == 0:
            alt = '.'
        else:
            alt = variant_position['ALT'][index_dominant_allele - 1]

    else:
        ad_idv = int(variant_position['info']['IDV'])

        if float(ad_idv) / float(dp) >= 0.5:
            if len([x for x in index_alleles_sorted_position if x[0] == index_alleles_sorted_position[0][0]]) > 1:
                index_alleles_sorted_position = sorted([x for x in index_alleles_sorted_position if
                                                        x[0] == index_alleles_sorted_position[0][0]])

            index_dominant_allele = index_alleles_sorted_position[0][1]
            if index_dominant_allele == 0:
                alt = '.'
            else:
                alt = variant_position['ALT'][index_dominant_allele - 1]
        else:
            ad_idv = int(variant_position['format']['AD'][0])
            alt = '.'

    return alt, dp, ad_idv, index_dominant_allele


def count_number_diferences(ref, alt):
    number_diferences = 0

    if len(ref) != len(alt):
        number_diferences += 1

    for i in range(0, min(len(ref), len(alt))):
        if alt[i] != 'N' and ref[i] != alt[i]:
            number_diferences += 1

    return number_diferences


def get_alt_correct(variant_position, alt_no_matter, dp, ad_idv, index_dominant_allele, minimum_depth_presence,
                    minimum_depth_call, minimum_depth_frequency_dominant_allele):
    alt = None
    low_coverage = False
    multiple_alleles = False

    if dp >= minimum_depth_presence:
        if dp < minimum_depth_call:
            alt = 'N' * len(variant_position['REF'])
            low_coverage = True
        else:
            if ad_idv < minimum_depth_call:
                alt = 'N' * len(variant_position['REF'])
                low_coverage = True
                if float(ad_idv) / float(dp) < minimum_depth_frequency_dominant_allele:
                    multiple_alleles = True
            else:
                if float(ad_idv) / float(dp) < minimum_depth_frequency_dominant_allele:
                    alt = 'N' * len(variant_position['REF'])
                    if index_dominant_allele is not None:
                        variants_coverage = [int(variant_position['format']['AD'][i]) for i in
                                             range(0, len(variant_position['ALT']) + 1) if i != index_dominant_allele]
                        if sum(variants_coverage) > 0:
                            if float(max(variants_coverage)) / float(sum(variants_coverage)) > 0.5:
                                multiple_alleles = True
                            elif float(max(variants_coverage)) / float(sum(variants_coverage)) == 0.5 and \
                                    len(variants_coverage) > 2:
                                multiple_alleles = True
                    else:
                        multiple_alleles = True
                else:
                    alt = alt_no_matter
    else:
        low_coverage = True

    return alt, low_coverage, multiple_alleles


def get_alt_alignment(ref, alt):
    if alt is None:
        alt = 'N' * len(ref)
    else:
        if len(ref) != len(alt):
            if len(alt) < len(ref):
                if alt == '.':
                    alt = ref
                alt += 'N' * (len(ref) - len(alt))
            else:
                if alt[:len(ref)] == ref:
                    alt = '.'
                else:
                    alt = alt[:len(ref)]

    return alt


def get_indel_more_likely(variant_position, indels_entry):
    indel_coverage = {}
    for i in indels_entry:
        indel_coverage[i] = int(variant_position['info']['IDV'])
    return indel_coverage.index(str(max(indel_coverage.values())))


def determine_variant(variant_position, minimum_depth_presence, minimum_depth_call,
                      minimum_depth_frequency_dominant_allele, indel_true):
    alt_no_matter, dp, ad_idv, index_dominant_allele = get_alt_no_matter(variant_position, indel_true)

    alt_correct, low_coverage, multiple_alleles = get_alt_correct(variant_position, alt_no_matter, dp, ad_idv,
                                                                  index_dominant_allele, minimum_depth_presence,
                                                                  minimum_depth_call,
                                                                  minimum_depth_frequency_dominant_allele)

    alt_alignment = get_alt_alignment(variant_position['REF'], alt_correct)

    return variant_position['REF'], alt_correct, low_coverage, multiple_alleles, alt_no_matter, alt_alignment


def confirm_nucleotides_indel(ref, alt, variants, position_start_indel, minimum_depth_presence, minimum_depth_call,
                              minimum_depth_frequency_dominant_allele, alignment_true):
    alt = list(alt)

    for i in range(0, len(alt) - 1):
        if len(alt) < len(ref):
            new_position = position_start_indel + len(alt) - i - 1
            alt_position = len(alt) - i - 1
        else:
            if i + 1 > len(ref):
                break
            new_position = position_start_indel + 1 + i
            alt_position = 1 + i

        if alt[alt_position] != 'N':
            if new_position not in variants:
                if alignment_true:
                    alt[alt_position] = 'N'
                else:
                    alt = alt[: alt_position]
                    break

            entry_with_indel, entry_with_snp = indel_entry(variants[new_position])
            new_ref, alt_correct, low_coverage, multiple_alleles, alt_no_matter, alt_alignment = \
                determine_variant(variants[new_position][entry_with_snp], minimum_depth_presence, minimum_depth_call,
                                  minimum_depth_frequency_dominant_allele, False)
            if alt_no_matter != '.' and alt[alt_position] != alt_no_matter:
                alt[alt_position] = alt_no_matter

    return ''.join(alt)


def snp_indel(variants, position, minimum_depth_presence, minimum_depth_call, minimum_depth_frequency_dominant_allele):
    entry_with_indel, entry_with_snp = indel_entry(variants[position])

    if len(entry_with_indel) == 0:
        ref, alt_correct, low_coverage, multiple_alleles, alt_no_matter, alt_alignment = \
            determine_variant(variants[position][entry_with_snp], minimum_depth_presence, minimum_depth_call,
                              minimum_depth_frequency_dominant_allele, False)
    else:
        ref_snp, alt_correct_snp, low_coverage_snp, multiple_alleles_snp, alt_no_matter_snp, alt_alignment_snp = \
            determine_variant(variants[position][entry_with_snp], minimum_depth_presence, minimum_depth_call,
                              minimum_depth_frequency_dominant_allele, False)

        indel_more_likely = entry_with_indel[0]
        if len(entry_with_indel) > 1:
            indel_more_likely = get_indel_more_likely(variants[position], entry_with_indel)

        ref, alt_correct, low_coverage, multiple_alleles, alt_no_matter, alt_alignment = \
            determine_variant(variants[position][indel_more_likely], minimum_depth_presence, minimum_depth_call,
                              minimum_depth_frequency_dominant_allele, True)

        if alt_no_matter == '.':
            ref, alt_correct, low_coverage, multiple_alleles, alt_no_matter, alt_alignment = \
                ref_snp, alt_correct_snp, low_coverage_snp, multiple_alleles_snp, alt_no_matter_snp, alt_alignment_snp
        else:
            if alt_correct is None and alt_correct_snp is not None:
                alt_correct = alt_correct_snp
            elif alt_correct is not None and alt_correct_snp is not None:
                if alt_correct_snp != '.' and alt_correct[0] != alt_correct_snp:
                    alt_correct = alt_correct_snp + alt_correct[1:] if len(alt_correct) > 1 else alt_correct_snp
            if alt_no_matter_snp != '.' and alt_no_matter[0] != alt_no_matter_snp:
                alt_no_matter = alt_no_matter_snp + alt_no_matter[1:] if len(alt_no_matter) > 1 else alt_no_matter_snp
            if alt_alignment_snp != '.' and alt_alignment[0] != alt_alignment_snp:
                alt_alignment = alt_alignment_snp + alt_alignment[1:] if len(alt_alignment) > 1 else alt_alignment_snp

            # if alt_no_matter != '.':
            #     alt_no_matter = confirm_nucleotides_indel(ref, alt_no_matter, variants, position, minimum_depth_presence, minimum_depth_call, minimum_depth_frequency_dominant_allele, False)
            # if alt_correct is not None and alt_correct != '.':
            #     alt_correct = confirm_nucleotides_indel(ref, alt_correct, variants, position, minimum_depth_presence, minimum_depth_call, minimum_depth_frequency_dominant_allele, False)
            # if alt_alignment != '.':
            #     alt_alignment = confirm_nucleotides_indel(ref, alt_alignment, variants, position, minimum_depth_presence, minimum_depth_call, minimum_depth_frequency_dominant_allele, True)

    return ref, alt_correct, low_coverage, multiple_alleles, alt_no_matter, alt_alignment


def get_true_variants(variants, minimum_depth_presence, minimum_depth_call, minimum_depth_frequency_dominant_allele,
                      sequence):
    variants_correct = {}
    variants_no_matter = {}
    variants_alignment = {}

    correct_absent_positions = {}
    correct_last_absent_position = ''

    no_matter_absent_positions = {}
    no_matter_last_absent_position = ''

    multiple_alleles_found = []

    counter = 1
    while counter <= len(sequence):
        if counter in variants:
            no_matter_last_absent_position = ''

            ref, alt_correct, low_coverage, multiple_alleles, alt_no_matter, alt_alignment = \
                snp_indel(variants, counter, minimum_depth_presence, minimum_depth_call,
                          minimum_depth_frequency_dominant_allele)

            if alt_alignment != '.':
                variants_alignment[counter] = {'REF': ref, 'ALT': alt_alignment}

            if alt_no_matter != '.':
                variants_no_matter[counter] = {'REF': ref, 'ALT': alt_no_matter}

            if alt_correct is None:
                if counter - len(correct_last_absent_position) in correct_absent_positions:
                    correct_absent_positions[counter - len(correct_last_absent_position)]['REF'] += ref
                else:
                    correct_absent_positions[counter] = {'REF': ref, 'ALT': ''}
                correct_last_absent_position += ref
            else:
                if alt_correct != '.':
                    if len(alt_correct) < len(ref):
                        if len(alt_correct) == 1:
                            correct_absent_positions[counter + 1] = {'REF': ref[1:], 'ALT': ''}
                        else:
                            correct_absent_positions[counter + 1] = {'REF': ref[1:], 'ALT': alt_correct[1:]}

                        correct_last_absent_position = ref[1:]
                    else:
                        variants_correct[counter] = {'REF': ref, 'ALT': alt_correct}
                        correct_last_absent_position = ''
                else:
                    correct_last_absent_position = ''

            if multiple_alleles:
                multiple_alleles_found.append(counter)

            counter += len(ref)
        else:
            variants_alignment[counter] = {'REF': sequence[counter - 1], 'ALT': 'N'}

            if counter - len(correct_last_absent_position) in correct_absent_positions:
                correct_absent_positions[counter - len(correct_last_absent_position)]['REF'] += sequence[counter - 1]
            else:
                correct_absent_positions[counter] = {'REF': sequence[counter - 1], 'ALT': ''}
            correct_last_absent_position += sequence[counter - 1]

            if counter - len(no_matter_last_absent_position) in no_matter_absent_positions:
                no_matter_absent_positions[counter - len(no_matter_last_absent_position)]['REF'] += \
                    sequence[counter - 1]
            else:
                no_matter_absent_positions[counter] = {'REF': sequence[counter - 1], 'ALT': ''}
            no_matter_last_absent_position += sequence[counter - 1]

            counter += 1

    for position in correct_absent_positions:
        if position == 1:
            variants_correct[position] = {'REF': correct_absent_positions[position]['REF'], 'ALT': 'N'}
        else:
            if position - 1 not in variants_correct:
                variants_correct[position - 1] = \
                    {'REF': sequence[position - 2] + correct_absent_positions[position]['REF'],
                     'ALT': sequence[position - 2] + correct_absent_positions[position]['ALT']}
            else:
                variants_correct[position - 1] = \
                    {'REF': variants_correct[position - 1]['REF'] +
                            correct_absent_positions[position]['REF'][len(variants_correct[position - 1]['REF']) - 1:],
                     'ALT': variants_correct[position - 1]['ALT'] +
                            correct_absent_positions[position]['ALT'][len(variants_correct[position - 1]['ALT']) - 1 if
                                                                      len(variants_correct[position - 1]['ALT']) > 0
                                                                      else 0:]}

    for position in no_matter_absent_positions:
        if position == 1:
            variants_no_matter[position] = {'REF': no_matter_absent_positions[position]['REF'], 'ALT': 'N'}
        else:
            if position - 1 not in variants_no_matter:
                variants_no_matter[position - 1] = \
                    {'REF': sequence[position - 2] + no_matter_absent_positions[position]['REF'],
                     'ALT': sequence[position - 2] + no_matter_absent_positions[position]['ALT']}
            else:
                variants_no_matter[position - 1] = \
                    {'REF': variants_no_matter[position - 1]['REF'] +
                            no_matter_absent_positions[position]['REF'][len(variants_no_matter[position - 1]['REF']) -
                                                                        1:],
                     'ALT': variants_no_matter[position - 1]['ALT'] +
                            no_matter_absent_positions[position]['ALT'][len(variants_no_matter[position - 1]['ALT']) -
                                                                        1 if
                                                                        len(variants_no_matter[position - 1]['ALT']) > 0
                                                                        else 0:]}

    return variants_correct, variants_no_matter, variants_alignment, multiple_alleles_found


def clean_variant_in_extra_seq_left(variant_dict, position, length_extra_seq, multiple_alleles_found,
                                    number_multi_alleles):
    number_diferences = 0

    if position + len(variant_dict[position]['REF']) - 1 > length_extra_seq:
        if multiple_alleles_found is not None and position in multiple_alleles_found:
            number_multi_alleles += 1

        temp_variant = variant_dict[position]
        del variant_dict[position]
        variant_dict[length_extra_seq] = {}
        variant_dict[length_extra_seq]['REF'] = temp_variant['REF'][length_extra_seq - position:]
        variant_dict[length_extra_seq]['ALT'] = temp_variant['ALT'][length_extra_seq - position:] if \
            len(temp_variant['ALT']) > length_extra_seq - position else \
            temp_variant['REF'][length_extra_seq - position]
        number_diferences = count_number_diferences(variant_dict[length_extra_seq]['REF'],
                                                    variant_dict[length_extra_seq]['ALT'])
    else:
        del variant_dict[position]

    return variant_dict, number_multi_alleles, number_diferences


def clean_variant_in_extra_seq_rigth(variant_dict, position, sequence_length, length_extra_seq):
    if position + len(variant_dict[position]['REF']) - 1 > sequence_length - length_extra_seq:
        variant_dict[position]['REF'] = \
            variant_dict[position]['REF'][: - (position - (sequence_length - length_extra_seq)) + 1]
        variant_dict[position]['ALT'] = \
            variant_dict[position]['ALT'][: - (position - (sequence_length - length_extra_seq)) + 1] if \
                len(variant_dict[position]['ALT']) >= - (position - (sequence_length - length_extra_seq)) + 1 else \
                variant_dict[position]['ALT']

    number_diferences = count_number_diferences(variant_dict[position]['REF'], variant_dict[position]['ALT'])

    return variant_dict, number_diferences


def cleanning_variants_extra_seq(variants_correct, variants_no_matter, variants_alignment, multiple_alleles_found,
                                 length_extra_seq, sequence_length):
    number_multi_alleles = 0
    number_diferences = 0

    counter = 1
    while counter <= sequence_length:
        if counter <= length_extra_seq:
            if counter in variants_correct:
                variants_correct, number_multi_alleles, number_diferences = \
                    clean_variant_in_extra_seq_left(variants_correct, counter, length_extra_seq, multiple_alleles_found,
                                                    number_multi_alleles)
            if counter in variants_no_matter:
                variants_no_matter, ignore, ignore = \
                    clean_variant_in_extra_seq_left(variants_no_matter, counter, length_extra_seq, None, None)
            if counter in variants_alignment:
                variants_alignment, ignore, ignore = \
                    clean_variant_in_extra_seq_left(variants_alignment, counter, length_extra_seq, None, None)
        elif sequence_length - length_extra_seq >= counter > length_extra_seq:
            if counter in variants_correct:
                if counter in multiple_alleles_found:
                    number_multi_alleles += 1
                variants_correct, number_diferences_found = \
                    clean_variant_in_extra_seq_rigth(variants_correct, counter, sequence_length, length_extra_seq)
                number_diferences += number_diferences_found
            if counter in variants_no_matter:
                variants_no_matter, ignore = \
                    clean_variant_in_extra_seq_rigth(variants_no_matter, counter, sequence_length, length_extra_seq)
            if counter in variants_alignment:
                variants_alignment, ignore = \
                    clean_variant_in_extra_seq_rigth(variants_alignment, counter, sequence_length, length_extra_seq)
        else:
            if counter in variants_correct:
                del variants_correct[counter]
            if counter in variants_no_matter:
                del variants_no_matter[counter]
            if counter in variants_alignment:
                del variants_alignment[counter]

        counter += 1

    return variants_correct, variants_no_matter, variants_alignment, number_multi_alleles, number_diferences


def get_coverage(gene_coverage):
    coverage = {}

    with open(gene_coverage, 'rtU') as reader:
        for line in reader:
            line = line.rstrip('\r\n')
            if len(line) > 0:
                line = line.split('\t')
                coverage[int(line[1])] = int(line[2])

    return coverage


def get_coverage_report(coverage, sequence_length, minimum_depth_presence, minimum_depth_call, length_extra_seq):
    if len(coverage) == 0:
        return sequence_length - 2 * length_extra_seq, 100.0, 0.0

    count_absent = 0
    count_low_coverage = 0
    sum_coverage = 0

    counter = 1
    while counter <= sequence_length:
        if sequence_length - length_extra_seq >= counter > length_extra_seq:
            if coverage[counter] < minimum_depth_presence:
                count_absent += 1
            else:
                if coverage[counter] < minimum_depth_call:
                    count_low_coverage += 1
                sum_coverage += coverage[counter]
        counter += 1

    mean_coverage = 0
    percentage_low_coverage = 0
    if sequence_length - 2 * length_extra_seq - count_absent > 0:
        mean_coverage = float(sum_coverage) / float(sequence_length - 2 * length_extra_seq - count_absent)
        percentage_low_coverage = \
            float(count_low_coverage) / float(sequence_length - 2 * length_extra_seq - count_absent) * 100

    return count_absent, percentage_low_coverage, mean_coverage


# Get genome coverage data
def compute_genome_coverage_data(alignment_file, sequence_to_analyse, outdir, counter):
    genome_coverage_data_file = os.path.join(outdir, 'samtools_depth.sequence_' + str(counter) + '.tab')
    command = ['samtools', 'depth', '-a', '-q', '0', '-r', sequence_to_analyse, alignment_file, '>',
               genome_coverage_data_file]
    run_successfully, stdout, stderr = utils.run_command_popen_communicate(command, True, None, False)
    return run_successfully, genome_coverage_data_file


def write_variants_vcf(variants, outdir, sequence_to_analyse, sufix):
    vcf_file = os.path.join(outdir, str(sequence_to_analyse + '.' + sufix + '.vcf'))
    with open(vcf_file, 'wt') as writer:
        writer.write('##fileformat=VCFv4.2' + '\n')
        writer.write('#' + '\t'.join(['SEQUENCE', 'POSITION', 'ID_unused', 'REFERENCE_sequence', 'ALTERNATIVE_sequence',
                                      'QUALITY_unused', 'FILTER_unused', 'INFO_unused', 'FORMAT_unused']) + '\n')
        for i in sorted(variants.keys()):
            writer.write('\t'.join([sequence_to_analyse, str(i), '.', variants[i]['REF'], variants[i]['ALT'], '.', '.',
                                    '.', '.']) + '\n')

    compressed_vcf_file = vcf_file + '.gz'
    command = ['bcftools', 'convert', '-o', compressed_vcf_file, '-O', 'z', vcf_file]
    run_successfully, stdout, stderr = utils.run_command_popen_communicate(command, False, None, False)
    if run_successfully:
        command = ['bcftools', 'index', compressed_vcf_file]
        run_successfully, stdout, stderr = utils.run_command_popen_communicate(command, False, None, False)

    if not run_successfully:
        compressed_vcf_file = None

    return run_successfully, compressed_vcf_file


def parse_fasta_in_memory(fasta_memory):
    fasta_memory = fasta_memory.splitlines()
    sequence_dict = {}
    for line in fasta_memory:
        if len(line) > 0:
            if line.startswith('>'):
                sequence_dict = {'header': line[1:], 'sequence': ''}
            else:
                sequence_dict['sequence'] += line

    return sequence_dict


def compute_consensus_sequence(reference_file, sequence_to_analyse, compressed_vcf_file, outdir):
    sequence_dict = None

    gene_fasta = os.path.join(outdir, str(sequence_to_analyse + '.fasta'))

    run_successfully, stdout = index_fasta_samtools(reference_file, sequence_to_analyse, gene_fasta, False)
    if run_successfully:
        command = ['bcftools', 'norm', '-c', 's', '-f', gene_fasta, '-Ov',  compressed_vcf_file]
        run_successfully, stdout, stderr = utils.run_command_popen_communicate(command, False, None, False)    
        if run_successfully:
            command = ['bcftools', 'consensus', '-f', gene_fasta, compressed_vcf_file, '-H', '1']
            run_successfully, stdout, stderr = utils.run_command_popen_communicate(command, False, None, False)
            if run_successfully:
                sequence_dict = parse_fasta_in_memory(stdout)

    return run_successfully, sequence_dict


def create_sample_consensus_sequence(outdir, sequence_to_analyse, reference_file, variants, minimum_depth_presence,
                                     minimum_depth_call, minimum_depth_frequency_dominant_allele, sequence,
                                     length_extra_seq):
    variants_correct, variants_noMatter, variants_alignment, multiple_alleles_found = \
        get_true_variants(variants, minimum_depth_presence, minimum_depth_call, minimum_depth_frequency_dominant_allele,
                          sequence)

    variants_correct, variants_noMatter, variants_alignment, number_multi_alleles, number_diferences = \
        cleanning_variants_extra_seq(variants_correct, variants_noMatter, variants_alignment, multiple_alleles_found,
                                     length_extra_seq, len(sequence))

    run_successfully = False
    consensus = {'correct': {}, 'noMatter': {}, 'alignment': {}}
    for variant_type in ['variants_correct', 'variants_noMatter', 'variants_alignment']:
        run_successfully, compressed_vcf_file = \
            write_variants_vcf(eval(variant_type), outdir, sequence_to_analyse, variant_type.split('_', 1)[1])
        if run_successfully:
            run_successfully, sequence_dict = \
                compute_consensus_sequence(reference_file, sequence_to_analyse, compressed_vcf_file, outdir)
            if run_successfully:
                consensus[variant_type.split('_', 1)[1]] = \
                    {'header': sequence_dict['header'],
                     'sequence': sequence_dict['sequence'][length_extra_seq:len(sequence_dict['sequence']) -
                                                                            length_extra_seq]}

    return run_successfully, number_multi_alleles, consensus, number_diferences


@utils.trace_unhandled_exceptions
def analyse_sequence_data(bam_file, sequence_information, outdir, counter, reference_file, length_extra_seq,
                          minimum_depth_presence, minimum_depth_call, minimum_depth_frequency_dominant_allele):
    count_absent = None
    percentage_low_coverage = None
    mean_coverage = None
    number_diferences = 0
    number_multi_alleles = 0
    consensus_sequence = {'correct': {}, 'noMatter': {}, 'alignment': {}}

    # Create vcf file (for multiple alleles check)
    run_successfully, gene_vcf = create_vcf(bam_file, sequence_information['header'], outdir, counter, reference_file)
    if run_successfully:
        # Create coverage tab file
        run_successfully, gene_coverage = \
            compute_genome_coverage_data(bam_file, sequence_information['header'], outdir, counter)

        if run_successfully:
            try:
                variants = get_variants(gene_vcf=gene_vcf, seq_name=sequence_information['header'],
                                        encoding=sys.getdefaultencoding())
            except UnicodeDecodeError:
                try:
                    print('It was found an enconding error while parsing the following VCF, but lets try forcing it to'
                          ' "utf_8" encoding: {}'.format(gene_vcf))
                    variants = get_variants(gene_vcf=gene_vcf, seq_name=sequence_information['header'],
                                            encoding='utf_8')
                except UnicodeDecodeError:
                    print('It was found an enconding error while parsing the following VCF, but lets try forcing it to'
                          ' "latin_1" encoding: {}'.format(gene_vcf))
                    variants = get_variants(gene_vcf=gene_vcf, seq_name=sequence_information['header'],
                                            encoding='latin_1')

            coverage = get_coverage(gene_coverage)

            run_successfully, number_multi_alleles, consensus_sequence, number_diferences = \
                create_sample_consensus_sequence(outdir, sequence_information['header'], reference_file, variants,
                                                 minimum_depth_presence, minimum_depth_call,
                                                 minimum_depth_frequency_dominant_allele,
                                                 sequence_information['sequence'], length_extra_seq)

            try:
                count_absent, percentage_low_coverage, mean_coverage = \
                    get_coverage_report(coverage, sequence_information['length'], minimum_depth_presence,
                                        minimum_depth_call, length_extra_seq)
            except KeyError:
                print('ERROR: KeyError')
                print(sequence_information)
                raise KeyError

    utils.save_variable_to_pickle([run_successfully, counter, number_multi_alleles, count_absent,
                                   percentage_low_coverage, mean_coverage, consensus_sequence, number_diferences],
                                  outdir, str('coverage_info.' + str(counter)))


def clean_header(header):
    problematic_characters = ["|", " ", ",", ".", "(", ")", "'", "/", ":"]
    new_header = str(header)
    if any(x in header for x in problematic_characters):
            for x in problematic_characters:
                new_header = new_header.replace(x, '_')
    return header, new_header


def get_sequence_information(fasta_file, length_extra_seq):
    sequence_dict = {}
    headers = {}
    headers_changed = False

    with open(fasta_file, 'rtU') as reader:
        blank_line_found = False
        sequence_counter = 0
        temp_sequence_dict = {}
        for line in reader:
            line = line.rstrip('\r\n')
            if len(line) > 0:
                if not blank_line_found:
                    if line.startswith('>'):
                        if len(temp_sequence_dict) > 0:
                            if list(temp_sequence_dict.values())[0]['length'] - 2 * length_extra_seq > 0:
                                sequence_dict[list(temp_sequence_dict.keys())[0]] = list(temp_sequence_dict.values())[0]
                            else:
                                print('{header} sequence ignored due to'
                                      ' length = 0'.format(header=list(temp_sequence_dict.values())[0]['header']))
                                del headers[list(temp_sequence_dict.values())[0]['header']]
                            temp_sequence_dict = {}

                        original_header, new_header = clean_header(line[1:])
                        if new_header in headers:
                            sys.exit('Found duplicated sequence'
                                     ' headers: {original_header}'.format(original_header=original_header))

                        sequence_counter += 1
                        temp_sequence_dict[sequence_counter] = {'header': new_header, 'sequence': '', 'length': 0}
                        headers[new_header] = str(original_header)
                        if new_header != original_header:
                            headers_changed = True
                    else:
                        temp_sequence_dict[sequence_counter]['sequence'] += line.replace(' ', '')
                        temp_sequence_dict[sequence_counter]['length'] += len(line.replace(' ', ''))
                else:
                    sys.exit('It was found a blank line between the fasta file above line ' + line)
            else:
                blank_line_found = True

        if len(temp_sequence_dict) > 0:
            if list(temp_sequence_dict.values())[0]['length'] - 2 * length_extra_seq > 0:
                sequence_dict[list(temp_sequence_dict.keys())[0]] = list(temp_sequence_dict.values())[0]
            else:
                print('{header} sequence ignored due to'
                      ' length <= 0'.format(header=list(temp_sequence_dict.values())[0]['header']))
                del headers[list(temp_sequence_dict.values())[0]['header']]

    return sequence_dict, headers, headers_changed


def sequence_data(sample, reference_file, bam_file, outdir, threads, length_extra_seq, minimum_depth_presence,
                  minimum_depth_call, minimum_depth_frequency_dominant_allele, debug_mode_true, not_write_consensus):
    sequence_data_outdir = os.path.join(outdir, 'sequence_data', '')
    utils.remove_directory(sequence_data_outdir)
    os.mkdir(sequence_data_outdir)

    sequences, headers, headers_changed = get_sequence_information(reference_file, length_extra_seq)

    pool = multiprocessing.Pool(processes=threads)
    for sequence_counter in sequences:
        sequence_dir = os.path.join(sequence_data_outdir, str(sequence_counter), '')
        utils.remove_directory(sequence_dir)
        os.makedirs(sequence_dir)
        pool.apply_async(analyse_sequence_data, args=(bam_file, sequences[sequence_counter], sequence_dir,
                                                      sequence_counter, reference_file, length_extra_seq,
                                                      minimum_depth_presence, minimum_depth_call,
                                                      minimum_depth_frequency_dominant_allele,))
    pool.close()
    pool.join()

    run_successfully, sample_data, consensus_files, consensus_sequences = \
        gather_data_together(sample, sequence_data_outdir, sequences, outdir.rsplit('/', 2)[0], debug_mode_true,
                             length_extra_seq, not_write_consensus)

    return run_successfully, sample_data, consensus_files, consensus_sequences


def chunkstring(string, length):
    return (string[0 + i:length + i] for i in range(0, len(string), length))


def write_consensus(outdir, sample, consensus_sequence):
    consensus_files = {}
    for consensus_type in ['correct', 'noMatter', 'alignment']:
        consensus_files[consensus_type] = os.path.join(outdir, str(sample + '.' + consensus_type + '.fasta'))
        with open(consensus_files[consensus_type], 'at') as writer:
            writer.write('>' + consensus_sequence[consensus_type]['header'] + '\n')
            fasta_sequence_lines = chunkstring(consensus_sequence[consensus_type]['sequence'], 80)
            for line in fasta_sequence_lines:
                writer.write(line + '\n')
    return consensus_files


def gather_data_together(sample, data_directory, sequences_information, outdir, debug_mode_true, length_extra_seq,
                         not_write_consensus):
    run_successfully = True
    counter = 0
    sample_data = {}

    consensus_files = None
    consensus_sequences_together = {'correct': {}, 'noMatter': {}, 'alignment': {}}

    write_consensus_first_time = True

    genes_directories = [d for d in os.listdir(data_directory) if
                         not d.startswith('.') and
                         os.path.isdir(os.path.join(data_directory, d, ''))]
    for gene_dir in genes_directories:
        gene_dir_path = os.path.join(data_directory, gene_dir, '')

        files = [f for f in os.listdir(gene_dir_path) if
                 not f.startswith('.') and
                 os.path.isfile(os.path.join(gene_dir_path, f))]
        for file_found in files:
            if file_found.startswith('coverage_info.') and file_found.endswith('.pkl'):
                file_path = os.path.join(gene_dir_path, file_found)

                if run_successfully:
                    run_successfully, sequence_counter, multiple_alleles_found, count_absent, percentage_low_coverage, \
                        mean_coverage, consensus_sequence, \
                        number_diferences = utils.extract_variable_from_pickle(file_path)

                    if not not_write_consensus:
                        for consensus_type in consensus_sequence:
                            consensus_sequences_together[consensus_type][sequence_counter] = \
                                {'header': consensus_sequence[consensus_type]['header'],
                                 'sequence': consensus_sequence[consensus_type]['sequence']}

                        if write_consensus_first_time:
                            for consensus_type in ['correct', 'noMatter', 'alignment']:
                                file_to_remove = os.path.join(outdir, str(sample + '.' + consensus_type + '.fasta'))
                                if os.path.isfile(file_to_remove):
                                    os.remove(file_to_remove)
                            write_consensus_first_time = False
                        consensus_files = write_consensus(outdir, sample, consensus_sequence)

                    gene_identity = 0
                    if sequences_information[sequence_counter]['length'] - 2 * length_extra_seq - count_absent > 0:
                        gene_identity = 100 - \
                                        (float(number_diferences) /
                                         (sequences_information[sequence_counter]['length'] - 2 * length_extra_seq -
                                          count_absent)) * 100

                    sample_data[sequence_counter] = \
                        {'header': sequences_information[sequence_counter]['header'],
                         'gene_coverage': 100 - (float(count_absent) /
                                                 (sequences_information[sequence_counter]['length'] - 2 *
                                                  length_extra_seq)) * 100,
                         'gene_low_coverage': percentage_low_coverage,
                         'gene_number_positions_multiple_alleles': multiple_alleles_found,
                         'gene_mean_read_coverage': mean_coverage,
                         'gene_identity': gene_identity}
                    counter += 1

        if not debug_mode_true:
            utils.remove_directory(gene_dir_path)

    if counter != len(sequences_information):
        run_successfully = False

    return run_successfully, sample_data, consensus_files, consensus_sequences_together


rematch_timer = functools.partial(utils.timer, name='ReMatCh module')


@rematch_timer
def run_rematch_module(sample, fastq_files, reference_file, threads, outdir, length_extra_seq, minimum_depth_presence,
                       minimum_depth_call, minimum_depth_frequency_dominant_allele, minimum_gene_coverage,
                       debug_mode_true, num_map_loc, minimum_gene_identity, rematch_run,
                       soft_clip_base_quality, soft_clip_recode_run, reference_dict, soft_clip_cigar_flag_recode,
                       bowtie_algorithm, bowtie_opt, gene_list_reference, not_write_consensus, clean_run=True):
    rematch_folder = os.path.join(outdir, 'rematch_module', '')

    utils.remove_directory(rematch_folder)
    os.mkdir(rematch_folder)

    # Map reads
    run_successfully, bam_file, reference_file = mapping_reads(fastq_files=fastq_files, reference_file=reference_file,
                                                               threads=threads, outdir=rematch_folder,
                                                               num_map_loc=num_map_loc, rematch_run=rematch_run,
                                                               soft_clip_base_quality=soft_clip_base_quality,
                                                               soft_clip_recode_run=soft_clip_recode_run,
                                                               reference_dict=reference_dict,
                                                               soft_clip_cigar_flag_recode=soft_clip_cigar_flag_recode,
                                                               bowtie_algorithm=bowtie_algorithm, bowtie_opt=bowtie_opt,
                                                               clean_run=clean_run)
    if run_successfully:
        # Index reference file
        run_successfully, stdout = index_fasta_samtools(reference_file, None, None, True)
        if run_successfully:
            print('Analysing alignment data')
            run_successfully, sample_data, consensus_files, consensus_sequences = \
                sequence_data(sample, reference_file, bam_file, rematch_folder, threads, length_extra_seq,
                              minimum_depth_presence, minimum_depth_call, minimum_depth_frequency_dominant_allele,
                              debug_mode_true, not_write_consensus)

            if run_successfully:
                print('Writing report file')
                number_absent_genes = 0
                number_genes_multiple_alleles = 0
                mean_sample_coverage = 0
                with open(os.path.join(outdir, 'rematchModule_report.txt'), 'wt') as writer:
                    print('\n'.join(outdir, 'rematchModule_report.txt'))
                    writer.write('\t'.join(['#gene', 'percentage_gene_coverage', 'gene_mean_read_coverage',
                                            'percentage_gene_low_coverage', 'number_positions_multiple_alleles',
                                            'percentage_gene_identity']) + '\n')
                    for i in range(1, len(sample_data) + 1):
                        writer.write('\t'.join([gene_list_reference[sample_data[i]['header']],
                                                str(round(sample_data[i]['gene_coverage'], 2)),
                                                str(round(sample_data[i]['gene_mean_read_coverage'], 2)),
                                                str(round(sample_data[i]['gene_low_coverage'], 2)),
                                                str(sample_data[i]['gene_number_positions_multiple_alleles']),
                                                str(round(sample_data[i]['gene_identity'], 2))]) + '\n')

                        if sample_data[i]['gene_coverage'] < minimum_gene_coverage or \
                                sample_data[i]['gene_identity'] < minimum_gene_identity:
                            number_absent_genes += 1
                        else:
                            mean_sample_coverage += sample_data[i]['gene_mean_read_coverage']
                            if sample_data[i]['gene_number_positions_multiple_alleles'] > 0:
                                number_genes_multiple_alleles += 1

                    if len(sample_data) - number_absent_genes > 0:
                        mean_sample_coverage = \
                            float(mean_sample_coverage) / float(len(sample_data) - number_absent_genes)
                    else:
                        mean_sample_coverage = 0

                    writer.write('\n'.join(['#general',
                                            '>number_absent_genes', str(number_absent_genes),
                                            '>number_genes_multiple_alleles', str(number_genes_multiple_alleles),
                                            '>mean_sample_coverage', str(round(mean_sample_coverage, 2))]) + '\n')

                    print('\n'.join([str('number_absent_genes: ' + str(number_absent_genes)),
                                     str('number_genes_multiple_alleles: ' + str(number_genes_multiple_alleles)),
                                     str('mean_sample_coverage: ' + str(round(mean_sample_coverage, 2)))]))

    if not debug_mode_true:
        utils.remove_directory(rematch_folder)

    return run_successfully, sample_data if 'sample_data' in locals() else None, \
           {'number_absent_genes': number_absent_genes if 'number_absent_genes' in locals() else None,
            'number_genes_multiple_alleles': number_genes_multiple_alleles if
            'number_genes_multiple_alleles' in locals() else None,
            'mean_sample_coverage': round(mean_sample_coverage, 2) if 'mean_sample_coverage' in locals() else None}, \
           consensus_files if 'consensus_files' in locals() else None,\
           consensus_sequences if 'consensus_sequences' in locals() else None