def main(config_file):
#Greeting
now_time('Beginning EndClip Prep run (v0.1.0)')
print("-"*50)
#Parse configure file
now_time("Parsing configure file...")
config_dict = Input_config_file(config_file)
#Check configure file
if not 'gene_gtf_file' in config_dict:
sys.exit("ERROR: Gene GTF file does not exist...")
if not 'gene_bed_file' in config_dict:
sys.exit("ERROR: Gene BED file does not exist...")
if not 'pA_site_file' in config_dict:
sys.exit("ERROR: pA site file does not exist...")
if not 'output_kfXref_file' in config_dict:
sys.exit("ERROR: output_kfXref_file was not designated...")
if not 'output_utr_file' in config_dict:
sys.exit("ERROR: output_utr_file does not exist...")
if not 'output_initial_UTR_database_file' in config_dict:
sys.exit("ERROR: output_initial_UTR_database_file was not designated...")
#Extract information
gene_gtf_file = config_dict['gene_gtf_file']
gene_bed_file = config_dict['gene_bed_file']
pA_site_file = config_dict['pA_site_file']
output_kfXref_file = config_dict['output_kfXref_file']
output_utr_file = config_dict['output_utr_file']
output_initial_UTR_database_file = config_dict['output_initial_UTR_database_file']
#Prepare symbol_refid_map_file(kfXref file)
'''
now_time("Prepare kfXref file...")
symbol_refid_map = Extract_gene_symbol_map_kfXref_file(gene_gtf_file)
map_file = open(output_kfXref_file, 'w')
for refid in symbol_refid_map.keys():
symbol = symbol_refid_map[refid]
print(refid, symbol, sep="\t", end="\n", file=map_file)
'''
#Make initial 3'UTR database
now_time("Prepare initial 3'UTR database...")
test = Extract_3UTR_from_bed(gene_bed_file, output_kfXref_file, output_utr_file)
##Prepare 3UTR database with pA site information
#now_time("Prepare 3'UTR database with pA site information...")
#temp_file = output_initial_UTR_database_file + '.tmp'
#cmd = 'bedtools intersect -a %s -b %s -wa -wb > %s' % (output_utr_file, pA_site_file, temp_file)
#os.system(cmd)
#Merge_pA_site_infor(temp_file, output_initial_UTR_database_file)
now_time("Completely finished!!")
def main():
'''Input_configure_file
Annotated_3UTR=data/hg19_refseq_extracted_3UTR_PTEN_ELAVL1.bed
PolyA_site_infor=data/polyA_DB_hg19.bed
Group1_Tophat_aligned_Wig=data/siCTRL_S_accepted_hits_PTEN_ELAVL1.bam.wig
Group2_Tophat_aligned_Wig=data/siCFIm25-1_accepted_hits_PTEN_ELAVL1.bam.wig
Output_directory=DaPars_Test_data/
Output_result_file=DaPars_Test_data
#Parameters
Num_least_in_group1=1
Num_least_in_group2=1
Coverage_cutoff=30
FDR_cutoff=0.05
PDUI_cutoff=0.5
Fold_change_cutoff=0.59
'''
start_time = time.time()
now_time("Beginning EndSnip run (v0.1.0)")
print("-"*50)
if len(sys.argv) == 1:
sys.exit("ERROR: Please provide the configure file...")
cfg_file = sys.argv[1]
now_time("Parsing configure file...")
config_dict = Input_config_file(cfg_file)
#Check configure file
if not 'Group1_Tophat_aligned_Wig' in config_dict:
sys.exit("ERROR: No Tophat aligned BAM file for group 1...")
if not 'Group2_Tophat_aligned_Wig' in config_dict:
sys.exit("ERROR: No Tophat aligned BAM file for group 2...")
if not 'Output_directory' in config_dict:
sys.exit("ERROR: No output directory...")
if not 'Annotated_3UTR' in config_dict:
sys.exit("ERROR: No annotated 3'UTR file...")
if not 'Output_result_file' in config_dict:
sys.exit("ERROR: No result file name...")
#File/Directory
Group1_Tophat_aligned_file = config_dict['Group1_Tophat_aligned_Wig'].split(',')
Group2_Tophat_aligned_file = config_dict['Group2_Tophat_aligned_Wig'].split(',')
output_directory = config_dict['Output_directory']
if output_directory[-1] != '/':
output_directory += '/'
Annotated_3UTR_file = config_dict['Annotated_3UTR']
Output_result_file = config_dict['Output_result_file']
#Default parameters
global Num_least_in_group1
global Num_least_in_group2
global Coverage_cutoff
global FDR_cutoff
global Fold_change_cutoff
global PDUI_cutoff
global Coverage_pPAS_cutoff
Num_least_in_group1 = 1
Num_least_in_group2 = 1
Coverage_cutoff = 30
FDR_cutoff = 0.05
Fold_change_cutoff = 0.59 #1.5-fold change
PDUI_cutoff = 0.2
Coverage_pPAS_cutoff = 5.0
#Check parameters
if not 'Num_least_in_group1' in config_dict:
print(" Num_least_in_group1: Default parameter(1) was designated.")
else:
Num_least_in_group1 = float(config_dict['Num_least_in_group1'])
if not 'Num_least_in_group2' in config_dict:
print(" Num_least_in_group2: Default parameter(1) was designated.")
else:
Num_least_in_group2 = float(config_dict['Num_least_in_group2'])
if not 'Coverage_cutoff' in config_dict:
print(" Coverage_cutoff: Default parameter(30) was designated.")
else:
Coverage_cutoff = float(config_dict['Coverage_cutoff'])
if not 'FDR_cutoff' in config_dict:
print(" FDR_cutoff: Default parameter(0.05) was designated.")
else:
FDR_cutoff = float(config_dict['FDR_cutoff'])
if not 'Fold_change_cutoff' in config_dict:
print(" Fold_change_cutoff: Default parameter(0.59[log2]/1.5-fold) was designated.")
else:
Fold_change_cutoff = config_dict['Fold_change_cutoff']
if not 'PDUI_cutoff' in config_dict:
print(" PDUI_cutoff: Default parameter(0.2) was designated.")
else:
PDUI_cutoff = float(config_dict['PDUI_cutoff'])
if not 'Coverage_pPAS_cutoff' in config_dict:
print(" Coverage_pPAS_cutoff: Default parameter(5.0) was designated.")
else:
Coverage_pPAS_cutoff = float(config_dict['Coverage_pPAS_cutoff'])
#Collect sample files
num_group_1 = len(Group1_Tophat_aligned_file)
num_group_2 = len(Group2_Tophat_aligned_file)
All_Sample_files = Group1_Tophat_aligned_file[:]
All_Sample_files.extend(Group2_Tophat_aligned_file)
num_samples = len(All_Sample_files)
#Prepare output directory
d = os.path.dirname(output_directory)
if not os.path.exists(d):
os.makedirs(d)
#Prepare temp directory
temp_dir = d + '/tmp/'
if not os.path.exists(temp_dir):
os.makedirs(temp_dir)
Output_all_prediction_file = output_directory + Output_result_file + '_result_temp.txt'
Output_result = open(Output_all_prediction_file, 'w')
#Load coverage
now_time("Loading coverage...")
All_samples_Target_3UTR_coverages, All_samples_sequencing_depths, UTR_events_dict = Load_Target_Wig_files(All_Sample_files, Annotated_3UTR_file)
#Depth(Coverage) weight for each sample
All_sample_coverage_weights = All_samples_sequencing_depths / np.mean(All_samples_sequencing_depths)
now_time("Loading coverage finished.")
#Prepare header information for output file
first_line = ['Gene','Predicted_Proximal_APA','loci']
for i in range(num_group_1):
curr_long_exp = 'A_%s_long_exp' % str(i+1)
curr_short_exp = 'A_%s_short_exp' % str(i+1)
curr_ratio = 'A_%s_PDUI' % str(i+1)
curr_score = 'A_%s_EndClip_Score' % str(i+1)
first_line.extend([curr_long_exp, curr_short_exp, curr_ratio, curr_score])
for i in range(num_group_2):
curr_long_exp = 'B_%s_long_exp' % str(i+1)
curr_short_exp = 'B_%s_short_exp' % str(i+1)
curr_ratio = 'B_%s_PDUI' % str(i+1)
curr_score = 'B_%s_EndClip_Score' % str(i+1)
first_line.extend([curr_long_exp, curr_short_exp, curr_ratio, curr_score])
first_line.extend(['A_PDUI_mean','B_PDUI_mean','PDUI_Group_diff[B_PDUI_mean - A_PDUI_mean]','Fold-change[A_PDUI_mean/B_PDUI_mean]','Fold-change[B_short_exp_mean / A_short_exp_mean]','Fold-change[B_score_mean / A_score_mean]'])
print("\t".join(first_line), end="\n", file=Output_result)
#Test APA event for each 3UTR
now_time("Testing APA events for each 3UTR region...")
#Wig file preparation
Wig_sample_files = []
for name in All_Sample_files:
#name = 'data/NoCTRL_accepted_hits_PTEN_ELAVL1.bam.wig'
name = os.path.basename(name)
name = str(os.path.splitext(name)[0])
name = str(os.path.splitext(name)[0])
name += ".bg"
name_file = open(name, 'w')
print_write = "track type=bedGraph name=EndSnip_test_%s description=EndSnip_test_%s visibility=2 maxHeightPixels=40:40:20" % (name, name)
print(print_write, end="\n",file=name_file)
Wig_sample_files.append(name_file)
#Bed file preparation
Bed_sample_files = []
for name in All_Sample_files:
#name = 'data/NoCTRL_accepted_hits_PTEN_ELAVL1.bam.wig'
name = os.path.basename(name)
name = str(os.path.splitext(name)[0])
name = str(os.path.splitext(name)[0])
name += ".bed"
name_file = open(name, 'w')
print_write = "track type=bed name=EndSnip_UTRIsoform_%s description=EndSnip_UTRIsoform_%s" % (name, name)
print(print_write, end="\n",file=name_file)
Bed_sample_files.append(name_file)
for curr_3UTR_id in UTR_events_dict:
#3UTR region information for each gene
curr_3UTR_structure = UTR_events_dict[curr_3UTR_id]
chrom = curr_3UTR_structure[0]
region_start = curr_3UTR_structure[1] #region start
region_end = curr_3UTR_structure[2] #region end
curr_strand = curr_3UTR_structure[3] #strand
UTR_pos = curr_3UTR_structure[4] #UTR position information
#pA_site = curr_3UTR_structure[5].split('|') #pA_site list
#pA_site = list(map(int,pA_site))
print(curr_3UTR_id)
#If gene names exist in coverage dict(for each gene)
if curr_3UTR_id in All_samples_Target_3UTR_coverages:
#3UTR coverage for each gene
curr_3UTR_coverage_wig = All_samples_Target_3UTR_coverages[curr_3UTR_id] #List of 3UTR coverage for each sample
curr_3UTR_all_samples_bp_coverage = []
curr_3UTR_all_samples_bp_chrom_site = []
for curr_sample_curr_3UTR_coverage_wig in curr_3UTR_coverage_wig: #3UTR coverage for each sample
bp_resolution_data = Convert_wig_into_bp_coverage(curr_sample_curr_3UTR_coverage_wig[0], #List of coverage
curr_sample_curr_3UTR_coverage_wig[1], #List of 3UTR region(1-base)
curr_strand) #strand
#test = Convert_wig_into_bp_coverage(curr_sample_curr_3UTR_coverage_wig[0],curr_sample_curr_3UTR_coverage_wig[1],curr_strand) #test
curr_3UTR_curr_samples_bp_coverage = bp_resolution_data[0]
curr_3UTR_curr_samples_bp_chrom_site = bp_resolution_data[1]
curr_3UTR_all_samples_bp_coverage.append(curr_3UTR_curr_samples_bp_coverage) #List of bp_coverage for each sample
curr_3UTR_all_samples_bp_chrom_site.append(curr_3UTR_curr_samples_bp_chrom_site) #List of bp chromosome site for each sample
#TODO: TEST: Coverage in 3'UTR region for PTEN, ELAVL1
# plt.plot(curr_3UTR_curr_samples_bp_coverage)
# #plt.show()
global test_name
test_name = curr_3UTR_id.split('|')[1]
# filename = "data/output_coverage_" + test_name + ".png"
# plt.savefig(filename)
#TODO: TEST: Coverage in 3'UTR region for PTEN, ELAVL1
#plt.close()
#De novo identification of APA event for each 3UTR region
curr_3UTR_all_samples_bp_coverage = np.array(curr_3UTR_all_samples_bp_coverage)
#select_mean_squared_error, selected_break_point, UTR_abundance = De_Novo_3UTR_all_samples_bp_extimation(curr_3UTR_all_samples_bp_coverage,
# region_start,
# region_end,
# curr_strand,
# All_sample_coverage_weights,
# Coverage_pPAS_cutoff,
# test_name)
#coverage_comparison_with_pA_site(curr_3UTR_all_samples_bp_coverage, curr_3UTR_all_samples_bp_chrom_site, region_start, region_end, curr_strand, All_sample_coverage_weights, Coverage_pPAS_cutoff, pA_site,test_name)
de_novo_coverage_comparison_with_windows(curr_3UTR_all_samples_bp_coverage, curr_3UTR_all_samples_bp_chrom_site, region_start, region_end, curr_strand, All_sample_coverage_weights, Coverage_pPAS_cutoff, test_name, chrom, Wig_sample_files, Bed_sample_files, curr_3UTR_id, Output_result, num_group_1, num_group_2, UTR_pos)
#Elapsed time
end_time = time.time() - start_time
end_h = int(end_time/3600)
end_time -= 3600 * end_h
if end_h < 10:
end_h = "0" + str(end_h)
else:
end_h = str(end_h)
end_m = int(end_time/60)
end_time -= 60 * end_m
if end_m < 10:
end_m = "0" + str(end_m)
else:
end_m = str(end_m)
end_s = int(end_time)
if end_s < 10:
end_s = "0" + str(end_s)
else:
end_s = str(end_s)
run_time = "Completely finished: %s:%s:%s elapsed" % (end_h, end_m, end_s)
now_time(run_time)
def Load_Target_Wig_files(All_Wig_files, UTR_Annotation_file):
UTR_events_dict = {}
All_Samples_Total_depth = []
#Load UTR Annotation file
for line in open(UTR_Annotation_file, 'r'):
fields = line.rstrip().split("\t")
curr_chr = fields[0]
region_start = fields[1]
region_end = fields[2]
name = fields[3]
curr_strand = fields[5]
#pA_site = fields[6]
UTR_pos = "%s:%s-%s" % (curr_chr, region_start, region_end)
#Define 3'UTR Annotation regions
#end_shift = int(round(abs(int(region_start) - int(region_end)) * 0.2)) # TODO: 2割の領域でいいか確認する。
end_shift = 0
if curr_strand == '+':
region_end = str(int(region_end) - end_shift)
elif curr_strand == '-':
region_start = str(int(region_start) + end_shift)
else:
sys.exit("ERROR: Strand column in your UTR annotation file is wrong...")
region_start = int(region_start) + 1 #0-base => 1-base
region_end = int(region_end)
if (region_end - region_start) >= 500: #Min 3UTR length(Default: 500bp)
#UTR_events_dict => [chrom, start, end, strand, UTR_position(chrom:start-end)]
UTR_events_dict[name] = [curr_chr, region_start, region_end, curr_strand, UTR_pos]
# TODO: Isoformごとに判断する場合を考慮に入れる
# 終止コドンが異なるケースでは、Isoformごとに判断する。
# 3'UTR中でスプライシングを受けている場合も考慮する。
#Load coverage for all samples
All_samples_extracted_3UTR_coverage_dict = {}
for curr_wig_file in All_Wig_files:
curr_sample_All_chroms_coverage_dict = {}
num_line = 0
curr_sample_total_depth = 0
for line in open(curr_wig_file, 'r'):
if '#' in line and line[0:3] != 'chr':
continue
fields = line.strip().split("\t")
#Load wig file
chrom_name = fields[0]
region_start = int(fields[1])
region_end = int(fields[2])
read_depth = int(float(fields[-1]))
#Initialize coverage data in each chromosome
if chrom_name not in curr_sample_All_chroms_coverage_dict:
curr_sample_All_chroms_coverage_dict[chrom_name] = [[0],[0]] #[[region_site], [depth]]
#Add coverage data in each region on each chromosome
if region_start > curr_sample_All_chroms_coverage_dict[chrom_name][0][-1]: #if gap region exists
curr_sample_All_chroms_coverage_dict[chrom_name][0].append(region_start) #Region end => Region start #1-based
curr_sample_All_chroms_coverage_dict[chrom_name][1].append(0) #Read depth => 0
curr_sample_All_chroms_coverage_dict[chrom_name][0].append(region_end) #Region end
curr_sample_All_chroms_coverage_dict[chrom_name][1].append(read_depth) #Read depth
#Total coverage and read count in each sample
curr_sample_total_depth += read_depth * (region_end - region_start)
num_line += 1
#Collect total depth for each sample
curr_sample_All_chroms_coverage_dict[chrom_name][1].append(0) #slicing your list(extracted_coverage): 185 line
All_Samples_Total_depth.append(curr_sample_total_depth) #Collection of total depth for each sample
#print(curr_sample_All_chroms_coverage_dict[chrom_name][0][1:20])
#print(curr_sample_All_chroms_coverage_dict[chrom_name][1][1:20])
#print(len(curr_sample_All_chroms_coverage_dict[chrom_name][0]))
#print(len(curr_sample_All_chroms_coverage_dict[chrom_name][1]))
now_time(curr_wig_file + ": Total depth were loaded.")
#Define each depth for each 3'UTR
for curr_3UTR_event_id in UTR_events_dict.keys():
#Each transcript information
curr_3UTR_structure = UTR_events_dict[curr_3UTR_event_id]
curr_chr = curr_3UTR_structure[0] #Chromosome number
region_start = curr_3UTR_structure[1] #3'UTR region start
region_end = curr_3UTR_structure[2] #3'UTR region end
#Call current chromosome from dictionary
if curr_chr in curr_sample_All_chroms_coverage_dict.keys():
#Region and Depth for current chromosome
curr_chr_coverage = curr_sample_All_chroms_coverage_dict[curr_chr]
#TEST:
#Raw_data
#chrom_site = [1, 2, 3, 4, 5, 6, 7, 8, 9, 10]
#chr_coverage = [1, 4, 4, 0, 0, 0, 1, 2, 2, 0]
#Input_data
#chrom_site = [0, 1, 3, 6, 7, 9, 10]
#chr_coverage = [0, 1, 4, 0, 1, 2, 0]
#from bisect import bisect
#curr_chr_coverage = [[0, 1, 3, 6, 7, 9, 10], [0, 1, 4, 0, 1, 2, 0]]
#from bisect import bisect
#curr_chr_coverage = [[1,10,20,30,40,50,60], [1,10,10,10,30,30,50]] #[[Chrom_site],[Coverage]]
#NO1:
#region_start = 1
#region_end = 60
#NO2:
#region_start = 1
#region_end = 55
#NO3:
#region_start = 5
#region_end = 60
#NO4:
#region_start = 5
#region_end = 55
#NO5:
#region_start = 70
#region_end = 90
left_region_index = bisect(curr_chr_coverage[0], region_start) #Insertion site(index) of region start
right_region_index = bisect(curr_chr_coverage[0], region_end) #Insertion site(index) of region end
extracted_3UTR_region = []
extracted_coverage = []
#In the case of 0 coverage,
if left_region_index == right_region_index:
extracted_3UTR_region = [region_start, region_end]
extracted_coverage = [0, 0]
elif int(curr_chr_coverage[0][left_region_index-1]) == int(region_start) and int(curr_chr_coverage[0][right_region_index-1]) == int(region_end):
#print("1")
#List of 3UTR region
extracted_3UTR_region = curr_chr_coverage[0][left_region_index-1:right_region_index]
#List of depth(coverage) in 3'UTR region
extracted_coverage = curr_chr_coverage[1][left_region_index-1:right_region_index]
elif int(curr_chr_coverage[0][left_region_index-1]) == int(region_start):
#print("2")
#List of 3UTR region
extracted_3UTR_region = curr_chr_coverage[0][left_region_index-1:right_region_index]
extracted_3UTR_region.append(region_end)
#List of depth(coverage) in 3'UTR region
extracted_coverage = curr_chr_coverage[1][left_region_index-1:right_region_index+1]
elif int(curr_chr_coverage[0][right_region_index-1]) == int(region_end):
#print("3")
#List of 3UTR region
extracted_3UTR_region = curr_chr_coverage[0][left_region_index:right_region_index]
extracted_3UTR_region.insert(0,region_start)
#List of depth(coverage) in 3'UTR region
extracted_coverage = curr_chr_coverage[1][left_region_index:right_region_index]
extracted_coverage.insert(0,curr_chr_coverage[1][left_region_index])
else:
#print("4")
#List of 3UTR region
extracted_3UTR_region = curr_chr_coverage[0][left_region_index:right_region_index]
extracted_3UTR_region.insert(0,region_start)
extracted_3UTR_region.append(region_end)
#List of depth(coverage) in 3'UTR region
extracted_coverage = curr_chr_coverage[1][left_region_index:right_region_index+1]
extracted_coverage.insert(0,curr_chr_coverage[1][left_region_index])
'''
#Example
#Index: 0 1 2 3 4 5 6
#chrom_site = [1,10,20,30,40,50,60]
#coverage = [1,10,10,10,30,30,50]
#1bp => 1
#2-10bp => 10
#11-20bp => 10
#21-30bp => 10
#31-40bp => 30
#41-50bp => 30
#51-60bp => 50
###1-60[1,7]###
#bisect(chrom_site,1) => 1 => 0/0
#bisect(chrom_site,60) => 7 => 7/7
#chrom_site: [1,10,20,30,40,50,60] => [1,10,20,30,40,50,60]
#coverage: [1,10,10,10,30,30,50] => [1,10,10,10,30,30,50]
###1-55[1,6]###
#bisect(chrom_site,1) => 1 => 0/0
#bisect(chrom_site,55) => 6 => 6/7
#chrom_site: [1,10,20,30,40,50] => [1,10,20,30,40,50,"55"]
#coverage: [1,10,10,10,30,30,50] => [1,10,10,10,30,30, 50]
###5-60[1,7]###
#bisect(chrom_site,5) => 1 => 1/1
#bisect(chrom_site,60) => 7 => 7/7
#chrom_site: [10,20,30,40,50,60] => [ "5",10,20,30,40,50,60]
#coverage: [10,10,10,30,30,50] => ["10",10,10,10,30,30,50]
###5-55[1,7]###
#bisect(chrom_site,5) => 1 => 1/1
#bisect(chrom_site,55) => 6 => 6/7
#chrom_site: [10,20,30,40,50] => [ "5",10,20,30,40,50,"55"]
#coverage: [10,10,10,30,30,50] => ["10",10,10,10,30,30, 50 ]
'''
#List of depth(coverage) in 3'UTR region
#extracted_coverage = curr_chr_coverage[1][left_region_index:right_region_index+1]
#List of 3UTR region
#extracted_3UTR_region = curr_chr_coverage[0][left_region_index:right_region_index]
#extracted_3UTR_region.insert(0, region_start)
#extracted_3UTR_region.append(region_end)
###Example:
###chrom_site = [0,10,20,30,40,50,60,70,80,90,100]
###trx_exp =[0,5,5,5,5,5,1,1,0,0,0]
###bisect(chrom_site, 15)
###[2] => [0,10 | 20,30,40,50,60,70,80,90,100]
###bisect(chrom_site, 85)
###[9] => [0,10,20,30,40,50,60,70,80 | 90,100]
###chrom_site[2:9] => [20,30,40,50,60,70,80] (7 items) => [15,20,30,40,50,60,70,80,85] (8 items)
###trx_exp[2:9+1] => [5,5,5,5,1,1,0,0] (8 items)
#Initiate current 3UTR event id in All_samples_extracted_3UTR_coverage_dict
if not curr_3UTR_event_id in All_samples_extracted_3UTR_coverage_dict:
All_samples_extracted_3UTR_coverage_dict[curr_3UTR_event_id] = []
#Reserve Depth(Coverage) and 3UTR region information for each sample in dictonary
All_samples_extracted_3UTR_coverage_dict[curr_3UTR_event_id].append([extracted_coverage, extracted_3UTR_region])
#Gene information(curr_3UTR_event_id) =>
#[ [[Coverage list 1], [3UTR region list 1]], [[Coverage list 2], [3UTR region list 2]], ... , [[Coverage list N], [3UTR region list N]] ]
now_time(curr_wig_file + ": Each depth for each 3'UTR was loaded.")
#Reserve Depth(Coverage) and 3UTR region information for each 3UTR region | Total depth in samples | 3UTR region information
return All_samples_extracted_3UTR_coverage_dict, np.array(All_Samples_Total_depth), UTR_events_dict
