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check_ncss.py
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check_ncss.py
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### Boas Pucker ###
### [email protected] ###
### v0.1 ###
### MEMORY SAVING VERSION ###
__usage__ = """
python check_ncss.py
--cov <FULL_PATH_TO_INPUT_COVERAGE_FILE>
--doc <FULL_PATH_TO_INPUT_DOCUMENTATION_FILE>
--out <FULL_PATH_TO_OUTPUT_DIRECTORY>
bug reports and feature requests: [email protected]
"""
import glob, re, sys, os, shelve
import matplotlib.pyplot as plt
import numpy as np
from operator import itemgetter
# --- end of imports --- #
def load_pos_from_gff( gff_file ):
"""! @brief load positions from GFF3 file """
pos = {}
with open( gff_file, "r" ) as f:
line = f.readline()
while line:
if line[0] != '#':
parts = line.strip().split('\t')
if parts[2] in [ "gene", "mRNA" ]:
try:
ID = re.findall( "rna\d+", parts[-1] )[0]
parent = re.findall( "gene\d+", parts[-1] )[0]
except IndexError:
ID = re.findall( "gene\d+", parts[-1] )[0]
parent = re.findall( "gene\d+", parts[-1] )[0]
if parts[6]:
orientation = True
else:
orientation = False
try:
pos.update( { ID: { 'id': ID, 'parent': parent, 'chr': parts[0], 'start': parts[3], 'end': parts[4], 'orientation': orientation } } )
except:
pass
line = f.readline()
return pos
def load_all_ncss_regions( docf, transcript_gene_pos ):
"""! @brief load all ncss candidate regions from given documentation file """
ncss_regions = []
with open( docf, "r" ) as f:
line = f.readline()
while line:
parts = line.strip().split('\t')
if len( parts ) > 3 and "css" in line: #could be edited back to 'ncss' to focus on those only
info = transcript_gene_pos[ parts[0] ]
ex3_pos, in5_pos, in3_pos, ex5_pos = parts[4].split(',')
if info['orientation']:
ncss_regions.append( { 'id': info['parent'],
'chr': info['chr'],
'e3': int( ex3_pos.split('_')[1] )-1,
'i5': int( in5_pos.split('_')[0] ),
'i3': int( in3_pos.split('_')[1] )-1,
'e5': int( ex5_pos.split('_')[0] ),
'5prime': parts[1],
'3prime': parts[2]
} )
else:
ncss_regions.append( { 'id': info['parent'],
'chr': info['chr'],
'e3': int( ex3_pos.split('_')[0] ),
'i5': int( in5_pos.split('_')[1] )-1,
'i3': int( in3_pos.split('_')[0] ),
'e5': int( ex5_pos.split('_')[1] )-1,
'5prime': parts[1],
'3prime': parts[2]
} )
line = f.readline()
return sorted( ncss_regions, key=itemgetter( 'chr' ) )
def load_coverage( cov_file, chromosome ):
"""! @brief load coverage from given file """
with open( cov_file, "r" ) as f:
line = f.readline()
cov = []
while line:
parts = line.strip().split('\t')
if parts[0] == chromosome:
cov.append( float( parts[2] ) )
line = f.readline()
return cov
def analyze_cov_at_ncss( cov_file, ncss_regions, result_file ):
"""! @brief analyze coverage around ncss """
with open( result_file, "w", 0 ) as out:
out.write( "GeneID\tExon3prime\tIntron5prime\tIntron3prime\tExon5prime\t5prime_splice_site\t3pime_splice_site\n" )
current_chr = ""
for ncss in ncss_regions:
if current_chr != ncss['chr']:
chr_cov = load_coverage( cov_file, ncss['chr'] )
current_chr = "" + ncss['chr']
e3 = chr_cov[ ncss['e3'] ]
i5 = chr_cov[ ncss['i5'] ]
i3 = chr_cov[ ncss['i3'] ]
e5 = chr_cov[ ncss['e5'] ]
out.write( "\t".join( map( str, [ ncss['id'], e3, i5, i3, e5, ncss['5prime'], ncss['3prime'] ] ) ) + '\n' )
def get_cov_at_ncss( doc_file, cov_file, output_dir ):
"""! @brief get coverage at all putative ncss """
gff_file = doc_file.replace( ".txt", ".gff" )
transcript_gene_pos = load_pos_from_gff( gff_file )
ncss_regions = load_all_ncss_regions( doc_file, transcript_gene_pos )
print "number of ncss regions: " + str( len( ncss_regions ) )
result_file = output_dir + "splice_site_coverage_check.txt"
analyze_cov_at_ncss( cov_file, ncss_regions, result_file )
return result_file
def load_all_splice_sites( input_file ):
"""! @brief load all splice sits from given input file """
splice_sites = []
with open( input_file, "r" ) as f:
f.readline() #header
line = f.readline()
while line:
parts = line.strip().split('\t')
splice_sites.append( { 'id': parts[0], 'e3': float( parts[1] ), 'i5': float( parts[2] ), 'i3': float( parts[3] ), 'e5': float( parts[4] ), '5prime': parts[5], '3prime': parts[6] } )
line = f.readline()
return splice_sites
def analyze_splice_sites( splice_sites, output_dir, min_cov_cutoff ):
"""! @brief analyze splice sites by group """
GTAG_5prime_ratio = []
GTAG_3prime_ratio = []
GTAG_not_expressed = 0
GCAG_5prime_ratio = []
GCAG_3prime_ratio = []
GCAG_not_expressed = 0
ATAC_5prime_ratio = []
ATAC_3prime_ratio = []
ATAC_not_expressed = 0
others_5prime_ratio = []
others_3prime_ratio = []
others_not_expressed = 0
for site in splice_sites:
if site['5prime'] == "GT" and site['3prime'] == "AG":
if site['e3'] > 0 and site['e5'] > 0:
GTAG_5prime_ratio.append( ( site['e3']-site['i5'] ) / site['e3'] )
GTAG_3prime_ratio.append( ( site['e5']-site['i3'] ) / site['e5'] )
else:
GTAG_not_expressed += 1
elif site['5prime'] == "GC" and site['3prime'] == "AG":
if site['e3'] > 0 and site['e5'] > 0:
GCAG_5prime_ratio.append( ( site['e3']-site['i5'] ) / site['e3'] )
GCAG_3prime_ratio.append( ( site['e5']-site['i3'] ) / site['e5'] )
else:
GCAG_not_expressed += 1
elif site['5prime'] == "AT" and site['3prime'] == "AC":
if site['e3'] > 0 and site['e5'] > 0:
ATAC_5prime_ratio.append( ( site['e3']-site['i5'] ) / site['e3'] )
ATAC_3prime_ratio.append( ( site['e5']-site['i3'] ) / site['e5'] )
else:
ATAC_not_expressed += 1
else:
if site['e3'] > 0 and site['e5'] > 0:
others_5prime_ratio.append( ( site['e3']-site['i5'] ) / site['e3'] )
others_3prime_ratio.append( ( site['e5']-site['i3'] ) / site['e5'] )
else:
others_not_expressed += 1
# --- construct ratio distribution plots --- #
fig_5prime_out = output_dir + "5prime_ratios.png"
fig, ax = plt.subplots()
#ax.hist( GTAG_5prime_ratio, bins=10000, color="green", label="GT-AG" )
ax.hist( GCAG_5prime_ratio, bins=1000, color="blue", label="GC-AG" )
ax.hist( ATAC_5prime_ratio, bins=1000, color="purple", label="AT-AC" )
ax.hist( others_5prime_ratio, bins=1000, color="red", label="others" )
ax.set_ylim( 0, 100 )
ax.set_xlim( 0, 1 )
ax.set_xlabel("coverage ratio over 5prime intron border")
ax.set_ylabel("counts")
fig.savefig( fig_5prime_out, dpi=300 )
def get_supported_ncss( splice_sites, supported_ncss_file, cutoff ):
"""! @brief get supported ncss """
supported_ncss = []
with open( supported_ncss_file, "w" ) as out:
for site in splice_sites:
if site['5prime'] != "GT":
if site['e3'] > 0 and site['e5'] > 0:
if ( ( site['e3']-site['i5'] ) / site['e3'] ) > cutoff and ( ( site['e5']-site['i3'] ) / site['e5'] ) > cutoff:
out.write( "\t".join( [ site['id'], site['5prime'], site['3prime'] ] ) + '\n' )
supported_ncss.append( site['5prime']+".."+site['3prime'] )
elif site['3prime'] != "AG":
if site['e3'] > 0 and site['e5'] > 0:
if ( ( site['e3']-site['i5'] ) / site['e3'] ) > cutoff and ( ( site['e5']-site['i3'] ) / site['e5'] ) > cutoff:
out.write( "\t".join( [ site['id'], site['5prime'], site['3prime'] ] ) + '\n' )
supported_ncss.append( site['5prime']+".."+site['3prime'] )
else:
if site['e3'] > 0 and site['e5'] > 0:
if ( ( site['e3']-site['i5'] ) / site['e3'] ) > cutoff and ( ( site['e5']-site['i3'] ) / site['e5'] ) > cutoff:
out.write( "\t".join( [ site['id'], site['5prime'], site['3prime'] ] ) + '\n' )
supported_ncss.append( site['5prime']+".."+site['3prime'] )
print "number of supported ncss: " + str( len( supported_ncss ) )
overview_file = supported_ncss_file + "_overview.txt"
with open( overview_file, "w" ) as out:
for splice_site in list( set( supported_ncss ) ):
out.write( splice_site + "\t" + str( supported_ncss.count( splice_site ) ) + '\n' )
def investigate_ncss( input_file, output_dir, cutoff, min_cov_cutoff ):
"""! @brief check coverage drop at predicted ncss """
supported_ncss_file = output_dir + "supported_ncss.txt"
splice_sites = load_all_splice_sites( input_file )
#analyze_splice_sites( splice_sites, output_dir, min_cov_cutoff )
get_supported_ncss( splice_sites, supported_ncss_file, cutoff )
def main( arguments ):
"""! @brief get coverage at ncss and check all putative ncss if they are real """
output_dir = arguments[ arguments.index('--out')+1 ]
cov_file = arguments[ arguments.index('--cov')+1 ]
doc_file = arguments[ arguments.index('--doc')+1 ]
if output_dir[-1] != '/':
output_dir += "/"
if not os.path.exists( output_dir ):
os.makedirs( output_dir )
splice_site_support_cutoff = 0.2 #difference in coverage between exon and intron to call valid splice site
min_cov_cutoff = 3
result_file = get_cov_at_ncss( doc_file, cov_file, output_dir )
investigate_ncss( result_file, output_dir, splice_site_support_cutoff, min_cov_cutoff )
if __name__ == "__main__":
if '--out' in sys.argv and '--cov' in sys.argv and '--doc' in sys.argv:
main( sys.argv )
else:
sys.exit( __usage__ )