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sqanti_qc.py
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sqanti_qc.py
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#!/usr/bin/env python
# SQANTI: Structural and Quality Annotation of Novel Transcript Isoforms
# Authors: Lorena de la Fuente, Hector del Risco, Cecile Pereira and Manuel Tardaguila
from collections import defaultdict
import getopt
import sys
import os.path, os
import timeit
import subprocess
import argparse
utilitiesPath = os.path.dirname(os.path.realpath(__file__))+"/utilities/"
sys.path.insert(0, utilitiesPath)
from rt_switching import rts
from indels_annot import indels
import psutil
import gc
import distutils.spawn
class myQueryTranscripts:
def __init__(self, transcript, tss_diff, tts_diff, exon_number, length, str_class, subtype=None, genes=None, transcripts=None, chrom=None, strand=None, bite = "FALSE", RT_switching = "FALSE", canonical="canonical", min_cov = "NA", min_cov_pos = "NA", min_samp_cov="NA", sd = "NA", FL = "NA", nIndels = "NA", nIndelsJunc = "NA", proteinID=None, ORFlen="NA", CDS_start="NA", CDS_end="NA", isoExp = "NA", geneExp = "NA" , coding = "non_coding", refLen = "NA", refExons = "NA", FSM_class = None, percAdownTTS = None):
self.transcript = transcript
self.tss_diff = tss_diff
self.tts_diff = tts_diff
self.genes = []
self.transcripts = []
self.exon_number = exon_number
self.length = length
self.str_class = str_class # structural classification of the isoform.
self.chrom = chrom
self.strand = strand
self.subtype = subtype
self.RT_switching= RT_switching
self.canonical = canonical
self.min_samp_cov = min_samp_cov
self.min_cov = min_cov
self.min_cov_pos = min_cov_pos
self.sd = sd
self.proteinID = proteinID
self.ORFlen = ORFlen
self.CDS_start = CDS_start
self.CDS_end = CDS_end
self.coding = coding
self.FL = FL
self.nIndels = nIndels
self.nIndelsJunc = nIndelsJunc
self.isoExp = isoExp
self.geneExp = geneExp
self.refLen = refLen
self.refExons = refExons
self.FSM_class = FSM_class
self.bite = bite
self.percAdownTTS = percAdownTTS
def get_total_diff(self):
total_diff = abs(int(self.tss_diff))+abs(int(self.tts_diff))
return total_diff
def modify(self, transcript, gene, tss_diff, tts_diff, refLen, refExons):
self.transcript = transcript
self.genes.append(gene)
self.tss_diff = tss_diff
self.tts_diff = tts_diff
self.transcripts.append(self.transcript)
self.refLen = refLen
self.refExons = refExons
def geneName(self):
geneName = "_".join(set(self.genes))
return geneName
def ratioExp(self):
if self.geneExp == 0 or self.geneExp == "NA":
return "NA"
else:
ratio = float(self.isoExp)/float(self.geneExp)
return(ratio)
def CDSlen(self):
if self.coding == "coding":
return(str(int(self.CDS_end) - int(self.CDS_start) + 1))
else:
return("NA")
def __str__(self):
return "%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s" % (self.chrom, self.strand, str(self.length), str(self.exon_number), str(self.str_class), "_".join(set(self.genes)), self.transcript, str(self.refLen), str(self.refExons), str(self.tss_diff), str(self.tts_diff), self.subtype, self.RT_switching, self.canonical, str(self.min_samp_cov),str(self.min_cov), str(self.min_cov_pos), str(self.sd), str(self.FL), str(self.nIndels), str(self.nIndelsJunc), self.bite, str(self.isoExp), str(self.geneExp), str(self.ratioExp()), self.FSM_class, self.coding, str(self.ORFlen), str(self.CDSlen()), str(self.CDS_start), str(self.CDS_end), str(self.percAdownTTS))
class myQueryProteins:
def __init__(self, cds_start, cds_end, orf_length, proteinID="NA"):
self.orf_length = orf_length
self.cds_start = cds_start
self.cds_end = cds_end
self.proteinID = proteinID
class myQueryJunctions:
def __init__(self, transcript, junctionNumber, chrom, strand, junc_class, donor, acceptor, diff_start, diff_end, genomCoordStart, genomCoordEnd, transcriptCoord, spliceSite, canonical, indel="NA", coverage=None):
self.transcript = transcript
self.junctionNumber = junctionNumber
self.chrom = chrom
self.strand = strand
self.junc_class = junc_class
self.donor = donor
self.acceptor = acceptor
self.diff_start = diff_start
self.diff_end = diff_end
self.genomCoordStart = genomCoordStart
self.genomCoordEnd = genomCoordEnd
self.transcriptCoord = transcriptCoord
self.spliceSite = spliceSite
self.canonical = canonical
self.coverage = []
self.indel = indel
def totalCov(self):
if "NA" in self.coverage or len(self.coverage)==0:
totalCov = "NA"
repCov = "NA"
else:
totalCov = sum([int(i) for i in self.coverage])
repCov = len([i for i in self.coverage if int(i) > 0])
return(totalCov, repCov)
def coverage_files(self):
if len(self.coverage)==0:
return("")
else:
return("\t"+("\t").join(self.coverage))
def biteDef(self):
#if self.diff_donor != 0 and self.diff_acceptor != 0 and self.diff_donor%3 != 0 and self.diff_donor%3 != 0:
if self.diff_start not in [0, "NA"] and self.diff_end not in [0, "NA"]:
return ("TRUE")
else:
return ("FALSE")
def __str__(self):
return "%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s\t%s%s" % (self.transcript, self.chrom, self.strand, self.junctionNumber, self.genomCoordStart, self.genomCoordEnd, str(self.transcriptCoord), self.junc_class, self.donor, self.acceptor, self.diff_start, self.diff_end, self.biteDef(), self.spliceSite, self.canonical, self.indel, self.totalCov()[1], self.totalCov()[0], self.coverage_files())
class myQueries:
def __init__(self, line, coordToSort, assoc=None): #CoordToSort can be the start of the first exon or the start of the first junction
self.line = line
self.coordToSort = coordToSort
self.assoc = assoc
class refTranscripts:
def __init__(self, transcript, gene, chrom, strand, tss=None, tts=None):
self.transcript = transcript
self.chrom = chrom
self.strand = strand
self.junctions = [] #inside two list: starts of junctions and ends of junctions (sorted by coordinates)
self.tss = tss
self.tts = tts
self.gene = gene
self.exons = [] #inside two list: starts of exons and ends of exons (sorted by coordinates)
self.exons_coordinates = []
def add_information(self, line, strand):
exon_s = line[8].split(",")[0:-1]
exon_e = line[9].split(",")[0:-1]
exon_s = [int(i)+1 for i in exon_s]
#exons
self.exons.append(exon_s)
self.exons.append(exon_e)
# tss and tts
if strand == "+":
tss = exon_s[0]
tts = exon_e[-1]
else:
tts = exon_s[0]
tss = exon_e[-1]
self.tss = int(tss)
self.tts = int(tts)
#junctions
if len(exon_e) >= 2: #junctions only if multiple-exon isoforms
junctions_e = exon_s[1:]
junctions_e = [int(i)-1 for i in junctions_e]
junctions_s = exon_e[:-1]
junctions_s = [int(i)+1 for i in junctions_s]
self.junctions.append(junctions_s)
self.junctions.append(junctions_e)
def difference_fragment_transcript(self, coord_start, coord_end, ref_coord):
if int(coord_start) >= int(ref_coord[0]):
lack_start = ref_coord.index(int(coord_start))
else:
lack_start = int(coord_start) - int(ref_coord[0])
if int(coord_end) <= int(ref_coord[-1]):
lack_end = len(ref_coord) - ref_coord.index(int(coord_end)) -1
else:
lack_end = int(ref_coord[-1]) - int(coord_end)
return (lack_start, lack_end)
def transcriptLength(self):
length=0
for i in range(len(self.exons[0])):
length = length + (int(self.exons[1][i])-int(self.exons[0][i])+1)
return length
class refGenes:
def __init__(self, gene, chrom, strand):
self.chrom = chrom
self.junctions = [] #Here we'll save vectors of two elements (start and end of the junction). #The final junctions vector will have as many elements as different juctions exist in that gene.
self.strand = strand
self.tss = []
self.tts = []
self.exons = [] # inside each exon of the gene is in a list (start-end). #The final junctions vector will have as many elements as different exons exist in that gene.
self.last3junctions = [] #all last3junctions for that gene
self.last5junctions = [] #all last5junctions for that gene
self.last3exons = [] #all last3exons for that gene
self.last5exons = [] #all last3exons for that gene
self.gene = gene
self.transcripts = []
self.exonsTranscript = [] #[[[exon1_t1][exon2_t2]], [[exon1_t2][exon2_t2]],....) # Now you can see all the exons for each transcript of the gene
self.exonsTranscriptTog = []
def add_transcript(self, line, strand):
# exons in genomic coordinates
exon_s = line[8].split(",")[0:-1]
exon_e = line[9].split(",")[0:-1]
exon_s = [int(i)+1 for i in exon_s]
exon_e = [int(i) for i in exon_e]
subject = line[0]
exons_per_transcript = []
exons_per_transcript_total = []
for i in range(len(exon_s)):
self.exons.append([exon_s[i],exon_e[i]])
exons_per_transcript_total.append([exon_s[i],exon_e[i]]) #el primer y ultimo exon lo tenemos en cuenta. ##### Mirar para que lo necesitamos realmente
if i != 1 and i != len(exon_s):
exons_per_transcript.append([exon_s[i],exon_e[i]]) #el primer y ultimo exon no lo tenemos en cuenta. ##### Mirar para que lo necesitamos realmente
self.exonsTranscriptTog.append(exons_per_transcript_total) #el primer y ultimo exon se tienen en cuenta.
self.exonsTranscript.append(exons_per_transcript) #el primer y ultimo exon no se tienen en cuenta.
self.transcripts.append(subject) #adding transcripts names
if len(exon_s) >= 2: #last exons only if multiple-exon isoforms
if strand == "+":
self.last5exons.append([exon_s[0], exon_e[0]])
self.last3exons.append([exon_s[-1], exon_e[-1]])
if strand == "-":
self.last5exons.append([exon_s[-1], exon_e[-1]])
self.last3exons.append([exon_s[0], exon_e[0]])
# tss and tts
if strand == "+":
tss = exon_s[0]
tts = exon_e[-1]
else:
tts = exon_s[0]
tss = exon_e[-1]
self.tss.append(int(tss))
self.tts.append(int(tts))
#junctions
if len(exon_s) >= 2: #junctions only if multiple-exon isoforms
junctions_e = exon_s[1:]
junctions_e = [int(i)-1 for i in junctions_e]
junctions_s = exon_e[:-1]
junctions_s = [int(i)+1 for i in junctions_s]
for i in range(len(junctions_s)):
self.junctions.append([junctions_s[i],junctions_e[i]])
if strand == "+":
self.last5junctions.append([junctions_s[0], junctions_e[0]])
self.last3junctions.append([junctions_s[-1], junctions_e[-1]])
if strand == "-":
self.last5junctions.append([junctions_s[-1], junctions_e[-1]])
self.last3junctions.append([junctions_s[0], junctions_e[0]])
def __str__(self):
return "%s\t%s\t%s\t%s" % (self.gene, self.chrom, self.strand, self.exons)
def ReverseComplement(seq):
basecomplement = {'A': 'T', 'C': 'G', 'G': 'C', 'T': 'A', 'N': 'N'}
seq = seq.upper()
RCseq = ""
for base in seq:
if base not in basecomplement:
sys.stderr.write("Error: %s NOT a DNA sequence" %(seq))
return None
break
RCseq = basecomplement[base]+RCseq
return (RCseq)
def transcriptLength(startExons, endExons):
length=0
for i in range(len(startExons)):
length = length + (int(endExons[i])-int(startExons[i])+1)
return length
def exons_together(starts, ends): #arguments: list of starts of exons and list of ends of exons
exons_together = []
for i in range(len(starts)):
exons_together = exons_together + range(int(starts[i]), int(ends[i])+1)
return exons_together
def getTranscriptJunctCoordinates(exons_starts, exons_ends, strand, junctions=None):
exons_unknown_together = []
starts_transcript = []
ends_transcript = []
for i in range(len(exons_starts)):
exons_unknown_together = exons_unknown_together + range(int(exons_starts[i]), int(exons_ends[i])+1)
if junctions==None:
junctions = exons_ends[:-1]
if strand=="+":
exons_unknown_together_sort = sorted(exons_unknown_together)
for l in junctions:
for k in range(len(exons_unknown_together_sort)):
if exons_unknown_together_sort[k] == int(l):
starts_transcript.append(k+1)
ends_transcript.append(k+1+1) #k+1+1
if strand=="-":
exons_unknown_together_sort = sorted(exons_unknown_together, reverse=True)
for l in junctions:
for k in range(len(exons_unknown_together_sort)):
if exons_unknown_together_sort[k] == int(l):
starts_transcript.append(str(k+1))
ends_transcript.append(str(k)) #k+1-1
start = min(starts_transcript, ends_transcript)
end = max(starts_transcript, ends_transcript)
return [start, end]
def fasta_parser(fastaFile):
try:
fasta = open(fastaFile, "r")
except IOError:
sys.stderr.write('ERROR: Unable to read %s file\n' % fastaFile)
raise SystemExit(1)
try:
seqDicc = {}
index = 0
for line in fasta:
if line.startswith(">"):
if index > 0:
seqDicc[name] = seq
index+=1
name = line[1:].rstrip()
#name = line[1:].split()[0].rstrip()
seq = ''
elif line.strip()!="":
seq += line.rstrip()
if index>0:
seqDicc[name] = seq
fasta.close()
except (RuntimeError, TypeError, NameError):
sys.stderr.write('ERROR: File %s without fasta format.\n' % fastaFile)
raise SystemExit(1)
return(seqDicc)
def genome_parser(fastaFile):
try:
fasta = open(fastaFile, "r")
except IOError:
sys.stderr.write('ERROR: Unable to read %s file.\n' % fastaFile)
raise SystemExit(1)
try:
seqDicc = {}
index = 0
for line in fasta:
if line.startswith(">"):
if index > 0:
seqDicc[name] = seq
index+=1
name = line[1:].rstrip().split()[0]
#name = line[1:].split()[0].rstrip()
seq = ''
elif line.strip()!="":
seq += line.rstrip()
seqDicc[name] = seq
fasta.close()
except (RuntimeError, TypeError, NameError):
sys.stderr.write('ERROR: File %s without fasta format.\n' % fastaFile)
raise SystemExit(1)
return(seqDicc)
def correctionPlusORFpred(args, chrom_names):
global corrORF
global corrGTF
global corrSAM
global corrFASTA
corrPathPrefix = args.dir+"/"+os.path.splitext(os.path.basename(args.isoforms))[0]
corrGFF_gmap = corrPathPrefix +"_corrected_gmap.gff"
corrGFF = corrPathPrefix +"_corrected.gff"
corrGTF = corrPathPrefix +"_corrected.gtf"
corrSAM = corrPathPrefix +"_corrected.sam"
corrFASTA = corrPathPrefix +"_corrected.fasta"
ORF = corrPathPrefix +"_corrected.faa"
corrORF = os.path.splitext(ORF)[0]+'_ATG.faa'
if not args.gtf:
if (os.path.isdir(os.path.dirname(args.gmap_index))): # check index folder
index_dir = os.path.dirname(args.gmap_index)
prefix = os.path.basename(args.gmap_index)
# modifiying fasta header to keep just transcript ID.
fastaDicc = fasta_parser(args.isoforms)
tmpFasta = os.path.splitext(args.isoforms)[0]+'.tmp'
tmpFasta_file = open(tmpFasta, "w")
for ID in fastaDicc:
if len(ID.split("|"))>2 and ID[0:2]=="PB": # PacBio fasta header (including chained format)
ID_mod = ID.split("|")[0].split(" ")[0]
elif len(ID.split("|"))>4: # Refseq fasta header
ID_mod = ID.split("|")[3]
else:
ID_mod = ID.split()[0] # Ensembl fasta header
tmpFasta_file.write(">"+ID_mod+"\n"+fastaDicc[ID]+"\n")
tmpFasta_file.close()
sys.stdout.write("\n****Aligning reads with gmap...\n")
# aligning sequences
threads=str(args.gmap_threads)
sense = str(args.sense)
print sense
try:
#with open(corrGFF_gmap, 'w') as corrGFF_out:
# subprocess.call(['gmap','-n', '1', '-t', threads, '--cross-species', '--gff3-add-separators=0','-f', '2', '-z', 'sense_force','-D', index_dir,'-d', prefix, tmpFasta], stdout=corrGFF_out)
with open(corrSAM, 'w') as corrSAM_out:
subprocess.call(['gmap','-n', '1', '-t', threads, '--cross-species', '--gff3-add-separators=0','-f', 'samse', '-z', sense, '-D', index_dir,'-d', prefix, tmpFasta], stdout=corrSAM_out)
with open(corrGFF_gmap, 'w') as corrGFF_out:
subprocess.call(['gmap','-n', '1', '-t', threads, '--cross-species', '--gff3-add-separators=0','-f', '2', '-z', sense,'-D', index_dir,'-d', prefix, tmpFasta], stdout=corrGFF_out)
os.remove(tmpFasta)
except (RuntimeError, TypeError, NameError):
sys.stderr.write('ERROR: Problem during alignment of sequences.\n')
raise SystemExit(1)
#GFF GMAP to accurate GFF
with open(corrGFF, 'w') as corrGFF_out:
with open(corrGFF_gmap, 'r') as corrGFF_in:
for i in corrGFF_in:
if i[0] != "#" and i.split("\t")[2] != "CDS":
if (".mrna1" in i) or (".path1" in i):
j = i.replace(".mrna1","")
k = j.replace(".path1", ".gene")
corrGFF_out.write(k)
# GFF to GTF
subprocess.call([utilitiesPath+"gffread", corrGFF , '-T', '-o', corrGTF])
os.remove(corrGFF_gmap)
os.remove(corrGFF)
del fastaDicc
else:
sys.stderr.write("ERROR: '%s' directory containing gmap indexes doesn't exist.\n" % (os.path.dirname(args.gmap_index)))
sys.exit()
if args.gtf:
sys.stdout.write("\nSkipping aligning of sequences because gtf file was provided.\n")
ind = 0
with open(args.isoforms, 'r') as isoforms_gtf:
for line in isoforms_gtf:
if line[0] != "#" and len(line.split("\t"))!=9:
sys.stderr.write("\nERROR: input isoforms file with not gtf format.\n")
sys.exit()
elif len(line.split("\t"))==9:
ind += 1
if ind==0:
sys.stderr.write("\nERROR: gtf has not annotation lines.\n")
sys.exit()
# GFF to GTF (in case the user provides gff instead of gtf)
corrGTF_tpm = corrGTF+".tmp"
try:
subprocess.call([utilitiesPath+"gffread", args.isoforms , '-T', '-o', corrGTF_tpm])
except (RuntimeError, TypeError, NameError):
sys.stderr.write('ERROR: File %s without gtf/gff format.\n' % args.isoforms)
raise SystemExit(1)
# check if gtf chromosomes inside genome file
with open(corrGTF, 'w') as corrGTF_out:
with open(corrGTF_tpm, 'r') as isoforms_gtf:
for line in isoforms_gtf:
if line[0] != "#":
chrom = line.split("\t")[0]
type = line.split("\t")[2]
if chrom not in chrom_names:
sys.stderr.write("\nERROR: gtf \"%s\" chromosome not found in genome reference file.\n" % (chrom))
sys.exit()
elif type=="exon":
corrGTF_out.write(line)
os.remove(corrGTF_tpm)
if not os.path.exists(corrSAM):
sys.stdout.write("\nIndels will be not calculated since you ran SQANTI without alignment step (SQANTI with gtf format as transcriptome input).\n")
# Corrected gtf to fasta
subprocess.call([utilitiesPath+"gffread", corrGTF, '-g', args.genome, '-w', corrFASTA])
# ORF generation
sys.stdout.write("\n**** Predicting ORF sequences...\n")
gmst_dir = args.dir+"/GMST/"
if not os.path.exists(gmst_dir):
os.makedirs(gmst_dir)
subprocess.call(["perl", utilitiesPath+"gmst/gmst.pl", corrFASTA , '-faa', '--strand','direct', '--fnn', '--output', os.path.splitext(os.path.basename(args.isoforms))[0]+"_corrected"], cwd=gmst_dir)
if os.path.isfile(gmst_dir+os.path.basename(ORF)):
os.rename(gmst_dir+os.path.basename(ORF), ORF)
# Modifying ORF sequences by removing sequence before ATG
ORFdicc = fasta_parser(ORF)
corrORF_file = open(corrORF, "w")
orfLenDicc = {}
for ID in ORFdicc:
seq = ORFdicc[ID]
l = len(ID.split("\t"))
pos = seq.find('M')
if pos != -1:
corr_seq = seq[pos:]
cds_start = int(ID.split("\t")[l-1].split("|")[4])+pos*3
cds_end = int(ID.split("\t")[l-1].split("|")[5])
strand = ID.split("\t")[l-1].split("|")[3]
ID_mod = ID.split()[0].strip()+"\t"+"isoform|GeneMark.hmm|"+ str(len(corr_seq)) + "_aa|" + strand + "|" + str(cds_start) +"|" + str(cds_end)
corrORF_file.write(">"+ID_mod+"\n"+corr_seq+"\n")
orfLenDicc[ID.split()[0].strip()] = myQueryProteins(cds_start, (cds_start+len(corr_seq)*3-1), len(corr_seq))
corrORF_file.close()
del ORFdicc
os.remove(ORF)
else:
corrORF_file = open(corrORF, "w")
corrORF_file.close()
orfLenDicc={}
if len(orfLenDicc)==0:
sys.stderr.write("WARNING: All input isoforms were predicted as non-coding")
os.rename(corrORF, ORF)
gc.collect()
return(orfLenDicc)
def reference_parser(args, chrom_names):
global referenceFiles
referenceFiles = args.dir+"/refAnnotation_"+args.output+".genePred"
sys.stdout.write("\n**** Parsing Reference Transcriptome...\n")
ind = 0
with open(args.annotation, 'r') as reference_gtf:
for line in reference_gtf:
if line[0] != "#" and len(line.split("\t"))!=9:
sys.stderr.write("\nERROR: reference annotation file with not gtf/gff format.\n")
sys.exit()
elif len(line.split("\t"))==9:
ind += 1
if ind==0:
sys.stderr.write("\nERROR: reference annotation file has not annotation lines.\n")
sys.exit()
## gtf to genePred
if args.name:
subprocess.call([utilitiesPath+"gtfToGenePred", args.annotation, referenceFiles, '-genePredExt', '-allErrors', '-ignoreGroupsWithoutExons', '-geneNameAsName2'])
else:
subprocess.call([utilitiesPath+"gtfToGenePred", args.annotation, referenceFiles, '-genePredExt', '-allErrors', '-ignoreGroupsWithoutExons'])
if not os.path.exists(referenceFiles):
sys.exit()
## parsing annotation while filtering miRNA RNAs
# dicc of transcripts. We differenciate between single-exon transcripts and multi-exon transcripts.
transcripts_chrom_1exon = {}
transcripts_chrom_exons = {}
# dicc of genes
transcripts_gene = {}
missingChroms = []
referenceFiles_tpm = referenceFiles+"notmiRNAtmp"
flag=True
with open(referenceFiles_tpm, 'w') as inputAnnotationNotmiRNAs:
with open(referenceFiles, 'r') as inputAnnotation:
for i in inputAnnotation:
line_split = i.split("\t")
if len(line_split[8].split(",")) < 3:
if (int(line_split[9].split(",")[0]) - int(line_split[8].split(",")[0])) > 200:
inputAnnotationNotmiRNAs.write(i)
flag=True
else:
flag=False
else:
inputAnnotationNotmiRNAs.write(i)
flag=True
if flag==True:
transcript_id = line_split[0]
chrom = line_split[1]
if (chrom not in chrom_names) and (chrom not in missingChroms):
missingChroms.append(chrom)
strand = line_split[2]
gene = line_split[11]
if gene == "":
continue
#add gene info
if gene not in transcripts_gene:
transcripts_gene[gene] = refGenes(gene, chrom, strand)
transcripts_gene[gene].add_transcript(line_split, strand)
#add transcript info
if len(line_split[8].split(",")) > 2: # multiple-exon isoform
if chrom not in transcripts_chrom_exons:
transcripts_chrom_exons[chrom]=[]
a = refTranscripts(transcript_id, gene, chrom, strand)
a.add_information(line_split, strand)
transcripts_chrom_exons[chrom].append(a)
else: #single-exon isoform
if chrom not in transcripts_chrom_1exon:
transcripts_chrom_1exon[chrom]=[]
a = refTranscripts(transcript_id, gene, chrom, strand)
a.add_information(line_split, strand)
transcripts_chrom_1exon[chrom].append(a)
os.rename(referenceFiles_tpm, referenceFiles)
if len(missingChroms)>0:
sys.stderr.write("\nWARNING: %s sequences not found in genome but containing annotated genes\n" %(",".join(missingChroms)))
## sorting transcript information by first junction (if mono-exon transcript we sort by first exon)
sum=0
for chrom in transcripts_chrom_exons:
transcripts_chrom_exons[chrom] = sorted(transcripts_chrom_exons[chrom], key = lambda tup:int(tup.junctions[0][0]))
sum = len(transcripts_chrom_exons[chrom]) + sum
for chrom in transcripts_chrom_1exon:
transcripts_chrom_1exon[chrom] = sorted(transcripts_chrom_1exon[chrom], key = lambda tup:int(tup.exons[0][0]))
sum = len(transcripts_chrom_1exon[chrom]) + sum
## dicc for chomosome and inside gene information. Got from transcripts_gene dicc.
transcripts_gene_chrom = {}
for gene in transcripts_gene.keys():
chrom = transcripts_gene[gene].chrom
if chrom not in transcripts_gene_chrom:
transcripts_gene_chrom[chrom]=[]
transcripts_gene_chrom[chrom].append(transcripts_gene[gene])
for chrom in transcripts_gene_chrom:
transcripts_gene_chrom[chrom] = sorted(transcripts_gene_chrom[chrom], key = lambda tup:min(tup.tss + tup.tts)) ##### COMPROBAR QUE ESTA BIEN!!!!
return (transcripts_chrom_1exon, transcripts_chrom_exons, transcripts_gene, transcripts_gene_chrom)
def isoforms_parser(args):
global queryFile
queryFile = os.path.splitext(corrGTF)[0] +".genePred"
sys.stdout.write("\n**** Parsing Isoforms...\n")
# gtf to genePred
try:
subprocess.call([utilitiesPath+"gtfToGenePred", corrGTF, queryFile , '-genePredExt', '-allErrors', '-ignoreGroupsWithoutExons'])
except (RuntimeError, TypeError, NameError):
sys.stderr.write('ERROR: Check isoforms file.\n' )
raise SystemExit(1)
## analysis of query transcripts
queryAnnotation = open(queryFile, 'r')
myQueryTranscripts_list = []
for line in queryAnnotation:
exon_s = line.split("\t")[8].split(",")[0:-1]
exon_e = line.split("\t")[9].split(",")[0:-1]
if len(exon_s) >= 2: #junctions only if multiple-exon isoforms
coordToSort = int(exon_e[0]) #first genomic splice site
else:
coordToSort = int(exon_s[0]) #first exon position
a = myQueries(line, coordToSort)
myQueryTranscripts_list.append(a)
queryAnnotation.close()
# sorting the subject transcript by start junction or start exon (depending if one or more exons)
myQueryTranscripts_list = sorted(myQueryTranscripts_list, key = lambda tup:int(tup.coordToSort))
return(myQueryTranscripts_list)
def STARcov_parser(coverageFiles): # just valid with unstrand-specific RNA-seq protocols.
#may be one file, files separated by comma or a directory where file are.
if os.path.isdir(coverageFiles)==True:
cov_paths = [os.path.join(coverageFiles,fn) for fn in next(os.walk(coverageFiles))[2]]
else:
cov_paths = coverageFiles.split(",")
cov_list_dicc = {}
for path in cov_paths:
dicc = {}
p = open(path.strip(), "r")
for line in p:
j = "_".join(line.split()[0:3])
dicc[j] = str(int(line.split()[6])+int(line.split()[7]))
cov_list_dicc[os.path.basename(path)] = dicc
p.close()
return(cov_list_dicc)
def expression_parser_old(expressionFile):
try:
p = open(expressionFile, "r")
except IOError:
sys.stderr.write('ERROR: Unable to read %s expression file\n' % expressionFile)
raise SystemExit(1)
try:
header = p.readline()
exp_dicc = {}
for line in p:
pbid = line.split()[0]
mean = sum([float(i) for i in line.rstrip().split()[1:]])/len(line.rstrip().split()[1:])
exp_dicc[pbid] = mean
p.close()
except IOError:
sys.stderr.write('File %s without expression matrix format' % expressionFile)
raise SystemExit(1)
return(exp_dicc)
def expression_parser(expressionFile):
try:
p = open(expressionFile, "r")
except IOError:
sys.stderr.write('ERROR: Unable to read %s expression file\n' % expressionFile)
raise SystemExit(1)
try:
header = p.readline()
exp_dicc = {}
for line in p:
pbid = line.split()[0]
if len(pbid.split("|"))>2 and pbid[0:2]=="PB": # PacBio fasta header (including chained format)
pbid_mod = pbid.split("|")[0].split(" ")[0]
elif len(pbid.split("|"))>4: # Refseq fasta header
pbid_mod = pbid.split("|")[3]
else:
pbid_mod = pbid.split()[0] # Ensembl fasta header
mean = sum([float(i) for i in line.rstrip().split()[1:]])/len(line.rstrip().split()[1:])
exp_dicc[pbid_mod] = mean
p.close()
except IOError:
sys.stderr.write('File %s without expression matrix format' % expressionFile)
raise SystemExit(1)
return(exp_dicc)
def transcriptsKnownSpliceSites(transcripts_chrom_1exon, transcripts_chrom_exons, line_split, index_chrom_exons, index_chrom_1exon, transcripts_gene, seq, nPolyA):
# Transcript information for a single query transcript and comparison with reference.
query_transcript = line_split[0]
CHROM = line_split[1]
STRAND = line_split[2]
EXON_s = [int(i)+1 for i in line_split[8].split(",")[0:-1]]
EXON_e = [int(i) for i in line_split[9].split(",")[0:-1]]
isoLength = transcriptLength(EXON_s, EXON_e)
JUNCTIONS_e = [int(i)-1 for i in EXON_s[1:] ]
JUNCTIONS_s = [int(i)+1 for i in EXON_e[:-1]]
ends_positions = []
myTranscript_Assoc=""
JUNCTIONS = []
[JUNCTIONS.append([JUNCTIONS_s[m],JUNCTIONS_e[m]]) for m in range(len(JUNCTIONS_s))]
EXONS_COORD = exons_together(EXON_s, EXON_e)
# # Intra-priming
# if STRAND == "+":
# TTS = int(EXON_e[-1])
# downTTS = str(seq[CHROM].seq[TTS:(int(EXON_e[-1])+nPolyA)])
# downTTS = downTTS.upper()
# if any([base not in ["A","T","C","G","N"] for base in downTTS]):
# sys.stderr.write("Error: %s in 3' TTS genomic downstream region is NOT a DNA sequence in genome file" %(downTTS))
# percA = 0
# elif STRAND == "-":
# TTS = int(EXON_s[0])
# downTTS = ReverseComplement(str(seq[CHROM].seq[int(EXON_s[0]-nPolyA):TTS]))
# percA = downTTS.count('A')/nPolyA*100
if STRAND == "+":
TTS = int(EXON_e[-1])
downTTS = str(seq[CHROM][TTS:(int(EXON_e[-1])+nPolyA)])
downTTS = downTTS.upper()
if any([base not in ["A","T","C","G","N"] for base in downTTS]):
sys.stderr.write("Error: %s in 3' TTS genomic downstream region is NOT a DNA sequence in genome file" %(downTTS))
percA = 0
elif STRAND == "-":
TTS = int(EXON_s[0])
downTTS = ReverseComplement(str(seq[CHROM][int(EXON_s[0]-(nPolyA+1)):(TTS-1)]))
percA = float(downTTS.count('A'))/nPolyA*100
myTranscript_Assoc = myQueryTranscripts("", "NA","NA",len(EXON_s),isoLength, "", chrom=CHROM, strand=STRAND, subtype="no_subcategory", percAdownTTS=str(percA))
##***************************************##
########### SPLICED TRANSCRIPTS ###########
##***************************************##
if len(EXON_s) >= 2: #junctions only if multiple-exon isoforms
if CHROM in transcripts_chrom_exons:
if CHROM not in index_chrom_exons:
index_chrom_exons[CHROM] = 0
for k in range(index_chrom_exons[CHROM], len(transcripts_chrom_exons[CHROM])):
subtype = "no_category"
ref_transcript = transcripts_chrom_exons[CHROM][k]
junctions_s = ref_transcript.junctions[0]
junctions_e = ref_transcript.junctions[1]
junctions = []
[junctions.append([junctions_s[n],junctions_e[n]]) for n in range(len(junctions_s))]
ends_positions.append(int(junctions_e[-1]))
########### SAME STRAND ###########
if ref_transcript.strand == STRAND:
if all(item < int(JUNCTIONS_s[0]) for item in ends_positions) :
index_chrom_exons[CHROM] = index_chrom_exons[CHROM] + 1
elif int(junctions_s[0]) > int(JUNCTIONS_e[-1]):
break
else:
#######################################
##### 1. Check full-splice match ######
#######################################
if (JUNCTIONS_e==junctions_e and JUNCTIONS_s==junctions_s): #splice junctions are identical
tss = int(ref_transcript.tss)
tts = int(ref_transcript.tts)
if STRAND == "+":
TSS = EXON_s[0]
TTS = EXON_e[-1]
tss_diff = int(TSS) - tss
tts_diff = tts - int(TTS)
else:
TTS = EXON_s[0]
TSS = EXON_e[-1]
tss_diff = tss - int(TSS)
tts_diff = int(TTS) - tts
if myTranscript_Assoc.str_class!="full-splice_match":
myTranscript_Assoc = myQueryTranscripts(ref_transcript.transcript, tss_diff, tts_diff, len(EXON_s), isoLength, "full-splice_match", subtype="multi-exon", chrom=CHROM, strand=STRAND, refLen = ref_transcript.transcriptLength(), refExons= len(ref_transcript.exons[0]), percAdownTTS=str(percA))
myTranscript_Assoc.transcripts = []
myTranscript_Assoc.transcripts.append(ref_transcript.transcript)
myTranscript_Assoc.genes = []
myTranscript_Assoc.genes.append(ref_transcript.gene)
elif (abs(tss_diff) + abs(tts_diff)) < myTranscript_Assoc.get_total_diff():
myTranscript_Assoc.genes = []
myTranscript_Assoc.modify(ref_transcript.transcript, ref_transcript.gene, tss_diff, tts_diff, ref_transcript.transcriptLength(), len(ref_transcript.exons[0]))
else:
myTranscript_Assoc.transcripts.append(ref_transcript.transcript)
####################################################################
##### 2. Check fragment-splice match if none full-splice match ######
####################################################################
elif myTranscript_Assoc.str_class!="full-splice_match" and all(s in junctions for s in JUNCTIONS): #check if it's a fragment of a reference transcript if not perfect match found. Just in case you have not found before another perfect match (because we don't break the loop if found a perfect match since we want the similar one (utr differences))
exons_coord = exons_together(ref_transcript.exons[0], ref_transcript.exons[1])
differential_nt = list(set(EXONS_COORD) - set(exons_coord))
longerUTRs = all((int(d) < int(ref_transcript.exons[0][0]) or int(d) > int(ref_transcript.exons[1][-1]) ) for d in differential_nt) # extra-nt in query are for differences in utrs
if len(differential_nt)==0 or longerUTRs:
if STRAND == "+":
(tss_diff, tts_diff) = ref_transcript.difference_fragment_transcript(EXON_s[0], EXON_e[-1], exons_coord)
LAST5JUNCTION = [JUNCTIONS_s[0], JUNCTIONS_e[0]]
LAST3JUNCTION = [JUNCTIONS_e[-1], JUNCTIONS_e[-1]]
last5junction = [junctions_s[0], junctions_e[0]]
last3junction = [junctions_s[-1], junctions_e[-1]]
else:
(tts_diff, tss_diff) = ref_transcript.difference_fragment_transcript(EXON_s[0], EXON_e[-1], exons_coord)
LAST3JUNCTION = [JUNCTIONS_s[0], JUNCTIONS_e[0]]
LAST5JUNCTION = [JUNCTIONS_e[-1], JUNCTIONS_e[-1]]
last3junction = [junctions_s[0], junctions_e[0]]
last5junction = [junctions_s[-1], junctions_e[-1]]
if (LAST5JUNCTION!=last5junction) and (LAST3JUNCTION != last3junction):
subtype = "internal_fragment"
elif LAST5JUNCTION != last5junction:
subtype = "5prime_fragment"
elif LAST3JUNCTION != last3junction:
subtype = "3prime_fragment"
else:
subtype = "complete"
if myTranscript_Assoc.str_class!="incomplete-splice_match":
myTranscript_Assoc = myQueryTranscripts(ref_transcript.transcript, tss_diff, tts_diff, len(EXON_s), isoLength,"incomplete-splice_match", subtype=subtype, chrom=CHROM, strand=STRAND, refLen = ref_transcript.transcriptLength(), refExons= len(ref_transcript.exons[0]), percAdownTTS=str(percA))
myTranscript_Assoc.transcripts.append(ref_transcript.transcript)
myTranscript_Assoc.genes = []
myTranscript_Assoc.genes.append(ref_transcript.gene)
elif (abs(tss_diff) + abs(tts_diff)) < myTranscript_Assoc.get_total_diff():
myTranscript_Assoc.genes = []
myTranscript_Assoc.modify(ref_transcript.transcript, ref_transcript.gene, tss_diff, tts_diff, ref_transcript.transcriptLength(), len(ref_transcript.exons[0]))
else:
myTranscript_Assoc.transcripts.append(ref_transcript.transcript)
########################################################################
##### 3. Association of transcripts to genes by sharing junctions ######
########################################################################