def make_tRNA_fasta_dict(tRNAdf): """ similar to make_fasta_dict, but for the tRNA database """ tRNA_fasta_outdict = OrderedDict() for i in tRNAdf.index: if tRNAdf.loc[i,'feature'] == 'tRNA': chrom = tRNAdf.loc[i,'#chrom'] chrStart = int(tRNAdf.loc[i,'chromStart']) chrEnd = int(tRNAdf.loc[i,'chromEnd']) strand = tRNAdf.loc[i,'strand'] if strand == "+": chrStart = chrStart-1 ### gtf files are 1-based, convert to 0-based trSeq = SeqIO.Seq(genome[chrom][chrStart:chrEnd]) trdict = parse_entry(tRNAdf.loc[i,'transcript_id']) else: # for neg strand chrStart = chrStart-1 trSeq = SeqIO.Seq(genome[chrom][chrStart:chrEnd]) trSeq = trSeq.reverse_complement() trdict = parse_entry(tRNAdf.loc[i,'transcript_id']) trID = "tRNA_"+trdict['gene_id'][0] desc = "| tRNA | "+trdict['gene_type'][0] + " | %s; %s; %s:%s" % (chrom, strand, chrStart, chrEnd) trSeqRec = SeqRecord(trSeq, id=trID, name=trdict['gene_name'][0], description=desc) tRNA_fasta_outdict[trID] = trSeqRec return tRNA_fasta_outdict
def make_fasta_dict(ncdf): fasta_outdict = OrderedDict() for i in ncdf.index: if ncdf.loc[i,'feature'] == 'transcript': chrom = ncdf.loc[i,'#chrom'] chrStart = int(ncdf.loc[i,'chromStart']) chrEnd = int(ncdf.loc[i,'chromEnd']) strand = ncdf.loc[i,'strand'] if strand == "+": chrStart = chrStart-1 ## gtf files are 1 based, convert to 0-based for python trSeq = SeqIO.Seq(genome[chrom][chrStart:chrEnd]) trdict = parse_mod_entry(ncdf.loc[i,'transcript_id']) else: # for neg strand chrStart = chrStart-1 trSeq = SeqIO.Seq(genome[chrom][chrStart:chrEnd]) trSeq = trSeq.reverse_complement() # negative strand trdict = parse_mod_entry(ncdf.loc[i,'transcript_id']) ### add output annotation line features trID = trdict['ID'][0] desc = "| "+trdict['gene_type'][0]+" | "+trdict['gene_name'][0]+ " | %s; %s; %s:%s" % (chrom, strand, chrStart, chrEnd) trSeqRec = SeqRecord(trSeq, id=trID, name=trdict['gene_name'][0], description=desc) fasta_outdict[trID] = trSeqRec return fasta_outdict
def write_consensus_seqs(refseq, contrib_props, contrib_reads, args):
"""
Generates consensus sequences for each contributor from the assigned reads
for output in FASTA format and writes them out.
Args:
refseq: The reference sequence to which the fragments were aligned.
contrib_props: A list of lists containing for each contributor
- contributor ID (hap#)
- haplogroup
- proportion in mixture (not used).
contrib_reads: A table mapping hap# IDs to lists of pysam
AlignedSegments + an entry of unassigned.
args: The argument values from mixemt's argparse results.
Returns:
nothing
"""
with open("%s.fa" % (args.cons_prefix), 'w') as fa_out:
seqs_to_write = list()
for con, hap, _ in contrib_props:
seq = call_consensus(refseq,
contrib_reads[con],
1,
args,
strict=False)
rec = SeqIO.SeqRecord(SeqIO.Seq(seq), id=con, description=hap)
seqs_to_write.append(rec)
if 'unassigned' in contrib_reads:
seq = call_consensus(refseq,
contrib_reads['unassigned'],
1,
args,
strict=False)
rec = SeqIO.SeqRecord(SeqIO.Seq(seq),
id='unassigned',
description='')
seqs_to_write.append(rec)
SeqIO.write(seqs_to_write, fa_out, 'fasta')
return
def parse_result(self, genome_path):
result_path = genome_path + '.gmhmm'
reading_gene = False
with open(result_path) as f:
for line in f:
if line.startswith('>gene'):
reading_gene = True
seq = []
seq_id = re.sub(r'[\s>]', '', line)
# >gene_2|GeneMark.hmm|57_nt|+|1|57 >NODE_3_length_713_cov_1.25228
elif reading_gene:
if line.isspace():
reading_gene = False
seq = SeqIO.Seq(''.join(seq))
#genes.append(Gene(contig_id, strand, left_index, right_index, str_seq))
yield SeqIO.SeqRecord(seq,
id='>' + seq_id,
description='',
name='')
else:
seq.append(line.strip())
def build_utr3_stop_positions(GFFlist):
"""
This is a function to get the cds and utr sizes for an mRNA from a GFF file
returns a list with: #transcript,chrom,featnum,strand,mrna_len,cds_len,5utr_len,3utr_len,gene_name
Includes most of the functions from densebuilder_main but does not return counts
"""
# GFFlist = GFFinput
transcriptdict = {}
ucscIDlist = []
total_transcripts = 0
nonvalidchorms = 0
nonATGstart = 0
wrongstopcodon = 0
shortcontext = 0
validchroms = 0
excluded_chroms = []
included_chroms = []
for chrom in GFFlist:
if not chrom in validChrs:
excluded_chroms.append(chrom)
nonvalidchorms += 1
# print chrom
continue # check that only valid choromosomes are used
validchroms += 1
included_chroms.append(chrom)
transcriptnum = -1 # set to negative one so first transcript is == to 0
for transcript in GFFlist[
chrom].features: # this is where the SeqFeatures are actually stored
tr_attribute_list = []
transcriptnum += 1
trsp_id = transcript.id # it is a number
trsp_strand = transcript.strand
trsp_genename = transcript.qualifiers['Name'][0]
trsp_chromstart = int(
transcript.location.start.position) # 0-based
trsp_chromend = int(transcript.location.end.position)
transcriptlist = [
0.0 for x in range(abs(trsp_chromend - trsp_chromstart))
] # a list for transcript (pre-mRNA), not CDS
exonsplicedseq = SeqIO.Seq('')
transcriptseq = SeqIO.Seq(
genome[chrom][trsp_chromstart:trsp_chromend])
startCodonMrnaList = []
stopCodonMrnaList = []
for item in GFFlist[chrom].features[transcriptnum].sub_features:
if trsp_strand == 1:
if item.type == 'exon': # or item.type== 'CDS': # For yeast, use 'CDS'
exonstart = int(
item.location.start.position) # 0-based position
exonend = int(
item.location.end.position) # not 0-based
exonstart_feat = exonstart - trsp_chromstart
exonend_feat = exonend - trsp_chromstart # Not 0-based, it is fine for length....next line.
exonsplicedseq += transcriptseq[
exonstart_feat:
exonend_feat] # takes from exonstart to exonend-1
if item.type == 'start_codon':
startcodonpos = item.location.start.position # 0-based position
# startcodonmrnapos= chrpostomrnapos(startcodonpos,chrom,transcriptnum,GFFlist) # spliced mRNA position
startCodonMrnaList.append(
chrpostomrnapos(startcodonpos, chrom,
transcriptnum,
GFFlist)) # spliced mRNA position
if item.type == 'stop_codon':
stopcodonpos = item.location.end.position - 1 # 0-based position
# stopcodonmrnapos= chrpostomrnapos(stopcodonpos,chrom,transcriptnum,GFFlist)
stopCodonMrnaList.append(
chrpostomrnapos(stopcodonpos, chrom, transcriptnum,
GFFlist))
if trsp_strand == -1:
# reverse_complement() # this comes from seqIO
transcriptseq_rev = transcriptseq.reverse_complement()
if item.type == 'exon': # or item.type== 'CDS': # For yeast, use 'CDS'
exonstart = int(
item.location.start.position) # 0-based position
exonend = int(
item.location.end.position) # not 0-based
exonstart_feat = (trsp_chromend - 1) - (exonend - 1
) # 0-based
exonend_feat = (trsp_chromend -
1) - exonstart # 0-based
exonseq = transcriptseq_rev[
exonstart_feat:exonend_feat + 1]
exonsplicedseq = exonseq + exonsplicedseq
if item.type == 'start_codon':
startcodonpos = item.location.end.position - 1 # Need to -1 to be 0-based.
# startcodonmrnapos= chrpostomrnapos(startcodonpos,chrom,transcriptnum,GFFlist)
startCodonMrnaList.append(
chrpostomrnapos(startcodonpos, chrom,
transcriptnum, GFFlist))
if item.type == 'stop_codon':
stopcodonpos = item.location.start.position # start.position is 0-based already.
# stopcodonmrnapos= chrpostomrnapos(stopcodonpos,chrom,transcriptnum,GFFlist)
stopCodonMrnaList.append(
chrpostomrnapos(stopcodonpos, chrom, transcriptnum,
GFFlist))
### choose start and stop codons
if len(startCodonMrnaList) > 0:
# print "MORE THAN 1 START", startCodonMrnaList
startcodonmrnapos = min(startCodonMrnaList)
else:
print "!!! no start codon for %s" % (trsp_id)
# if len(stopCodonMrnaList)
if len(stopCodonMrnaList) > 0:
stopcodonmrnapos = max(stopCodonMrnaList)
else:
print "!!! no stop codon for %s" % (trsp_id)
mRNAseq = exonsplicedseq
cdsseq = exonsplicedseq[
startcodonmrnapos:stopcodonmrnapos +
1] # take from startcodonmrnapos to stopcodonmrnapos
utr5seq = exonsplicedseq[:startcodonmrnapos]
utr3seq = exonsplicedseq[stopcodonmrnapos + 1:]
if str(cdsseq[:3].upper()) != "ATG":
nonATGstart += 1
continue # ignore non-AUG start codons
### stopcodon is included in cdsseq, represnted by the last 3nt's
stopcodon = str(cdsseq[-3:].upper())
if stopcodon != "TGA" and stopcodon != "TAG" and stopcodon != "TAA":
wrongstopcodon += 1
continue # ignore weird stop codons
# build itmes in transcript attribute list
mRNAlen = len(exonsplicedseq)
cdslen = len(cdsseq)
utr5len = len(utr5seq)
utr3len = len(utr3seq)
assert mRNAlen == utr3len + cdslen + utr5len # check that sum of features equals mRNA length
###### Finding inframe stop codons ######
### Frame zero for loop,
### count each codon into 3'UTR using 0-based counting
### With zero-based counting, next stopcodon * 3 == adjusted 3'UTR length
frameZeroTrans = utr3seq.translate()
frameZeroStopPositions = []
frameZeroStopPositionsMRNA = []
frameZeroPos = -1
frameZeroStopCounter = 0
frameZeroUtr3LenAdj = 0
for codon in frameZeroTrans:
frameZeroPos += 1
if codon == '*':
frameZeroStopPositions.append(
frameZeroPos * 3) # get utr3position in nucleotides
frameZeroStopPositionsMRNA.append((utr5len + cdslen) +
(frameZeroPos * 3))
### check mRNA position to make sure stop codons are all valid
sc = str(mRNAseq[(utr5len + cdslen) +
(frameZeroPos * 3):(utr5len + cdslen) +
(frameZeroPos * 3) + 3].upper())
if sc != "TAA" and sc != "TAG" and sc != "TGA":
print "stop codon in frame 0 for %s is non correct!" % trsp_id
print "stopcodon is: %s" % sc
sys.exit()
frameZeroStopCounter += 1
if codon == '*' and frameZeroStopCounter == 1:
frameZeroUtr3LenAdj = frameZeroPos * 3
if frameZeroUtr3LenAdj == 0 and frameZeroStopCounter == 0:
frameZeroUtr3LenAdj = len(utr3seq)
### Frame +1 for loop,
framePlusOneTrans = utr3seq[1:].translate(
) # start one nucleotide into 3'UTR for +1 frameshift
framePlusOneStopPositions = []
framePlusOneStopPositionsMRNA = []
framePlusOnePos = -1
framePlusOneStopCounter = 0
framePlusOneUtr3LenAdj = 0
for codon in framePlusOneTrans:
framePlusOnePos += 1
if codon == '*':
framePlusOneStopPositions.append(
(framePlusOnePos * 3) +
1) # get utr3position in nucleotides
framePlusOneStopPositionsMRNA.append((utr5len + cdslen) +
(framePlusOnePos *
3) + 1)
### check mRNA position to make sure stop codons are all valid
sc = str(
mRNAseq[((utr5len + cdslen) + (framePlusOnePos * 3) +
1):((utr5len + cdslen) +
(framePlusOnePos * 3) + 1) + 3].upper())
if sc != "TAA" and sc != "TAG" and sc != "TGA":
print "stop codon in frame +1 for %s is non correct!" % trsp_id
print "stopcodon is: %s" % sc
sys.exit()
framePlusOneStopCounter += 1
if codon == '*' and framePlusOneStopCounter == 1:
framePlusOneUtr3LenAdj = (framePlusOnePos * 3) + 1
if framePlusOneUtr3LenAdj == 0 and frameZeroStopCounter == 0:
framePlusOneUtr3LenAdj = len(utr3seq[1:])
### Frame -1 for loop,
frameMinusOneTrans = (cdsseq[-1] + utr3seq).translate(
) # include last nucleotide of cds for -1 frameshift
frameMinusOneStopPositions = []
frameMinusOneStopPositionsMRNA = []
frameMinusOnePos = -1
frameMinusOneStopCounter = 0
frameMinusOneUtr3LenAdj = 0
for codon in frameMinusOneTrans:
frameMinusOnePos += 1
if codon == '*':
frameMinusOneStopPositions.append(
(frameMinusOnePos * 3) -
1) # get utr3position in nucleotides
frameMinusOneStopPositionsMRNA.append((utr5len + cdslen) +
(frameMinusOnePos *
3) - 1)
### check mRNA position to make sure stop codons are all valid
sc = str(
mRNAseq[((utr5len + cdslen) + (frameMinusOnePos * 3) -
1):((utr5len + cdslen) +
(frameMinusOnePos * 3) - 1) + 3].upper())
if sc != "TAA" and sc != "TAG" and sc != "TGA":
print "stop codon in frame -1 for %s is non correct!" % trsp_id
print "stopcodon is: %s" % sc
sys.exit()
frameMinusOneStopCounter += 1
if codon == '*' and frameMinusOneStopCounter == 1:
frameMinusOneUtr3LenAdj = (frameMinusOnePos * 3) - 1
if frameMinusOneUtr3LenAdj == 0 and frameZeroStopCounter == 0:
frameMinusOneUtr3LenAdj = len(cdsseq[-1] + utr3seq)
####
trsp_attr_list = [
trsp_id, trsp_genename, frameZeroStopPositions,
frameZeroStopPositionsMRNA, framePlusOneStopPositions,
framePlusOneStopPositionsMRNA, frameMinusOneStopPositions,
frameMinusOneStopPositionsMRNA
]
ucscIDlist.append(trsp_attr_list[0])
transcriptdict[trsp_id] = trsp_attr_list
total_transcripts += 1
#transcript,chrom,featnum,strand,mrna_len,cds_len,5utr_len,3utr_len,gene_name,stopcodon,stop4nt
print "total number of transcripts in data table: %s" % total_transcripts
print "Number of included chromosomes chr: %s" % validchroms
print "Number of excluded chromosomes chr: %s" % nonvalidchorms
print "included chroms: ", included_chroms
print "excluded chroms: ", excluded_chroms
print "transcripts discarded due to non-AUG start codon %s" % nonATGstart
print "transcripts discarded due to noncanonical stop codon %s" % wrongstopcodon
return ucscIDlist, transcriptdict
def builddense(self):
transcriptdict = {}
mappedlocalreads = 0
dumppedreads = 0
illegalreads = 0
tooshortlongreads = 0
wrongstrandreads = 0
noStartOrStop = 0
noStartCodon = 0
noStopCodon = 0
totalreads = 0 # not totreads
GFFlist = self.makeGFFlist(self.GTFgen)
# validChrs = 'chrLUC' # for building a single chromosome
validChrs = [
'chr1', 'chr2', 'chr3', 'chr4', 'chr5', 'chr6', 'chr7', 'chr8',
'chr9', 'chr10', 'chr11', 'chr12', 'chr13', 'chr14', 'chr15',
'chr16', 'chr17', 'chr18', 'chr19', 'chr20', 'chr21', 'chr22',
'chrX', 'chrY', 'chrM', 'chrSinV', 'chrLUC'
]
for chrom in GFFlist:
if not chrom in validChrs: continue
transcriptnum = -1
for transcript in GFFlist[chrom].features:
transcriptnum += 1
trsp_id = transcript.id # it is a number
trsp_strand = transcript.strand
# if transcript.type == 'inferred_parent': # this is a hack to deal with improperly formatted gtf files, will return strand == 0
# trsp_strand = transcript.sub_features[0].strand # use the first subfeature entry to get strand instead
trsp_chromstart = int(
transcript.location.start.position) # 0-based
trsp_chromend = int(transcript.location.end.position)
transcriptlist = [
0.0 for x in range(abs(trsp_chromend - trsp_chromstart))
] # a list for transcript (pre-mRNA), not CDS
gb = self.getbam_5or3counts(
self.bamfile, transcriptlist, chrom, transcriptnum,
trsp_chromstart, trsp_chromend, trsp_id, trsp_strand,
self.riboshiftdict, self.assignment, self.bamfileout
) # return a riboshifted list (0-based) of unspliced counts
mappedlocalreads += gb[1]
dumppedreads += gb[2]
illegalreads += gb[3]
tooshortlongreads += gb[4]
wrongstrandreads += gb[5]
totalreads += gb[6]
exonsplicedseq = SeqIO.Seq('')
exonsplicedcounts = []
transcriptseq = SeqIO.Seq(
genome[chrom][trsp_chromstart:trsp_chromend])
# For EGFP
#transcriptseq= genome
#if transcript.type== 'gene': # For yeast
# if trsp_strand== 1:
# startcodonpos= transcript.location.start.position
# startcodonmrnapos= seqtools.chrpostomrnapos(startcodonpos,chrom,transcriptnum,GFFlist)
# stopcodonpos= transcript.location.end.position- 1# 0-based position
# stopcodonmrnapos= seqtools.chrpostomrnapos(stopcodonpos,chrom,transcriptnum,GFFlist)
# if trsp_strand== -1:
# startcodonpos= transcript.location.end.position- 1
# startcodonmrnapos= seqtools.chrpostomrnapos(startcodonpos,chrom,transcriptnum,GFFlist)
# stopcodonpos= transcript.location.start.position # 0-based position, the first nt of stop codon
# stopcodonmrnapos= seqtools.chrpostomrnapos(stopcodonpos,chrom,transcriptnum,GFFlist)
### handling transcripts with no start or stop codon:
# startcodonmrnapos = 'absent'
# stopcodonmrnapos = 'absent'
startCodonMrnaList = []
stopCodonMrnaList = []
for item in GFFlist[chrom].features[
transcriptnum].sub_features:
if trsp_strand == 1:
if item.type == 'exon': # or item.type== 'CDS': # For yeast, use 'CDS'
exonstart = int(item.location.start.position
) # 0-based position
exonend = int(
item.location.end.position) # not 0-based
exonstart_feat = exonstart - trsp_chromstart
exonend_feat = exonend - trsp_chromstart # Not 0-based, it is fine for length....next line.
exonsplicedcounts += gb[0][
exonstart_feat:
exonend_feat] # takes from exonstart to exonend-1
exonsplicedseq += transcriptseq[
exonstart_feat:
exonend_feat] # takes from exonstart to exonend-1
if item.type == 'start_codon':
startcodonpos = item.location.start.position # 0-based position
# startcodonmrnapos= self.chrpostomrnapos(startcodonpos,chrom,transcriptnum,GFFlist) # spliced mRNA position
startCodonMrnaList.append(
self.chrpostomrnapos(
startcodonpos, chrom, transcriptnum,
GFFlist)) # spliced mRNA position
if item.type == 'stop_codon':
stopcodonpos = item.location.end.position - 1 # 0-based position
# stopcodonmrnapos= self.chrpostomrnapos(stopcodonpos,chrom,transcriptnum,GFFlist)
stopCodonMrnaList.append(
self.chrpostomrnapos(stopcodonpos, chrom,
transcriptnum, GFFlist))
if trsp_strand == -1:
transcriptseq_rev = transcriptseq.reverse_complement()
if item.type == 'exon': # or item.type== 'CDS': # For yeast, use 'CDS'
exonstart = int(item.location.start.position
) # 0-based position
exonend = int(
item.location.end.position) # not 0-based
exonstart_feat = (trsp_chromend - 1) - (
exonend - 1) # 0-based
exonend_feat = (trsp_chromend -
1) - exonstart # 0-based
exoncounts = gb[0][
exonstart_feat:exonend_feat +
1] # both 0-based, need to +1 for length
exonsplicedcounts = exoncounts + exonsplicedcounts # exoncounts added to the upstream of existing counts, so don't flip again.
exonseq = transcriptseq_rev[
exonstart_feat:exonend_feat + 1]
exonsplicedseq = exonseq + exonsplicedseq
if item.type == 'start_codon':
startcodonpos = item.location.end.position - 1 # Need to -1 to be 0-based.
# startcodonmrnapos= self.chrpostomrnapos(startcodonpos,chrom,transcriptnum,GFFlist)
startCodonMrnaList.append(
self.chrpostomrnapos(startcodonpos, chrom,
transcriptnum, GFFlist))
if item.type == 'stop_codon':
stopcodonpos = item.location.start.position # start.position is 0-based already.
# stopcodonmrnapos= self.chrpostomrnapos(stopcodonpos,chrom,transcriptnum,GFFlist)
stopCodonMrnaList.append(
self.chrpostomrnapos(stopcodonpos, chrom,
transcriptnum, GFFlist))
if len(startCodonMrnaList) > 0:
# print "MORE THAN 1 START", startCodonMrnaList
startcodonmrnapos = min(startCodonMrnaList)
else:
noStartCodon += 1
startcodonmrnapos = 0 ### adding for transcripts without start codon
# print "!!! no start codon for %s" % (trsp_id)
if len(stopCodonMrnaList) > 0:
stopcodonmrnapos = max(stopCodonMrnaList)
else:
noStopCodon += 1
stopcodonmrnapos = len(
exonsplicedseq) - 3 ### leave 3nt's in "3'UTR"
# print "!!! no stop codon for %s" % (trsp_id)
# if startcodonmrnapos == 'absent' or stopcodonmrnapos == 'absent':
# noStartOrStop +=1
# continue
cdsseq = exonsplicedseq[
startcodonmrnapos:stopcodonmrnapos +
1] # take from startcodonmrnapos to stopcodonmrnapos
cdscounts = exonsplicedcounts[
startcodonmrnapos:stopcodonmrnapos +
1] # take from startcodonmrnapos to stopcodonmrnapos
# if str(cdsseq[:3].upper())!= "ATG": continue # ignore non-AUG start codons
# stopcodon= str(cdsseq[-3:].upper())
# if stopcodon!= "TGA" and stopcodon!= "TAG" and stopcodon!= "TAA": continue # ignore weird stop codons
#utr5len= startcodonmrnapos
#utr3len= len(exonsplicedseq)- stopcodonmrnapos- 1
if sum(cdscounts) >= float(
self.threshold): # thresholding minimal reads per CDS.
transcriptdict[trsp_id] = exonsplicedcounts
if self.totreads == '-1':
print str(
totalreads) + " total mapped reads used for normalization."
self.norm_m(
transcriptdict, totalreads
) # Normalzied by total reads mapped to transcriptdict only... but not total mapped reads.
else:
print str(
self.totreads
) + " total mapped reads from STAR alignment used for normalization."
self.norm_m(transcriptdict, self.totreads)
### disable writing counts to file here
# self.writecountsf(transcriptdict, self.outputdata)
### assemble dataframe here
outdict = OrderedDict()
for key, val in transcriptdict.items():
outdict[key] = [val]
df = pd.DataFrame.from_dict(outdict, orient='index')
df.columns = ['density']
print df.head()
df.to_csv('%s.csv.gz' % (self.outputdata), compression='gzip')
# Write output file of comments.
fc = open(self.outputdata + "output.txt", "w")
fc.write("Density was built with parameters:\n")
fc.write("riboshiftdict=" + str(self.riboshiftdict) + "\n")
fc.write("threshold=" + str(self.threshold) + "\n")
fc.write("assignment=" + str(self.assignment) + "\n")
fc.write("reads mapped to known canonical coding transcripts: " +
str(mappedlocalreads) + "\n")
fc.write("reads are dumpped, due to weird cigar codes: " +
str(dumppedreads) + "\n")
fc.write(
"reads are illegal, mapped outside of annotated transcripts: " +
str(illegalreads) + "\n")
fc.write("reads are too short/long: " + str(tooshortlongreads) + "\n")
fc.write("reads are on the wrong strand: " + str(wrongstrandreads) +
"\n")
fc.write("total mapped reads from aligner: " + str(totalreads))
fc.close()
print str(
mappedlocalreads
) + " reads within length limitation mapped to known canonical coding transcripts. "
print str(
dumppedreads) + " reads are dumpped, due to weird cigar codes."
print str(
illegalreads
) + " reads are illegal, mapped outside of annotated transcripts."
print str(tooshortlongreads) + " reads are too short/long."
print str(wrongstrandreads) + " reads are on the wrong strand."
print str(totalreads) + " total mapped reads from aligner. "
def build_utr_table(GFFlist, inculde_noncanon_start, include_noncanon_stop):
"""
This is a function to get the cds and utr sizes for an mRNA from a GFF file
returns a list with: #transcript,chrom,featnum,strand,mrna_len,cds_len,5utr_len,3utr_len,gene_name
Includes most of the functions from densebuilder_main but does not return counts
"""
# GFFlist = GFFinput
transcriptdict = {}
ucscIDlist = []
total_transcripts = 0
nonvalidchorms = 0
nonATGstart = 0
wrongstopcodon = 0
validchroms = 0
excluded_chroms = []
included_chroms = []
for chrom in GFFlist:
if not chrom in validChrs:
excluded_chroms.append(chrom)
nonvalidchorms += 1
# print chrom
continue # check that only valid choromosomes are used
validchroms += 1
included_chroms.append(chrom)
transcriptnum = -1 # set to negative one so first transcript is == to 0
for transcript in GFFlist[
chrom].features: # this is where the SeqFeatures are actually stored
tr_attribute_list = []
transcriptnum += 1
trsp_id = transcript.id # it is a number
trsp_strand = transcript.strand
### changing this to be compatible with new hg38 annotation
# print transcript.qualifiers ### these are all of the fields parsed by the GTF parser from column 8, output is a dictionary {'key':['item1', 'item2', 'ect']}
trsp_genename = transcript.qualifiers['Name'][0]
trsp_chromstart = int(
transcript.location.start.position) # 0-based
trsp_chromend = int(transcript.location.end.position)
transcriptlist = [
0.0 for x in range(abs(trsp_chromend - trsp_chromstart))
] # a list for transcript (pre-mRNA), not CDS
exonsplicedseq = SeqIO.Seq('')
transcriptseq = SeqIO.Seq(
genome[chrom][trsp_chromstart:trsp_chromend])
### use lists to handle transcripts with multiple start and stop codons
startCodonMrnaList = []
stopCodonMrnaList = []
for item in GFFlist[chrom].features[transcriptnum].sub_features:
if trsp_strand == 1:
### dealing with transcripts having multiple start or stop codon entries, if spaning splice junctions
if item.type == 'exon': # or item.type== 'CDS': # For yeast, use 'CDS'
exonstart = int(
item.location.start.position) # 0-based position
exonend = int(
item.location.end.position) # not 0-based
exonstart_feat = exonstart - trsp_chromstart
exonend_feat = exonend - trsp_chromstart # Not 0-based, it is fine for length....next line.
exonsplicedseq += transcriptseq[
exonstart_feat:
exonend_feat] # takes from exonstart to exonend-1
if item.type == 'start_codon':
startcodonpos = item.location.start.position # 0-based position
# startcodonmrnapos= chrpostomrnapos(startcodonpos,chrom,transcriptnum,GFFlist) # spliced mRNA position
startCodonMrnaList.append(
chrpostomrnapos(startcodonpos, chrom,
transcriptnum,
GFFlist)) # spliced mRNA position
# print startcodonmrnapos
if item.type == 'stop_codon':
stopcodonpos = item.location.end.position - 1 # 0-based position
# stopcodonmrnapos= chrpostomrnapos(stopcodonpos,chrom,transcriptnum,GFFlist)
stopCodonMrnaList.append(
chrpostomrnapos(stopcodonpos, chrom, transcriptnum,
GFFlist))
# print stopcodonmrnapos
if trsp_strand == -1:
# print 'neg_strand'
# reverse_complement() # this comes from seqIO
transcriptseq_rev = transcriptseq.reverse_complement()
if item.type == 'exon': # or item.type== 'CDS': # For yeast, use 'CDS'
exonstart = int(
item.location.start.position) # 0-based position
exonend = int(
item.location.end.position) # not 0-based
exonstart_feat = (trsp_chromend - 1) - (exonend - 1
) # 0-based
exonend_feat = (trsp_chromend -
1) - exonstart # 0-based
exonseq = transcriptseq_rev[
exonstart_feat:exonend_feat + 1]
exonsplicedseq = exonseq + exonsplicedseq
if item.type == 'start_codon':
startcodonpos = item.location.end.position - 1 # Need to -1 to be 0-based.
# print startcodonpos
# startcodonmrnapos= chrpostomrnapos(startcodonpos,chrom,transcriptnum,GFFlist)
startCodonMrnaList.append(
chrpostomrnapos(startcodonpos, chrom,
transcriptnum, GFFlist))
# print "start codon: ", startcodonmrnapos
if item.type == 'stop_codon':
stopcodonpos = item.location.start.position # start.position is 0-based already.
# print stopcodonpos
# stopcodonmrnapos= chrpostomrnapos(stopcodonpos,chrom,transcriptnum,GFFlist)
stopCodonMrnaList.append(
chrpostomrnapos(stopcodonpos, chrom, transcriptnum,
GFFlist))
# print "stop codon: ", stopcodonmrnapos
if len(startCodonMrnaList) > 0:
# print "MORE THAN 1 START", startCodonMrnaList
startcodonmrnapos = min(startCodonMrnaList)
else:
print "!!! no start codon for %s" % (trsp_id)
startcodonmrnapos = 0 ### adding for transcripts without start codon
# if len(stopCodonMrnaList)
if len(stopCodonMrnaList) > 0:
stopcodonmrnapos = max(stopCodonMrnaList)
else:
print "!!! no stop codon for %s" % (trsp_id)
stopcodonmrnapos = len(
exonsplicedseq) - 3 ### leave 3nt's in "3'UTR"
cdsseq = exonsplicedseq[
startcodonmrnapos:stopcodonmrnapos +
1] # take from startcodonmrnapos to stopcodonmrnapos
utr5seq = exonsplicedseq[:startcodonmrnapos]
utr3seq = exonsplicedseq[stopcodonmrnapos + 1:]
# print trsp_id
# # print transcript.qualifiers['transcript_name']
# print trsp_strand
# print utr5seq
# print " - - - "
# print cdsseq
# print " - - - "
# print utr3seq
# # print utr5seq+cdsseq+utr3seq
# print ""
# # print transcriptseq
if inculde_noncanon_start == False:
if str(cdsseq[:3].upper()) != "ATG":
nonATGstart += 1
print "non canon start"
print trsp_id
print cdsseq
print ""
continue # ignore non-AUG start codons
stopcodon = str(cdsseq[-3:].upper())
if len(utr3seq) > 0:
stop4nt = stopcodon + str(utr3seq[0].upper())
elif len(utr3seq) == 0:
stop4nt = '0'
else:
print "there is a 3'UTR with negative length..."
sys.exit()
if include_noncanon_stop == False:
if stopcodon != "TGA" and stopcodon != "TAG" and stopcodon != "TAA":
wrongstopcodon += 1
print "wrong stop!"
print trsp_id
print cdsseq
print ""
continue # ignore weird stop codons
# build itmes in transcript attribute list
mRNAlen = len(exonsplicedseq)
cdslen = len(cdsseq)
utr5len = len(utr5seq)
utr3len = len(utr3seq)
assert mRNAlen == utr3len + cdslen + utr5len # check that sum of features equals mRNA length
trsp_attr_list = [
trsp_id, chrom, transcriptnum, trsp_strand, mRNAlen, cdslen,
utr5len, utr3len, trsp_genename, stopcodon, stop4nt
]
ucscIDlist.append(trsp_attr_list[0])
transcriptdict[trsp_id] = trsp_attr_list
total_transcripts += 1
#transcript,chrom,featnum,strand,mrna_len,cds_len,5utr_len,3utr_len,gene_name,stopcodon,stop4nt
print "total number of transcripts in data table: %s" % total_transcripts
print "Number of included chromosomes chr: %s" % validchroms
print "Number of excluded chromosomes chr: %s" % nonvalidchorms
print "included chroms: ", included_chroms
print "excluded chroms: ", excluded_chroms
print "transcripts discarded due to non-AUG start codon %s" % nonATGstart
print "transcripts discarded due to noncanonical stop codon %s" % wrongstopcodon
return ucscIDlist, transcriptdict
def get_Prot_sequence(GFFlist):
transcriptdict = {}
ucscIDlist = []
total_transcripts = 0
nonvalidchorms = 0
nonATGstart = 0
wrongstopcodon = 0
validchroms = 0
excluded_chroms = []
included_chroms = []
for chrom in GFFlist:
if not chrom in validChrs:
excluded_chroms.append(chrom)
nonvalidchorms += 1
# print chrom
continue # check that only valid choromosomes are used
validchroms += 1
included_chroms.append(chrom)
transcriptnum = -1 # set to negative one so first transcript is == to 0
for transcript in GFFlist[
chrom].features: # this is where the SeqFeatures are actually stored
tr_attribute_list = []
transcriptnum += 1
trsp_id = transcript.id # it is a number
trsp_strand = transcript.strand
trsp_genename = transcript.qualifiers['Name'][0]
trsp_chromstart = int(
transcript.location.start.position) # 0-based
trsp_chromend = int(transcript.location.end.position)
transcriptlist = [
0.0 for x in range(abs(trsp_chromend - trsp_chromstart))
] # a list for transcript (pre-mRNA), not CDS
exonsplicedseq = SeqIO.Seq('')
transcriptseq = SeqIO.Seq(
genome[chrom][trsp_chromstart:trsp_chromend])
### use lists to handle transcripts with multiple start and stop codons
startCodonMrnaList = []
stopCodonMrnaList = []
for item in GFFlist[chrom].features[transcriptnum].sub_features:
if trsp_strand == 1:
if item.type == 'exon': # or item.type== 'CDS': # For yeast, use 'CDS'
exonstart = int(
item.location.start.position) # 0-based position
exonend = int(
item.location.end.position) # not 0-based
exonstart_feat = exonstart - trsp_chromstart
exonend_feat = exonend - trsp_chromstart # Not 0-based, it is fine for length....next line.
exonsplicedseq += transcriptseq[
exonstart_feat:
exonend_feat] # takes from exonstart to exonend-1
if item.type == 'start_codon':
startcodonpos = item.location.start.position # 0-based position
# startcodonmrnapos= chrpostomrnapos(startcodonpos,chrom,transcriptnum,GFFlist) # spliced mRNA position
startCodonMrnaList.append(
chrpostomrnapos(startcodonpos, chrom,
transcriptnum,
GFFlist)) # spliced mRNA position
if item.type == 'stop_codon':
stopcodonpos = item.location.end.position - 1 # 0-based position
# stopcodonmrnapos= chrpostomrnapos(stopcodonpos,chrom,transcriptnum,GFFlist)
stopCodonMrnaList.append(
chrpostomrnapos(stopcodonpos, chrom, transcriptnum,
GFFlist))
if trsp_strand == -1:
# reverse_complement() # this comes from seqIO
transcriptseq_rev = transcriptseq.reverse_complement()
if item.type == 'exon': # or item.type== 'CDS': # For yeast, use 'CDS'
exonstart = int(
item.location.start.position) # 0-based position
exonend = int(
item.location.end.position) # not 0-based
exonstart_feat = (trsp_chromend - 1) - (exonend - 1
) # 0-based
exonend_feat = (trsp_chromend -
1) - exonstart # 0-based
exonseq = transcriptseq_rev[
exonstart_feat:exonend_feat + 1]
exonsplicedseq = exonseq + exonsplicedseq
if item.type == 'start_codon':
startcodonpos = item.location.end.position - 1 # Need to -1 to be 0-based.
# startcodonmrnapos= chrpostomrnapos(startcodonpos,chrom,transcriptnum,GFFlist)
startCodonMrnaList.append(
chrpostomrnapos(startcodonpos, chrom,
transcriptnum, GFFlist))
if item.type == 'stop_codon':
stopcodonpos = item.location.start.position # start.position is 0-based already.
# stopcodonmrnapos= chrpostomrnapos(stopcodonpos,chrom,transcriptnum,GFFlist)
stopCodonMrnaList.append(
chrpostomrnapos(stopcodonpos, chrom, transcriptnum,
GFFlist))
### choose start and stop codons
if len(startCodonMrnaList) > 0:
# print "MORE THAN 1 START", startCodonMrnaList
startcodonmrnapos = min(startCodonMrnaList)
else:
print "!!! no start codon for %s" % (trsp_id)
# if len(stopCodonMrnaList)
if len(stopCodonMrnaList) > 0:
stopcodonmrnapos = max(stopCodonMrnaList)
else:
print "!!! no stop codon for %s" % (trsp_id)
mRNAseq = exonsplicedseq
cdsseq = exonsplicedseq[
startcodonmrnapos:stopcodonmrnapos +
1] # take from startcodonmrnapos to stopcodonmrnapos
utr5seq = exonsplicedseq[:startcodonmrnapos]
utr3seq = exonsplicedseq[stopcodonmrnapos + 1:]
cdsProt = cdsseq.translate()
# outseq = utr5seq.lower()+cdsseq.upper()+utr3seq.lower()
if str(cdsseq[:3].upper()) != "ATG":
nonATGstart += 1
continue # ignore non-AUG start codons
### stopcodon is included in cdsseq, represnted by the last 3nt's
stopcodon = str(cdsseq[-3:].upper())
if stopcodon != "TGA" and stopcodon != "TAG" and stopcodon != "TAA":
wrongstopcodon += 1
continue # ignore weird stop codons
# build itmes in transcript attribute list
mRNAlen = len(exonsplicedseq)
cdslen = len(cdsseq)
utr5len = len(utr5seq)
utr3len = len(utr3seq)
assert mRNAlen == utr3len + cdslen + utr5len # check that sum of features equals mRNA length
trsp_attr_list = [trsp_id, trsp_genename, cdsProt]
ucscIDlist.append(trsp_attr_list[0])
transcriptdict[trsp_id] = trsp_attr_list
total_transcripts += 1
print "total number of transcripts in data table: %s" % total_transcripts
print "Number of included chromosomes chr: %s" % validchroms
print "Number of excluded chromosomes chr: %s" % nonvalidchorms
print "included chroms: ", included_chroms
print "excluded chroms: ", excluded_chroms
print "transcripts discarded due to non-AUG start codon %s" % nonATGstart
print "transcripts discarded due to noncanonical stop codon %s" % wrongstopcodon
return ucscIDlist, transcriptdict
def find_uORFs(GFFlist):
"""
using the same basic structure as denesbuilder_main, this function identifies all uORFs and write csv files
"""
### define start codon
## could possibly change this to look at non canonical start codons
startCodon = Seq('ATG')
### build empty data frames, rows will be appended as function iterates over transcripts
dfCols = [
'trxname', 'symbol', 'strand', 'uORFCounter', 'startPosition',
'cdsExtension', 'utr5len', 'cdslen', 'utr3len', 'uORFlen', 'uORFseq',
'uORFaa'
]
uORFdf = pd.DataFrame(columns=dfCols)
summaryCols = [
'trxname', 'symbol', 'chr', 'tr_number', 'strand', 'uORFCounter',
'cdsExtension'
]
summarydf = pd.DataFrame(columns=summaryCols)
####
total_transcripts = 0
nonvalidchorms = 0
nonATGstart = 0
wrongstopcodon = 0
validchroms = 0
excluded_chroms = []
included_chroms = []
for chrom in GFFlist:
if not chrom in validChrs:
excluded_chroms.append(chrom)
nonvalidchorms += 1
# print chrom
continue # check that only valid choromosomes are used
validchroms += 1
included_chroms.append(chrom)
transcriptnum = -1 # set to negative one so first transcript is == to 0
# print chrom
for transcript in GFFlist[
chrom].features: # this is where the SeqFeatures are actually stored
# print transcript
tr_attribute_list = []
transcriptnum += 1
trsp_id = transcript.id # it is a number
trsp_strand = transcript.strand
trsp_genename = transcript.qualifiers['Name'][0]
trsp_chromstart = int(
transcript.location.start.position) # 0-based
trsp_chromend = int(transcript.location.end.position)
transcriptlist = [
0.0 for x in range(abs(trsp_chromend - trsp_chromstart))
] # a list for transcript (pre-mRNA), not CDS
exonsplicedseq = SeqIO.Seq('')
transcriptseq = SeqIO.Seq(
genome[chrom][trsp_chromstart:trsp_chromend])
### handling transcripts with no start or stop codon:
# startcodonmrnapos = 'absent'
# stopcodonmrnapos = 'absent'
startCodonMrnaList = []
stopCodonMrnaList = []
for item in GFFlist[chrom].features[transcriptnum].sub_features:
if trsp_strand == 1:
if item.type == 'exon': # or item.type== 'CDS': # For yeast, use 'CDS'
exonstart = int(
item.location.start.position) # 0-based position
exonend = int(
item.location.end.position) # not 0-based
exonstart_feat = exonstart - trsp_chromstart
exonend_feat = exonend - trsp_chromstart # Not 0-based, it is fine for length....next line.
exonsplicedseq += transcriptseq[
exonstart_feat:
exonend_feat] # takes from exonstart to exonend-1
if item.type == 'start_codon':
startcodonpos = item.location.start.position # 0-based position
# startcodonmrnapos= chrpostomrnapos(startcodonpos,chrom,transcriptnum,GFFlist) # spliced mRNA position
startCodonMrnaList.append(
chrpostomrnapos(startcodonpos, chrom,
transcriptnum, GFFlist))
if item.type == 'stop_codon':
stopcodonpos = item.location.end.position - 1 # 0-based position
# stopcodonmrnapos= chrpostomrnapos(stopcodonpos,chrom,transcriptnum,GFFlist)
stopCodonMrnaList.append(
chrpostomrnapos(stopcodonpos, chrom, transcriptnum,
GFFlist))
if trsp_strand == -1:
# reverse_complement() # this comes from seqIO
transcriptseq_rev = transcriptseq.reverse_complement()
if item.type == 'exon': # or item.type== 'CDS': # For yeast, use 'CDS'
exonstart = int(
item.location.start.position) # 0-based position
exonend = int(
item.location.end.position) # not 0-based
exonstart_feat = (trsp_chromend - 1) - (exonend - 1
) # 0-based
exonend_feat = (trsp_chromend -
1) - exonstart # 0-based
exonseq = transcriptseq_rev[
exonstart_feat:exonend_feat + 1]
exonsplicedseq = exonseq + exonsplicedseq
if item.type == 'start_codon':
startcodonpos = item.location.end.position - 1 # Need to -1 to be 0-based.
# startcodonmrnapos= chrpostomrnapos(startcodonpos,chrom,transcriptnum,GFFlist)
startCodonMrnaList.append(
chrpostomrnapos(startcodonpos, chrom,
transcriptnum, GFFlist))
if item.type == 'stop_codon':
stopcodonpos = item.location.start.position # start.position is 0-based already.
# stopcodonmrnapos= chrpostomrnapos(stopcodonpos,chrom,transcriptnum,GFFlist)
stopCodonMrnaList.append(
chrpostomrnapos(stopcodonpos, chrom, transcriptnum,
GFFlist))
if len(startCodonMrnaList) > 0:
# print "MORE THAN 1 START", startCodonMrnaList
startcodonmrnapos = min(startCodonMrnaList)
else:
print "!!! no start codon for %s" % (trsp_id)
# if len(stopCodonMrnaList)
if len(stopCodonMrnaList) > 0:
stopcodonmrnapos = max(stopCodonMrnaList)
else:
print "!!! no stop codon for %s" % (trsp_id)
# if startcodonmrnapos == 'absent' or stopcodonmrnapos == 'absent':
# # print "no start of stop for trsp %s" % trsp_id
# continue
cdsseq = exonsplicedseq[
startcodonmrnapos:stopcodonmrnapos +
1] # take from startcodonmrnapos to stopcodonmrnapos
utr5seq = exonsplicedseq[:startcodonmrnapos]
utr3seq = exonsplicedseq[stopcodonmrnapos + 1:]
if str(cdsseq[:3].upper()) != "ATG":
nonATGstart += 1
continue # ignore non-AUG start codons
stopcodon = str(cdsseq[-3:].upper())
# if len(utr3seq) > 0:
# stop4nt = stopcodon +str(utr3seq[0].upper())
# elif len(utr3seq) == 0:
# stop4nt = '0'
# else:
# print "there is a 3'UTR with negative length..."
# sys.exit()
if stopcodon != "TGA" and stopcodon != "TAG" and stopcodon != "TAA":
wrongstopcodon += 1
continue # ignore weird stop codons
# build itmes in transcript attribute list
mRNAlen = len(exonsplicedseq)
cdslen = len(cdsseq)
utr5len = len(utr5seq)
utr3len = len(utr3seq)
assert mRNAlen == utr3len + cdslen + utr5len # check that sum of features equals mRNA length
#### Counting of uORFs ####
uORFcounter = 0
cdsExtension = 0
for i in range(len(utr5seq)):
### iterate over every nucleotide in the 5'UTR
codon = utr5seq[i:i + 3] # define the codon at each position
if str(codon) == str(
startCodon): # check if it is a start codon
uORFcounter += 1
startPosition = i
seqIndex = i
uORFaa = []
uORFseq = []
# print "found start codon at pos %s" % startPosition
aminoAcid = codon.translate()
uORFseq.append(str(codon))
uORFaa.append(str(aminoAcid))
while str(
aminoAcid
) != "*": # continue this loop until a stop codon is encoutered
seqIndex += 3 # advance by 3 nt's each time (1 codon)
nextCodon = utr5seq[seqIndex:seqIndex + 3]
aminoAcid = nextCodon.translate()
if len(
nextCodon
) == 3: # ensure that a full codon is still present, do not want 1 or 2 nts
uORFseq.append(str(nextCodon))
uORFaa.append(str(aminoAcid))
if seqIndex > len(
utr5seq
) - 2: # if uORF continues into cds, retreive sequences from here
# -2 is because this will not yeild a full codon (only 2 nt's)
# print "end of UTR"
cdsExtension = 1
utrCdsSeq = utr5seq + cdsseq
nextCodon = utrCdsSeq[seqIndex:seqIndex + 3]
aminoAcid = nextCodon.translate()
uORFseq.append(str(nextCodon))
uORFaa.append(str(aminoAcid))
# print nextCodon, aminoAcid
if seqIndex > len(
utrCdsSeq
): ## if uORF exceeds coding region, stop counting this,
### could eventually extend to the 3'UTR if any transcript exists here
print 'end of CDS for trsp %s' % trsp_id
break
uORFseqCat = "".join(
uORFseq
) # remove seperate list entries and concat to a string
uORFaaCat = "".join(uORFaa)
### save all uORF features to a list, and build into a dataframe
uORF_features = [
trsp_id, trsp_genename, trsp_strand, uORFcounter,
startPosition, cdsExtension,
len(utr5seq),
len(cdsseq),
len(utr3seq),
len(uORFseqCat), uORFseqCat, uORFaaCat
]
dftemp = pd.DataFrame([uORF_features], columns=dfCols) ##
# print dftemp
uORFdf = pd.concat([uORFdf, dftemp], ignore_index=True)
if i == (len(utr5seq) -
1): # at the end of the 5'UTR, do this ...
# print i
uORFsummary = [
trsp_id, trsp_genename, chrom, transcriptnum,
trsp_strand, uORFcounter, cdsExtension
]
# print uORFsummary
dfSummaryTemp = pd.DataFrame([uORFsummary],
columns=summaryCols)
# print dfSummaryTemp
summarydf = pd.concat([summarydf, dfSummaryTemp],
ignore_index=True)
uORFdf.to_csv(uORFtableOutfile)
summarydf.to_csv(uORFsummaryOutfile)
print summarydf.head()
primerfile = "/home/pzs/primerdesign/primerdesign/tags/parallel/promoterprimers.csv" outfile = "processedprimers.csv" reader = csv.reader(open(primerfile, "r")) writer = csv.writer(open(outfile, "w")) for row in reader: rowlen = len(row) if rowlen == 4: assert(row[-1] == "site not present!") writer.writerow(row) elif rowlen == 6: assert(row[-1] == "None found!") writer.writerow(row) elif rowlen == 11: writer.writerow(row) continue elif rowlen == 10: left = row[5] right = SeqIO.Seq(row[6]) right = str(right.reverse_complement()) fullseq = row[4] leftindex = fullseq.index(left) rightindex = fullseq.index(right) + len(right) product = fullseq[leftindex:rightindex] row.insert(7, product) writer.writerow(row) else: print "unknown row type", row
