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MetaShot_Master_script.py
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MetaShot_Master_script.py
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__author__ = 'Bruno Fosso' # type: str
__version__ = 1.1 # type: float
__manteiner__ = "Bruno Fosso" # type: str
__mail__ = "b.fosso@ibiom.cnr.it" # type: str
import getopt
import gzip
import os
import psutil
from shlex import split
import sys
from string import strip
import subprocess
try:
from pysam import Samfile
except ValueError:
sys.exit("pysam is not installed")
try:
from Bio.SeqIO.QualityIO import FastqGeneralIterator
except ValueError:
sys.exit("biopython is not installed")
script_info = dict(Description="""
This script performs the whole MetaShot computation
MetaShot Computation can be distinguished in two principal steps:
1) Pre-processing procedures: by applying FaQCs, it removes low-quality, low complexity and short reads (under 50 nt)
2) Taxonomic investigation
""", usage="""
MetaShot is designed to analyse Illumina Paired-End (PE) data.
\n
Script Options:
\t-m\tA textual file containing the R1 and R2 PE reads file names, tab separated [MANDATORY].
\t \tIf a sample has been splitted in more flowcell LINES, please insert in the file a line per PE reads
\t \tExample: The sample1 has been sequenced in 3 flowcell lines. The read file content will be the following:
\t \tsample1_L001_R1_1.fastq\tsample1_L001_R2_1.fastq
\t \tsample1_L002_R1_2.fastq\tsample1_L002_R2_2.fastq
\t \tsample1_L003_R1_3.fastq\tsample1_L003_R2_3.fastq
\t-p\tparameters files: a file containing all the information required for the MetaShot application [MANDATORY]
\t-e\texclude the host mapping
\t-h\tprint this help
\n
Example:
\tMetaShot_Master_script.py -p param_file.txt -m read_files -s DNA
""")
multiple_input_data = []
working_directory = os.getcwd()
parameters_file = ""
exclude_host = False
try:
opts, args = getopt.getopt(sys.argv[1:], "m:p:h:e")
except getopt.GetoptError, err:
print str(err)
print script_info["Description"]
sys.exit()
if len(opts) != 0:
for o, a in opts:
if o == "-m":
with open(a) as read_file:
for line in read_file:
s = map(strip, line.split())
print s
if len(s) == 2:
multiple_input_data.append(s)
if len(multiple_input_data) == 0:
print "ERROR: not correctly formatted input file"
print script_info["usage"]
sys.exit()
elif o == "-p":
parameters_file = a
elif o == "-e":
exclude_host = True
elif o == "-h":
print script_info["usage"]
sys.exit()
else:
print script_info["usage"]
sys.exit()
else:
print script_info["usage"]
sys.exit()
#######################
# function definition #
#######################
# DEPRECATED
# def pid_status(process_pid):
# if psutil.pid_exists(process_pid):
# status = psutil.Process(process_pid).status()
# else:
# status = "finished"
# return status
def pid_status(process_pid):
"""This function controls the status of a specific process
:type process_pid: int
"""
try:
status = psutil.Process(process_pid).status()
# raise psutil.NoSuchProcess(process_pid)
except psutil.NoSuchProcess:
status = "finished"
return status
def verify_input_parameters(p_file):
if p_file == "":
print "Error!!! The -p option has not been inserted."
print script_info["usage"]
sys.exit()
else:
if os.path.exists(p_file):
if os.stat(p_file)[6] == 0:
print "Error!!! The indicated parameter file is empty"
print script_info["usage"]
sys.exit()
else:
print "Error. The selected parameter file doesn't exist"
print script_info["usage"]
sys.exit()
def control_mapping_procedure(sam_file):
if os.path.exists(os.path.join(sam_file)) and os.stat(os.path.join(sam_file))[6] != 0:
try:
controllo_sam = Samfile(os.path.join(sam_file))
controllo_sam.close()
result_number = 1
except:
result_number = 0
else:
result_number = 0
return result_number
def verify_fastq(fastq1, fastq2):
count_1 = 0
if os.path.exists(fastq1) is False:
sys.exit('file %s is missing' % fastq1)
if os.path.exists(fastq2) is False:
sys.exit('file %s is missing' % fastq2)
if fastq1.endswith("gz"):
l1 = gzip.open(fastq1)
else:
l1 = open(fastq1)
for title, seq, qual in FastqGeneralIterator(l1): # type: (str, str, str)
count_1 += 1
l1.close()
count_2 = 0
if fastq2.endswith("gz"):
l2 = gzip.open(fastq2)
else:
l2 = open(fastq2)
for acc, sequence, quality in FastqGeneralIterator(l2): # type: (str, str, str)
count_2 += 1
l2.close()
if count_1 == count_2:
fastq_result = 1
else:
fastq_result = 0
return fastq_result
def file_dimension(path2file):
dim = os.stat(path2file)[6]
return dim
def parameter_file_paser(param_file):
params = {}
with open(param_file) as c:
for stringa in c:
if stringa.startswith("#") is False:
lista = map(strip, stringa.split(":"))
if lista[0] == "script_path":
params["script"] = lista[1]
if os.path.exists(lista[1]) is False:
sys.exit("The MetaShot path is inexistent")
if lista[0] == "reference_path":
params["reference"] = lista[1]
if os.path.exists(lista[1]) is False:
sys.exit("The reference path is inexistent")
if lista[0] == "bacterial_split_file":
params["bacterial_split"] = lista[1]
if lista[0] == "virus_bowtie_index":
params["virus"] = lista[1]
if lista[0] == "fungi_bowtie_index":
params["fungi"] = lista[1]
if lista[0] == "protist_bowtie_index":
params["protist"] = lista[1]
return params
#####################
# option definition #
#####################
verify_input_parameters(parameters_file)
parameters_data = parameter_file_paser(parameters_file)
script_path = parameters_data["script"]
print script_path
reference_path = parameters_data["reference"]
print reference_path
bacterial_split_file = os.path.join(reference_path, parameters_data["bacterial_split"])
print bacterial_split_file
if os.path.exists(bacterial_split_file) is False:
sys.exit("The bacterial split file is inexistent")
working_directory = os.getcwd()
###################################
# PRE-PROCESSING PROCEDURE #
###################################
# STDOUT and STDERR are redirected to the same file
# Removing PhiX contaminants
cleaned_multiple_input_data = []
data_processing_list = {}
std_out_file = open("phix_removal_log.out", "w")
index = 1
for data in multiple_input_data:
data_processing_list.setdefault(index, [])
R1 = data[0]
R2 = data[1]
process_iteration = 0
output_folder = os.path.join(working_directory, "phix_removal_" + str(index))
cmd = split("python %s -1 %s -2 %s -o %s -p %s" % (
os.path.join(script_path, "Phix_cleaner.py"), R1, R2, output_folder, reference_path))
# print cmd
p = subprocess.Popen(cmd, stdout=std_out_file, stderr=std_out_file)
data_processing_list[index].append(p.pid)
data_processing_list[index].append(output_folder)
data_processing_list[index].append(R1)
data_processing_list[index].append(R2)
data_processing_list[index].append(process_iteration)
index += 1
completed = set()
while len(completed) != len(data_processing_list):
for index in data_processing_list.keys():
if index not in completed:
# print split
proc_id = data_processing_list[index][0]
output_folder = data_processing_list[index][1]
process_iteration = data_processing_list[index][4]
try:
process_status = pid_status(proc_id)
except:
process_status = ""
if psutil.pid_exists(proc_id) is False or process_status.lower() in ["finished", "zombie"]:
phix_data_1 = os.path.join(output_folder, "R1_no_phix.fastq")
phix_data_2 = os.path.join(output_folder, "R2_no_phix.fastq")
result = verify_fastq(phix_data_1, phix_data_2)
if result == 0:
process_iteration += 1
R1 = data_processing_list[index][2]
R2 = data_processing_list[index][3]
if process_iteration < 5:
# print output_folder
del data_processing_list[index]
data_processing_list.setdefault(index, [])
cmd = split("python %s -1 %s -2 %s -o %s -p %s" % (
os.path.join(script_path, "Phix_cleaner.py"), R1, R2, output_folder, reference_path))
p = subprocess.Popen(cmd, stdout=std_out_file, stderr=std_out_file)
data_processing_list[index].append(p.pid)
data_processing_list[index].append(output_folder)
data_processing_list[index].append(R1)
data_processing_list[index].append(R2)
data_processing_list[index].append(process_iteration)
else:
sys.exit("the Phix cleaning procedure for %s and %s data failed after 5 attemps" % (R1, R2))
elif result >= 1:
cleaned_multiple_input_data.append([phix_data_1, phix_data_2])
completed.add(index)
std_out_file.close()
# low_quality, low-complexity removal
# STDOUT and STDERR are redirected to the same file
print "Cleaning data"
data_processing_list = {}
std_out_file = open("faqcs_stdout.out", "w")
index = 1
process_iteration = 0
for data in cleaned_multiple_input_data:
data_processing_list.setdefault(index, [])
R1 = data[0]
R2 = data[1]
output_folder = os.path.join(working_directory, "trimmed_data_" + str(index))
cmd = split("FaQCs -1 %s -2 %s -mode BWA_plus -q 25 -min_L 50 -n 2 -lc 0.70 -t 10 -d %s" % (
R1, R2, output_folder))
p = subprocess.Popen(cmd, stdout=std_out_file, stderr=std_out_file)
data_processing_list[index].append(p.pid)
data_processing_list[index].append(output_folder)
data_processing_list[index].append(R1)
data_processing_list[index].append(R2)
data_processing_list[index].append(process_iteration)
index += 1
completed = set()
while len(completed) != len(data_processing_list):
for index in data_processing_list.keys():
exec_folder = data_processing_list[index][1]
if index not in completed:
# print split
proc_id = data_processing_list[index][0]
exec_folder = data_processing_list[index][1]
try:
process_status = pid_status(proc_id)
except:
process_status = ""
if psutil.pid_exists(proc_id) is False or process_status.lower() in ["finished", "zombie"]:
trimmed_data_1 = os.path.join(exec_folder, "QC.1.trimmed.fastq")
trimmed_data_2 = os.path.join(exec_folder, "QC.2.trimmed.fastq")
result = verify_fastq(trimmed_data_1, trimmed_data_2)
if result == 0:
process_iteration += 1
R1 = data_processing_list[index][2]
R2 = data_processing_list[index][3]
if process_iteration < 5:
# print exec_folder
del data_processing_list[index]
data_processing_list.setdefault(index, [])
cmd = split("FaQCs -p %s %s -mode BWA_plus -q 25 -min_L 50 -n 2 -lc 0.70 -t 10 -d %s" % (
R1, R2, exec_folder))
p = subprocess.Popen(cmd, stdout=std_out_file, stderr=std_out_file)
data_processing_list[index].append(p.pid)
data_processing_list[index].append(exec_folder)
data_processing_list[index].append(R1)
data_processing_list[index].append(R2)
data_processing_list[index].append(process_iteration)
else:
sys.exit("the FaQCs cleaning procedure for %s and %s data failed after 5 attemps" % (R1, R2))
elif result >= 1:
completed.add(index)
std_out_file.close()
###################################
# FIND MICROBIAL CANDIDATES #
###################################
for i in range(len(multiple_input_data)):
i += 1
folder = os.path.join(working_directory, "trimmed_data_%i" % i)
os.chdir(folder)
tmp = open("read_list", "w")
tmp.write("QC.1.trimmed.fastq\tQC.2.trimmed.fastq\n")
tmp.close()
split_file = os.path.join(reference_path, "find_microbiome_index.tsv")
cmd = split("python %s -i read_list -r %s" % (os.path.join(script_path, "find_microbiome.py"), split_file))
p = subprocess.Popen(cmd)
p.wait()
os.chdir(working_directory)
###################################
# CLEANED READ-LIST FILE CREATION #
###################################
# Annotazione dei file fastq contenenti le read denoised in un nuovo file read list
with open("read_list_cleaned", "w") as tmp:
for i in range(len(multiple_input_data)):
i += 1
for line in open(os.path.join(working_directory, "trimmed_data_%i" % i, "read_list")):
cleaned_list_file = map(strip, line.split("\t"))
r1_dim = None
r2_dim = None
if os.path.exists(os.path.join(working_directory, "trimmed_data_%i" % i, cleaned_list_file[0])):
r1_dim = file_dimension(os.path.join(working_directory, "trimmed_data_%i" % i, cleaned_list_file[0]))
if os.path.exists(os.path.join(working_directory, "trimmed_data_%i" % i, cleaned_list_file[1])):
r2_dim = file_dimension(
os.path.join(working_directory, "trimmed_data_%i" % i, cleaned_list_file[1]))
if r1_dim is not None and r2_dim is not None and r1_dim != 0 and r2_dim != 0:
tmp.write(
"%s\t%s\n" % (os.path.join(working_directory, "trimmed_data_%i" % i, cleaned_list_file[0]),
os.path.join(working_directory, "trimmed_data_%i" % i, cleaned_list_file[1])))
if file_dimension("read_list_cleaned") == 0:
sys.exit("There are no trimmed data")
###################################
# MICROBIAL CANDIDATES READ LIST #
###################################
# Annotazione dei file fastq contenenti le read denoised in un nuovo file read list
with open("candidate_microbial_list", "w") as tmp:
for i in range(len(multiple_input_data)):
i += 1
trimmed = os.path.join(working_directory, "trimmed_data_%i" % i)
r1_dim = None
r2_dim = None
if os.path.exists(os.path.join(working_directory, "trimmed_data_%i" % i, "R1_micro_candidates.fastq")):
r1_dim = file_dimension(os.path.join(working_directory, "trimmed_data_%i" % i, "R1_micro_candidates.fastq"))
if os.path.exists(os.path.join(working_directory, "trimmed_data_%i" % i, "R2_micro_candidates.fastq")):
r2_dim = file_dimension(os.path.join(working_directory, "trimmed_data_%i" % i, "R2_micro_candidates.fastq"))
if r1_dim is not None and r2_dim is not None and r1_dim != 0 and r2_dim != 0:
tmp.write("%s\t%s\n" % (os.path.join(working_directory, "trimmed_data_%i" % i, "R1_micro_candidates.fastq"),
os.path.join(working_directory, "trimmed_data_%i" % i,
"R2_micro_candidates.fastq")))
if file_dimension("candidate_microbial_list") == 0:
sys.exit("in your sample there are not microbial candidates")
###############################
# MAPPING ON THE HUMAN GENOME #
###############################
# to map all the human sequence against the human genome, first load the star reference genome
if exclude_host is False:
cmd = split("STAR --genomeDir %s --genomeLoad LoadAndExit" % os.path.join(reference_path, "Homo_sapiens"))
p = subprocess.Popen(cmd)
p.wait()
data_processing_list = {}
for i in range(len(multiple_input_data)):
i += 1
folder = os.path.join(working_directory, "trimmed_data_%i" % i)
# print folder
data_processing_list.setdefault(i, [])
os.chdir(folder)
process_iteration = 0
mapper = split(
"python %s -s human -i read_list -g -r %s" % (os.path.join(script_path, "host_mapper.py"), reference_path))
p = subprocess.Popen(mapper)
# print p.pid
data_processing_list[i].append(p.pid)
data_processing_list[i].append(os.path.join(folder, "mapping_on_human", "human_dataAligned.out.sam"))
data_processing_list[i].append(process_iteration)
os.chdir(working_directory)
completed = set()
while len(completed) != len(data_processing_list):
for index in data_processing_list.keys():
if index not in completed:
# print split
proc_id = data_processing_list[index][0]
sam_output = data_processing_list[index][1]
process_status = pid_status(proc_id)
if psutil.pid_exists(proc_id) is False or process_status.lower() in ["finished", "zombie"]:
# print sam_output
result = control_mapping_procedure(sam_output)
# print result
if result == 0:
folder = os.path.join(working_directory, "trimmed_data_%i" % index)
print folder
os.chdir(folder)
del data_processing_list[index]
data_processing_list.setdefault(index, [])
mapper = split(
"python %s -s human -i read_list -g -r %s" % (
os.path.join(script_path, "host_mapper.py"), reference_path))
p = subprocess.Popen(mapper)
data_processing_list[index].append(p.pid)
data_processing_list[index].append(
os.path.join(folder, "mapping_on_human", "human_dataAligned.out.sam"))
os.chdir(working_directory)
elif result == 1:
completed.add(index)
print len(completed), len(data_processing_list)
cmd = split("STAR --genomeDir %s --genomeLoad Remove" % os.path.join(reference_path, "Homo_sapiens"))
p = subprocess.Popen(cmd)
p.wait()
human_folder = os.path.join(working_directory, "mapping_on_human")
if os.path.exists(human_folder) is False:
os.mkdir(human_folder)
with open(os.path.join(human_folder, "mapped_on_host.lst"), "w") as tmp:
for i in completed:
data = os.path.join(working_directory, "trimmed_data_%i" % i, "mapping_on_human", "mapped_on_host.lst")
with open(data) as a:
for line in a:
tmp.write(line)
else:
human_folder = os.path.join(working_directory, "mapping_on_human")
if os.path.exists(human_folder) is False:
os.mkdir(human_folder)
tmp = open(os.path.join(human_folder, "mapped_on_host.lst"), "w")
tmp.close()
###################################
# DIVISION MAPPING #
###################################
# procedure di mapping sulle divisioni
# Esecuzione sui batteri
bacterial_mapper = split(
"python %s -i candidate_microbial_list -p 30 -s %s -r %s" % (
os.path.join(script_path, "bacterial_division_anlyser.py"), script_path, bacterial_split_file))
p = subprocess.Popen(bacterial_mapper)
p.wait()
multiple_division_process = {}
for division in ["virus", "fungi", "protist"]:
bowtie2_index = os.path.join(reference_path, parameters_data[division])
print bowtie2_index
multiple_division_process.setdefault(division, [])
division_mapper = split(
"python %s -i candidate_microbial_list -d %s -b %s" % (
os.path.join(script_path, "Division_analyser.py"), division, bowtie2_index))
p = subprocess.Popen(division_mapper, stdout=subprocess.PIPE, stderr=subprocess.STDOUT)
# print p.pid
multiple_division_process[division].append(p.pid)
multiple_division_process[division].append(
os.path.join(working_directory, division, "mapped_on_%s_total.txt" % division))
completed = set()
while len(completed) != len(multiple_division_process):
for division in multiple_division_process.keys():
if division not in completed:
# print split
proc_id = multiple_division_process[division][0]
mapped_reads_file = multiple_division_process[division][1]
try:
process_status = pid_status(proc_id)
except:
process_status = ""
if psutil.pid_exists(proc_id) is False or process_status.lower() in ["finished", "zombie"]:
if os.path.exists(mapped_reads_file):
completed.add(division)
else:
del multiple_division_process[division]
division_mapper = split(
"python %s -i candidate_microbial_list -d %s -b %s" % (
os.path.join(script_path, "Division_analyser.py"), division, bowtie2_index))
p = subprocess.Popen(division_mapper, stdout=subprocess.PIPE, stderr=subprocess.STDOUT)
multiple_division_process.setdefault(division, [])
multiple_division_process[division].append(p.pid)
multiple_division_process[division].append(
os.path.join(working_directory, division, "mapped_on_%s_total.txt" % division))
###################################
# PREPARE RESULT FILES #
###################################
classifier = split(
"python %s -s %s -r %s" % (os.path.join(script_path, "new_division_classifier.py"), script_path, reference_path))
p = subprocess.Popen(classifier)
p.wait()
krona = split(
"python %s " % (os.path.join(script_path, "krona_data_creator.py")))
p = subprocess.Popen(krona)
p.wait()