growth_rate.py

__author__ = 'alipirani'

import sys
import os
import argparse
import errno
from datetime import datetime
import ConfigParser
from config_settings import ConfigSectionMap
from modules.check_subroutines import *
from modules.stages import *
from modules.bedtools import *
from modules.qualimap import *
if sys.version_info < (3, 2):
    import subprocess32 as sp
else:
    import subprocess as sp
from modules.logging_subprocess import *
from modules.log_modules import *
from modules.generate_PTR_dataframe import *
from argparse import RawTextHelpFormatter

# Command Line Argument Parsing
def parser():
    parser = argparse.ArgumentParser(description='GROWTH RATE ANALYSIS PIPELINE. \n\nThis pipeline calculates peak to through ratio(PTR; ratio of copy numbers at origin of replication to terminus) using sequencing read coverage.', formatter_class=RawTextHelpFormatter)
    required = parser.add_argument_group('Required arguments')
    optional = parser.add_argument_group('Optional arguments')
    required.add_argument('-type', action='store', dest="type", help='Type of Sequencing Reads: SE or PE', required=True)
    optional.add_argument('-config', action='store', dest="config", help='Path to Config file', required=False)
    required.add_argument('-PE1', action='store', dest="forward_raw", help='Path to paired-end file 1', required=True)
    optional.add_argument('-PE2', action='store', dest="reverse_raw", help='Path to paired-end file 2', required=False)
    required.add_argument('-steps', action='store', dest="steps", help='Growth-Rate-Analysis Steps in sequential order.\n\n'
                                                                       '1. "All": This option will run all the steps starting from: \n\n - cleaning reads using Trimmomatic, \n - Aligning reads using Bowtie, \n - Post-align modifications using samtools, \n - Bedtools to extract coverage across the genome and \n - PTR calculation from Bed coverage;\n\n'
                                                                       '2. "clean,align,post-align,coverage,ptr" : This option will also run all steps starting from: \n\n - cleaning reads using Trimmomatic, \n - Aligning reads using Bowtie, \n - Post-align modifications using samtools, \n - Bedtools to extract coverage across the genome and \n - PTR calculation from Bed coverage;\n\nIMPORTANT: The order is required to be sequential while using this option. \nAlso, while skipping any of the step, make sure you have results for previous steps already present in your output folder.\n\n')
    required.add_argument('-o', action='store', dest="output_folder", help='Output Path ending with output directory name to save the results', required=True)
    required.add_argument('-analysis', action='store', dest="analysis_name", help='Unique analysis name to save the results', required=True)
    required.add_argument('-index', action='store', dest="index", help='Reference Index Name. Change this argument in config file and mention the reference header name such as KP_NTUH_chr/KPNIH1/KPNIH32.', required=True)
    optional.add_argument('-adapter', action='store', dest="adapter", help='Path to adapter fasta file')
    optional.add_argument('-c', action='store', dest="croplength", help='Crop Length in case needed')
    #optional.add_argument('-f', action='store', dest="bam_input", help='Input Bam')
    return parser

# Main Pipeline
def pipeline(args, logger):

    keep_logging('\nSTART: Pipeline\n', 'START: Pipeline\n', logger, 'info')

    """ SANITATION CHECKS """
    # Check Subroutines for Arguments, Input FASTQ files, Reference Index

    keep_logging('START: Checking Dependencies...', 'Checking Dependencies', logger, 'info')

    # Reference Genome file name
    reference = ConfigSectionMap(args.index, Config)['ref_path'] + "/" + ConfigSectionMap(args.index, Config)['ref_name']
    keep_logging('Getting Reference Genome name from config file: {}'.format(reference), 'Getting Reference Genome name from config file: {}'.format(reference), logger, 'info')

    # # Check IF FASTQ files exists
    # if args.type != "PE" and args.type != "BAM":
    #     reverse_raw = "None"
    #     file_exists(args.forward_raw, args.forward_raw, reference)
    # elif args.type != "PE" and args.type != "SE":
    #     print "BAM type... Not Integrated... continue"
    # else:
    #     file_exists(args.forward_raw, args.reverse_raw, reference)

    # Check Java Version
    java_check()
    keep_logging('END: Checking Dependencies...\n', 'END: Checking Dependencies\n', logger, 'info')

    """ INDIVIDUAL SUBPROCESS FOR EACH PIPELINE STEPS"""
    ## 1. Pre-Processing Raw reads using Trimmomatic
    def clean():
        keep_logging('START: Pre-Processing Raw reads using Trimmomatic',
                     'START: Pre-Processing Raw reads using Trimmomatic', logger, 'info')
        if args.type == "PE":
            # print "skip trimming"
            trimmomatic(args.forward_raw, args.reverse_raw, args.output_folder, args.croplength, args.adapter, logger, Config)
        else:
            reverse_raw = "None"
            # print "skip trimming"
            trimmomatic(args.forward_raw, reverse_raw, args.output_folder, args.croplength, args.adapter, logger, Config)
        keep_logging('END: Pre-Processing Raw reads using Trimmomatic\n',
                     'END: Pre-Processing Raw reads using Trimmomatic\n', logger, 'info')

    ## 2. Stages: prepare readgroups and map reads to a reference genome using user-provided aligner as set in config file.
    def align_reads():
        keep_logging('START: Mapping Reads using BWA', 'START: Mapping Reads using BWA', logger, 'info')
        split_field = prepare_readgroup(args.forward_raw, ConfigSectionMap("pipeline", Config)['aligner'], logger)
        out_sam = align(args.output_folder, args.index, split_field, args.analysis_name, files_to_delete, logger, Config, args.type)
        keep_logging('END: Mapping Reads using BWA', 'END: Mapping Reads using BWA', logger, 'info')
        return out_sam

    ## 3. Stages: Post-Alignment using SAMTOOLS, PICARD etc
    def post_align(out_sam):
        keep_logging('START: Post-Alignment using SAMTOOLS, PICARD etc...',
                     'START: Post-Alignment using SAMTOOLS, PICARD etc...', logger, 'info')
        out_sorted_bam = prepare_bam(out_sam, args.output_folder, args.analysis_name, files_to_delete, logger, Config)
        keep_logging('END: Post-Alignment using SAMTOOLS, PICARD etc...',
                     'END: Post-Alignment using SAMTOOLS, PICARD etc...', logger, 'info')
        # out_sorted_bam = "%s/%s_aln_sort.bam" % (args.output_folder, args.analysis_name)
        return out_sorted_bam

    ## 4. Stages: Generate Bedgrapg Coverage File for PTR calculation
    def bedgraph(out_sorted_bam):
        final_coverage_file = bedtools(out_sorted_bam, args.output_folder, args.analysis_name, logger, Config)
        #final_coverage_file = "%s/%s_coverage.bed" % (args.output_folder, args.analysis_name)
        return final_coverage_file

    ## 5. Stages: PTR Analysis
    def ptr(final_coverage_file):
        keep_logging('START: Analyzing Bedfiles for PTR analysis', 'START: Analyzing Bedfiles for PTR analysis', logger,
                     'info')
        generate_PTR_dataframe(final_coverage_file, args.output_folder, args.analysis_name, logger, Config)
        keep_logging('END: Analyzing Bedfiles for PTR analysis\n', 'END: Analyzing Bedfiles for PTR analysis\n', logger,
                     'info')

    ## 6. Stages: Statistics
    def stats(out_sorted_bam):
        keep_logging('START: Generating Statistics Reports', 'START: Generating Statistics Reports', logger, 'info')
        alignment_stats_file = alignment_stats(out_sorted_bam, args.output_folder, args.analysis_name, logger, Config)
        # gatk_DepthOfCoverage(out_sorted_bam, args.output_folder, args.analysis_name, reference, logger, Config)
        # final_coverage_file = "%s/%s_coverage.bed" % (args.output_folder, args.analysis_name)
        # keep_logging('END: Generating Statistics Reports\n', 'END: Generating Statistics Reports\n', logger, 'info')
        # final_coverage_file = "%s/%s_coverage.bed" % (args.output_folder, args.analysis_name)

    """ Start the pipeline: """
    # Split comma-seperated values provided with -steps argument and decide the starting point of pipeline.
    steps_list = args.steps.split(',')

    # Decide the starting point of pipeline and run accordingly.
    if len(steps_list) == 1:
        if steps_list[0] == "All":
            clean()
            out_sam = align_reads()
            out_sorted_bam = post_align(out_sam)
            final_coverage_file = bedgraph(out_sorted_bam)
            stats(out_sorted_bam)
            ptr(final_coverage_file)

        if steps_list[0] == "ptr":
            final_coverage_file = "%s/%s_coverage.bed" % (args.output_folder, args.analysis_name)
            out_sorted_bam = "%s/%s_aln_sort.bam" % (args.output_folder, args.analysis_name)
            final_coverage_file = "%s/%s_coverage.bed" % (args.output_folder, args.analysis_name)
            stats(out_sorted_bam)
            ptr(final_coverage_file)

    # Run individual pipeline steps based on the first value found in steps_list array: clean, align, post-align, bedgraph, ptr, stats etc
    else:

        if steps_list[0] == "clean":
            clean()
            out_sam = align_reads()
            out_sorted_bam = post_align(out_sam)
            final_coverage_file = bedgraph(out_sorted_bam)
            stats(out_sorted_bam)
            ptr(final_coverage_file)


        elif steps_list[0] == "align":
            out_sam = align_reads()
            out_sorted_bam = post_align(out_sam)
            final_coverage_file = bedgraph(out_sorted_bam)
            stats(out_sorted_bam)
            ptr(final_coverage_file)

        elif steps_list[0] == "post-align":
            out_sam = "%s/%s_aln.sam" % (args.output_folder, args.analysis_name)
            out_sorted_bam = post_align(out_sam)
            final_coverage_file = bedgraph(out_sorted_bam)
            stats(out_sorted_bam)
            ptr(final_coverage_file)


        elif steps_list[0] == "bedgraph":
            out_sorted_bam = "%s/%s_aln_sort.bam" % (args.output_folder, args.analysis_name)
            final_coverage_file = bedgraph(out_sorted_bam)
            stats(out_sorted_bam)
            ptr(final_coverage_file)


        elif steps_list[0] == "ptr":
            final_coverage_file = "%s/%s_coverage.bed" % (args.output_folder, args.analysis_name)
            out_sorted_bam = "%s/%s_aln_sort.bam" % (args.output_folder, args.analysis_name)
            stats(out_sorted_bam)
            ptr(final_coverage_file)


        elif steps_list[0] == "stats":
            out_sorted_bam = "%s/%s_aln_sort.bam" % (args.output_folder, args.analysis_name)
            stats(out_sorted_bam)
        else:
            keep_logging(
                'Seems like the Analysis Steps are not in sequential order. Please recheck the -steps argument and run the pipeline again',
                'Seems like the Analysis Steps are not in sequential order. Please recheck the -steps argument and run the pipeline again',
                logger, 'exception')
## Check Subroutines
def usage():
    print "Usage: python pipeline.py [-h] -PE1 path-to-forward-PE-read -PE2 path-to-reverse-PE-read -o path-to-OUTPUT_FOLDER -analysis ANALYSIS_NAME -index INDEX_NAME_as_per_config_file \n"

# Validate Filenames for any unsupported characters
def Validate_filename( name ):
    pattern_strings = ['\.', '\&', '\>', 'aaa', '\*']
    pattern_string = '|'.join(pattern_strings)
    searchobj = re.search(pattern_string, name, flags=0)
    if searchobj:
        print "The file " + name + " contains unsupported characters such as quotes, spaces, or &:%?*><\$. \nPlease Provide another file name.\n"
        exit()

def file_exists(path1, path2, reference):

    if not os.path.isfile(path1):
        file_basename = os.path.basename(path1)
        keep_logging('The input file {} does not exists. Please provide another file or check the files path.\n'.format(file_basename), 'The input file {} does not exists. Please provide another file or check the files path.\n'.format(file_basename), logger, 'exception')
        exit()
    if path2 is not None:
        if not os.path.isfile(path2):
            file_basename = os.path.basename(path2)
            keep_logging('The input file {} does not exists. Please provide another file or check the files path.\n'.format(file_basename), 'The input file {} does not exists. Please provide another file or check the files path.\n'.format(file_basename), logger, 'exception')
            exit()
    if not os.path.isfile(reference):
        file_basename = os.path.basename(reference)
        keep_logging('The reference fasta file {} does not exists. Please provide another file or check the files path.\n'.format(file_basename), 'The reference fasta file {} does not exists. Please provide another file or check the files path.\n'.format(file_basename), logger, 'exception')
        exit()
    if ConfigSectionMap("pipeline", Config)['aligner'] == "bwa":
        ref_index_suffix1 = reference + ".bwt"
        ref_index_suffix2 = reference + ".amb"
        ref_index_suffix3 = reference + ".ann"
        ref_index_suffix4 = reference + ".sa"
        ref_index_suffix5 = reference + ".pac"
    elif ConfigSectionMap("pipeline", Config)['aligner'] == "bowtie":
        ref_index_suffix1 = reference + ".1.bt2"
        ref_index_suffix2 = reference + ".2.bt2"
        ref_index_suffix3 = reference + ".3.bt2"
        ref_index_suffix4 = reference + ".4.ebwt"
        ref_index_suffix5 = reference + ".rev.1.bt2"
        ref_index_suffix6 = reference + ".rev.2.bt2"
    else:
        print "Please change the aligner section in config file."

        print "Different Aligner in config file"

    if not os.path.isfile(ref_index_suffix1):
        # file_basename = os.path.basename(reference)
        keep_logging('The reference index files given below does not exists:\n {}\n {}\n {}\n {}\n {}'.format(ref_index_suffix1, ref_index_suffix2, ref_index_suffix3, ref_index_suffix4, ref_index_suffix5), 'The reference index files given below does not exists:\n {}\n {}\n {}\n {}\n {}'.format(ref_index_suffix1, ref_index_suffix2, ref_index_suffix3, ref_index_suffix4, ref_index_suffix5), logger, 'warning')
        create_index(reference, ref_index_suffix1, ref_index_suffix2, ref_index_suffix3, ref_index_suffix4, ref_index_suffix5)
    else:
        keep_logging('Index file already exists.', 'Index file already exists.', logger, 'info')

    ref_fai_index = reference + ".fai"
    if not os.path.isfile(ref_fai_index):
        # file_basename = os.path.basename(reference)
        keep_logging('The reference fai index file {} required for samtools does not exists.'.format(ref_fai_index), 'The reference fai index file {} required for samtools does not exists.'.format(ref_fai_index), logger, 'warning')
        create_fai_index(reference, ref_fai_index)
    else:
        keep_logging('Samtools fai Index file already exists.', 'Samtools fai Index file already exists.', logger, 'info')

    dict_name = os.path.splitext(os.path.basename(reference))[0] + ".dict"
    if not os.path.isfile(ConfigSectionMap(args.index, Config)['ref_path'] + "/" + dict_name):
        keep_logging('The reference seq dict file {} required for GATK and PICARD does not exists.'.format(dict_name), 'The reference seq dict file {} required for GATK and PICARD does not exists.'.format(dict_name), logger, 'warning')
        picard_seqdict(dict_name, reference)
    else:
        keep_logging('The reference seq dict file required for GATK and PICARD exists.', 'The reference seq dict file required for GATK and PICARD exists.', logger, 'info')

def java_check():
    keep_logging('Checking Java Availability...', 'Checking Java Availability...', logger, 'info')
    jd = sp.check_output(["java", "-version"], stderr=sp.STDOUT)
    jd_version = jd.split('\n', 1)[0]
    if len(jd) < 1:
        keep_logging('Unable to find a java runtime environment. The pipeline requires java 6 or later.', 'Unable to find a java runtime environment. The pipeline requires java 6 or later.', logger, 'exception')
    else:
        keep_logging('Java Availability Check completed ...{}'.format(jd_version), 'Java Availability Check completed ...{}'.format(jd_version), logger, 'info')

def fileformat(file1, file2, final_out):
    print "Checking File format....\n"
    if not file1.endswith('.fastq.gz'):
        base = os.path.basename(file1)
        os.path.splitext(base)
        file_1 = os.path.splitext(base)[0]
        cmdstring = "gzip -d " + file1 + " > " + final_out + file_1
        print "Compressing input file " + base
        os.system(cmdstring)


    if not file2.endswith('.fastq.gz'):
        base = os.path.basename(file2)
        os.path.splitext(base)
        file_2 = os.path.splitext(base)[0]
        cmdstring = "gzip -d " + file2 + " > " + final_out + file_2
        print "Compressing input file " + base
        os.system(cmdstring)

def make_sure_path_exists(out_path):
    try:
        os.makedirs(out_path)
    except OSError as exception:
        if exception.errno != errno.EEXIST:
            keep_logging('Errors in output folder path! please change the output path or analysis name.', 'Errors in output folder path! please change the output path or analysis name', logger, 'exception')
            exit()

def create_index(reference,ref_index_suffix1, ref_index_suffix2, ref_index_suffix3, ref_index_suffix4, ref_index_suffix5):
    aligner = ConfigSectionMap("pipeline", Config)['aligner']
    keep_logging('Creating Index of reference fasta file for {} aligner.'.format(aligner), 'Creating Index of reference fasta file for {} aligner'.format(aligner), logger, 'info')
    if aligner == "bwa":
        cmd = "%s %s %s" % (ConfigSectionMap("bwa", Config)['base_cmd'], ConfigSectionMap("bwa", Config)['index'], reference)
        keep_logging(cmd, cmd, logger, 'debug')
        try:
            call(cmd, logger)
        except sp.CalledProcessError:
                keep_logging('Error in {} Indexer. Exiting.'.format(aligner), 'Error in {} Indexer. Exiting.'.format(aligner), logger, 'exception')
                sys.exit(1)
        if not os.path.isfile(ref_index_suffix1):
            keep_logging('The {} reference index files were not created properly. Please try to create the index files again or manually.'.format(aligner), 'The {} reference index files were not created properly. Please try to create the index files again or manually.'.format(aligner), logger, 'exception')
    elif aligner == "bowtie":
        cmd = "%s/%s/%s %s %s" % (ConfigSectionMap("bin_path", Config)['binbase'], ConfigSectionMap("bowtie", Config)['bowtie_bin'], ConfigSectionMap("bowtie", Config)['build_cmd'], reference, reference)
        keep_logging(cmd, cmd, logger, 'debug')
        try:
            call(cmd, logger)
        except sp.CalledProcessError:
                keep_logging('Error in {} Indexer. Exiting.'.format(aligner), 'Error in {} Indexer. Exiting.'.format(aligner), logger, 'exception')
                sys.exit(1)
        if not os.path.isfile(ref_index_suffix1):
            keep_logging('The {} reference index files were not created properly. Please try to create the index files again or manually.'.format(aligner), 'The {} reference index files were not created properly. Please try to create the index files again or manually.'.format(aligner), logger, 'exception')

    else:
        print "Different Aligner in config file"

def create_fai_index(reference, ref_fai_index):
    keep_logging('Creating FAI Index using Samtools.', 'Creating FAI Index using Samtools.', logger, 'info')
    cmd = "%s %s %s" % (ConfigSectionMap("samtools", Config)['base_cmd'], ConfigSectionMap("samtools", Config)['faiindex'], reference)
    keep_logging(cmd, cmd, logger, 'debug')
    try:
        call(cmd, logger)
    except sp.CalledProcessError:
        keep_logging('Error in Samtools FAI Indexing step. Exiting.', 'Error in Samtools FAI Indexing step. Exiting.', logger, 'exception')
        sys.exit(1)


    if not os.path.isfile(ref_fai_index):
        keep_logging('The reference fai index file {} was not created properly.\n Please try to create the samtools fai index files manually. \n'.format(ref_fai_index), 'The reference fai index file {} was not created properly.\n Please try to create the samtools fai index files manually. \n'.format(ref_fai_index), logger, 'exception')
    else:
        keep_logging('Samtools Fai Index file created.', 'Samtools Fai Index file created.', logger, 'info')

def picard_seqdict(dict_name, reference):
    #dict_name = os.path.splitext(os.path.basename(reference_filename))[0] + ".dict"
    keep_logging('Creating Sequence Dictionary using Picard.', 'Creating Sequence Dictionary using Picard.', logger, 'info')
    cmd = "java -jar %s/%s/%s CreateSequenceDictionary REFERENCE=%s OUTPUT=%s/%s" % (ConfigSectionMap("bin_path", Config)['binbase'], ConfigSectionMap("picard", Config)['picard_bin'], ConfigSectionMap("picard", Config)['base_cmd'], reference, ConfigSectionMap(args.index, Config)['ref_path'], dict_name)
    keep_logging(cmd, cmd, logger, 'debug')
    try:
        call(cmd, logger)
    except sp.CalledProcessError:
        keep_logging('Error in Picard Sequence Dictionary creation step. Exiting.', 'Error in Picard Sequence Dictionary creation step. Exiting.', logger, 'exception')
        sys.exit(1)

""" Start of Main Method/Pipeline """
if __name__ == '__main__':

    # Set up logging modules and config file
    start_time = datetime.now().strftime('%Y-%m-%d %H:%M:%S')
    start_time_2 = datetime.now()

    args = parser().parse_args()

    global config_file
    global log_unique_time
    global logger
    global files_to_delete
    files_to_delete = []

    if args.config:
        config_file = args.config
    else:
        config_file = os.path.dirname(os.path.abspath(__file__)) + "/config"


    if args.output_folder != '':
        args.output_folder += '/'

    make_sure_path_exists(args.output_folder)

    log_unique_time = datetime.now().strftime('%Y_%m_%d_%H_%M_%S')

    logger = generate_logger(args.output_folder, args.analysis_name, log_unique_time)

    global Config
    Config = ConfigParser.ConfigParser()
    Config.read(config_file)

    keep_logging('START: Pipeline', 'START: Pipeline', logger, 'info')

    pipeline(args, logger)

    keep_logging('END: Pipeline', 'End: Pipeline', logger, 'info')
    time_taken = datetime.now() - start_time_2
    keep_logging('Total Time taken: {}'.format(time_taken), 'Total Time taken: {}'.format(time_taken), logger, 'info')