global insertion_db

insertion_db={}

if len(symbols)>0:

### Search for genes not for coordinates

search_locations = geneCoordinates(species,symbols)

else:

### Search for coordinates and not genes

search_locations = variantCoordinates(variants)

### Discover the variants

variant_db = findVariants(bam_dir,search_locations)

variant_filtered_db={}

for var in variant_db:

#print var, variant_db[var]

if variant_db[var]>3:

#print var,variant_db[var]

variant_filtered_db[var] = variant_db[var]

### Quantify the variants versus background

search_locations=[]

contents = open(variants, "rU")

for line in contents:

line = line.rstrip()

chr,start,end,symbol = string.split(line,'\t')

if 'chr' not in chr: chr = 'chr'+chr

strand = 'NA'

search_locations.append([chr,strand,start,end,symbol])

genes=[]

import EnsemblImport

ensembl_annotation_db = EnsemblImport.reimportEnsemblAnnotations(species,symbolKey=True)

for symbol in symbols:

if symbol in ensembl_annotation_db:

ens_geneid = ensembl_annotation_db[symbol]

genes.append((ens_geneid,symbol))

else:

print symbol, 'not found'

### Get gene genomic locations

gene_location_db = EnsemblImport.getEnsemblGeneLocations(species,'RNASeq','key_by_array')

search_locations=[]

for (gene,symbol) in genes:

chr,strand,start,end = gene_location_db[gene]

#if symbol == 'SRSF10': chr = 'chr1'; strand = '-'; start = '24295573'; end = '24306953'

if len(chr)>6: print symbol, 'bad chromosomal reference:',chr

else:

search_locations.append([chr,strand,start,end,symbol])

start_time = time.time()

bamfile = pysam.Samfile(bam_dir, "rb" )

output_bed_rows=0

#https://www.biostars.org/p/76119/

variant_db={}

reference_rows=0

o = open (string.replace(bam_dir,'.bam','__variant.txt'),"w")

for (chr,strand,start,stop,symbol) in search_locations: ### read each line one-at-a-time rather than loading all in memory

read_count=0

reference_rows+=1

stop=int(stop)+100 ### buffer for single variants

start=int(start)-100 ### buffer for single variants

for alignedread in bamfile.fetch(chr, int(start),int(stop)):

md = alignedread.opt('MD')

omd = md

codes = map(lambda x: x[0],alignedread.cigar)

cigarstring = alignedread.cigarstring

#print symbol,cigarstring

if 1 in codes and alignedread.pos:

### Thus an insertion is present

cigarstring = alignedread.cigarstring

chr = bamfile.getrname(alignedread.rname)

pos = alignedread.pos

def getInsertions(cigarList,X):

cummulative=0

coordinates=[]

for (code,seqlen) in cigarList:

if code == 0 or code == 3:

cummulative+=seqlen

if code == 1:

coordinates.append(X+cummulative)

return coordinates

coordinates = getInsertions(alignedread.cigar,pos)

"""

print pos

print coordinates

print alignedread.seq

print codes

print alignedread.cigar

print cigarstring

print md;sys.exit()

"""

for pos in coordinates:

try: variant_db[chr,pos,symbol]+=1

except Exception: variant_db[chr,pos,symbol] = 1

insertion_db[chr,pos]=[]

continue

try:

int(md) ### If an integer, no mismatches or deletions present

continue

except Exception:

#print chr, int(start),int(stop)

#print alignedread.get_reference_sequence()

#print alignedread.seq

md = string.replace(md,'C','A')

md = string.replace(md,'G','A')

md = string.replace(md,'T','A')

md = string.split(md,'A')

pos = alignedread.pos

chr = bamfile.getrname(alignedread.rname)

#if omd == '34^GA16': print md, pos

for i in md[:-1]:

try:

pos+=int(i)+1

except Exception:

if i == '':

pos+=+1

elif '^' in i: ### position is equal to the last position

pos+=int(string.split(i,'^')[0])+1

#pass

#if 'CGGATCC' in alignedread.seq: print string.split(alignedread.seq,'CGGATCC')[1],[pos]

try: variant_db[chr,pos,symbol]+=1

except Exception: variant_db[chr,pos,symbol] = 1

#codes = map(lambda x: x[0],alignedread.cigar)

output_bed_rows+=1

o.close()

bamfile.close()

if multi==False:

print time.time()-start_time, 'seconds to assign reads for %d entries from %d reference entries' % (output_bed_rows,reference_rows)

#print variant_db;sys.exit()

start_time = time.time()

bamfile = pysam.Samfile(bam_dir, "rb" )

reference_rows=0

output_bed_rows=0

#https://www.biostars.org/p/76119/

variant_db={}

o = open (string.replace(bam_dir,'.bam','__variant.txt'),"w")

entries = ['chr','position','rare-allele frq','type','depth','gene']

o.write(string.join(entries,'\t')+'\n')

#print 'Analyzing',len(search_locations),'variants'

for (chr,pos,symbol) in search_locations: ### read each line one-at-a-time rather than loading all in memory

pos = int(pos)

read_count=0

reference_rows+=1

nucleotide_frequency={}

for pileupcolumn in bamfile.pileup(chr,pos,pos+1):

# Skip columns outside desired range

#print pos, pileupcolumn.pos, pileupcolumn.cigarstring, pileupcolumn.alignment.pos

if pileupcolumn.pos == (pos-1):

for pileupread in pileupcolumn.pileups:

try: nt = pileupread.alignment.query_sequence[pileupread.query_position]

except Exception,e:

if 'D' in pileupread.alignment.cigarstring:

nt = 'del'

else:

nt = 'ins'

try: nucleotide_frequency[nt]+=1

except Exception: nucleotide_frequency[nt]=1

nt_freq_list=[]

for nt in nucleotide_frequency:

nt_freq_list.append(nucleotide_frequency[nt])

s = sum(nt_freq_list)

nt_freq_list.sort()

try:

frq = float(search_locations[chr,pos,symbol])/s ### This fixes that (number of insertions from before)

except Exception: frq = '1.000000'; print symbol, pos, nucleotide_frequency, search_locations[chr,pos,symbol]

if (chr,pos) in insertion_db:

#print 'insertion', chr, pos

call = 'insertion'

### For insertions if the inserted base matches the reference base, incorrect freq will be reported

elif 'del' in nucleotide_frequency:

#frq = float(nt_freq_list[-2])/s

call = 'del'

else:

#frq = float(nt_freq_list[-2])/s

call = 'mismatch'

if len(nt_freq_list)>1 or call == 'insertion':

if frq>0.01:

frq = str(frq)[:4]

entries = [chr,str(pos),str(frq),call,str(s),symbol]

o.write(string.join(entries,'\t')+'\n')

output_bed_rows+=1

o.close()

bamfile.close()

if multi==False: