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summarise_snps.py
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summarise_snps.py
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#!/usr/bin/env python
#/usr/bin/env python
##################
# Import modules #
##################
import string, re
import os, sys, getopt, math
from random import *
from Bio import SeqIO
from Bio.Seq import Seq
from Bio.Alphabet import IUPAC, Gapped
from Bio.SeqRecord import SeqRecord
from Bio import AlignIO
from Bio.Align import Generic
from modules.Si_nexus import *
from modules.Si_SeqIO import *
#from scipy import stats
import fisher
#from guppy import hpy
from optparse import OptionParser, OptionGroup
##########################
# Error message function #
##########################
def DoError(errorstring):
print "\nError:", errorstring
print "\nFor help use -h or --help\n"
sys.exit()
##########################################
# Function to Get command line arguments #
##########################################
def get_user_options():
usage = "usage: %prog [options]"
version="%prog 1.0. Written by Simon Harris, Wellcome Trust Sanger Institute, 2010"
parser = OptionParser(usage=usage, version=version)
group = OptionGroup(parser, "Required")
group.add_option("-i", "--input", action="store", dest="inputfile", help="Input file name", default="")
group.add_option("-r", "--reference", action="store", dest="ref", help="Name of reference strain", default="")
parser.add_option_group(group)
group = OptionGroup(parser, "Optional")
group.add_option("-e", "--embl", action="store", dest="embl", help="Embl/genbank annotation file for reference strain (for dN/dS etc.)", default="")
group.add_option("-o", "--output", action="store", dest="outfile", help="Output file prefix", default="")
group.add_option("-t", "--tab", action="store_true", dest="tabfile", help="Produce tab files of snp locations", default=False)
group.add_option("-a", "--align", action="store_true", dest="align", help="Produce snp alignment file (in phylip format)", default=False)
group.add_option("-E", "--exclude", action="store", dest="exclude", help="Exclude sequences from SNP alignment if they are less thanINT% mapped [Default= %default]", default=50, type="float", metavar="int")
group.add_option("-p", "--phylogeny", action="store_true", dest="raxml", help="Run phylogeny with RAxML", default=False)
group.add_option("-v", "--asrv", action="store", dest="asrv", help="Method of correction for among site rate variation (optional) [Choices = GAMMA, CAT, CAT_GAMMA, MIX] [Default = %default]", default="GAMMA", type="choice", choices=["GAMMA","CAT", "CAT_GAMMA", "MIX"])
group.add_option("-I", "--pinvar", action="store_true", dest="pinvar", help="Use correction for proportion of invariant sites", default=False)
group.add_option("-F", "--frequencies", action="store_true", dest="f", help="Use empirical base frequencies (protein models only)", default=False)
group.add_option("-w", "--overwrite", action="store_true", dest="overwrite", help="Overwrite old RAxML files without warning", default=False)
group.add_option("-b", "--bootstrap", action="store", dest="bootstrap", help="Number of bootstrap replicates (0 = do not run bootstrap). [Default= %default]", default=100, type="int", metavar="int")
group.add_option("-f", "--fast", action="store_true", dest="fast", help="Use fast bootstrap method", default=False)
group.add_option("-l", "--LSF", action="store_true", dest="LSF", help="Run bootstrap replicates over LSF (does not apply with fast bootstrap method)", default=False)
group.add_option("-g", "--gaps", action="store_true", dest="gaps", help="Gaps (-) are real", default=False)
group.add_option("-G", "--genetic_code", action="store", dest="genetic_code_number", help="Genetic code number to use. Choose from 1: Standard, 4: Mycoplasma. Default is 1", default=False, type="choice", choices=[1, 4])
parser.add_option_group(group)
return parser.parse_args()
################################
# Check command line arguments #
################################
def check_input_validity(options, args):
if options.ref=='':
DoError('No reference selected')
elif options.inputfile=='':
DoError('No input file selected')
elif not os.path.isfile(options.inputfile):
DoError('Cannot find file '+options.inputfile)
elif options.embl!='' and not os.path.isfile(options.embl):
DoError('Cannot find file '+options.embl)
elif options.bootstrap>10000 or options.bootstrap<0:
DoError('Number of bootstrap replicates (-b) must be between 0 (do not run bootstrap) and 10,000')
elif options.exclude>=100 or options.exclude<0:
DoError('Exclude percentage must be an float between 0 and 100')
if options.outfile=='':
options.outfile=options.ref.split("/")[-1].split(".")[0]
if options.raxml and not options.align:
options.align=True
while os.path.isfile(options.outfile+".out") and options.overwrite==False:
outopt=""
outopt=raw_input('\nOutput files with chosen prefix already exist.\n\nWould you like to overwrite (o), choose a new output file prefix (n) or quit (Q): ')
if outopt=='Q':
sys.exit()
elif outopt=="o":
break
elif outopt=="n":
options.outfile=raw_input('Enter a new output file prefix: ')
return
def split_len(seq, length):
return [seq[i:i+length] for i in range(0, len(seq), length)]
#############################################
# Function to create and embl style ID line #
#############################################
def embl_style_id(sequence):
emblstring="ID XX000001; SV 1; circular; genomic DNA; STD; PRO; "+str(len(sequence.strip()))+" BP.\nXX"
return emblstring
#############################################
# Function to create and embl style ID line #
#############################################
def embl_style_header(sequence):
emblstring="FH Key Location/Qualifiers"
emblstring=emblstring+"\nFH"
emblstring=emblstring+"\nFT source 1.."+str(len(sequence.strip()))
return emblstring
####################################################
# Function to create an embl style sequence format #
####################################################
def embl_style_sequence(sequence):
emblstring="XX\n"
sequence=sequence.strip().lower()
basehash={"a":0,"c":0,"g":0,"t":0,"Other":0}
for x in sequence:
if basehash.has_key(x):
basehash[x]=basehash[x]+1
else:
basehash["Other"]=basehash["Other"]+1
emblstring=emblstring+"SQ Sequence "+str(len(sequence))+" BP; "+str(basehash["a"])+" A; "+str(basehash["c"])+" C; "+str(basehash["g"])+" G; "+str(basehash["t"])+" T; "+str(basehash["Other"])+" other;\n"
currentposition=0
for x in range(0,len(sequence), 60):
emblstring=emblstring+" "
charactersadded=0
for y in range(0,60,10):
if x+y+10<len(sequence):
emblstring=emblstring+sequence[x+y:x+y+10]+" "
charactersadded=charactersadded+11
currentposition=currentposition+10
elif x+y<len(sequence):
emblstring=emblstring+sequence[x+y:]+" "
charactersadded=charactersadded+(1+len(sequence))-(x+y)
currentposition=currentposition+len(sequence)-(x+y)
emblstring=emblstring+" "*(75-(charactersadded+len(str(currentposition))))+str(currentposition)+"\n"
emblstring=emblstring+"//"
return emblstring
#def embl_style_sequence(sequence):
#
# emblstring="XX\n"
# sequence=sequence.strip().upper()
# basehash={"A":0,"C":0,"G":0,"T":0,"Other":0}
#
# for x in sequence:
# if basehash.has_key(x):
# basehash[x]=basehash[x]+1
# else:
# basehash["Other"]=basehash["Other"]+1
#
#
# split_seq=split_len(sequence, 10)
#
# emblstring=emblstring+"SQ Sequence "+str(len(sequence))+" BP; "+str(basehash["A"])+" A; "+str(basehash["C"])+" C; "+str(basehash["G"])+" G; "+str(basehash["T"])+" T; "+str(basehash["Other"])+" other;\n"
#
# currentposition=0
# for x in range(0,len(split_seq), 6):
# emblstring=emblstring+" "
# charactersadded=0
#
# for y, subseq in enumerate(split_seq[x:x+6]):
# if len(subseq)==10:
# emblstring=emblstring+subseq+" "
# charactersadded=charactersadded+11
# currentposition=currentposition+10
# else:
# emblstring=emblstring+subseq+" "
# charactersadded=charactersadded+(1+len(sequence))-(x+y)
# currentposition=currentposition+len(sequence)-(x+y)
# emblstring=emblstring+" "*(68-charactersadded)+str(currentposition)+"\n"
#
# emblstring=emblstring+"\\\\"
#
# testout=open("test.embl","w")
# print >> testout, emblstring
##################################################################
# Function to count the minimum number of changes between codons #
##################################################################
def countcodonchanges(codon, SNPcodon, geneticcode, sd, nd, loopsd=0, loopnd=0, pathcount=0):
for x in range(3):
if codon[x]!=SNPcodon[x]:
newSNPcodon=SNPcodon[:x]+codon[x]+SNPcodon[x+1:]
#print SNPcodon, newSNPcodon, geneticcode[SNPcodon], geneticcode[newSNPcodon]
if geneticcode[newSNPcodon]=='*':
continue
elif geneticcode[SNPcodon]==geneticcode[newSNPcodon]:
newloopnd=loopnd
newloopsd=loopsd+1
else:
newloopnd=loopnd+1
newloopsd=loopsd
#print SNPcodon, newSNPcodon, codon, sd, nd, newloopsd, newloopnd, pathcount
if newSNPcodon!=codon:
sd, nd, pathcount=countcodonchanges(codon, newSNPcodon, geneticcode, sd, nd, newloopsd, newloopnd, pathcount)
else:
sd=sd+newloopsd
nd=nd+newloopnd
pathcount=pathcount+1
return sd, nd, pathcount
#####################################################
# Function to calculate dN/dS between two sequences #
#####################################################
def dnbyds(CDS, SNPseq, CDSbasenumbers, genetic_code_number=1):
#standard genetic code
geneticcode_1={'TTT':'F', 'TTC':'F', 'TTA':'L', 'TTG':'L', 'TCT': 'S', 'TCC': 'S','TCA': 'S','TCG': 'S', 'TAT': 'Y','TAC': 'Y', 'TAA': '*', 'TAG': '*', 'TGT': 'C', 'TGC': 'C', 'TGA': '*', 'TGG': 'W', 'CTT': 'L','CTC': 'L','CTA': 'L','CTG': 'L', 'CCT': 'P', 'CCC': 'P', 'CCA': 'P', 'CCG': 'P', 'CAT': 'H', 'CAC': 'H', 'CAA': 'Q', 'CAG': 'Q', 'CGT': 'R', 'CGC': 'R', 'CGA': 'R', 'CGG': 'R', 'ATT': 'I', 'ATC': 'I', 'ATA': 'I', 'ATG': 'M', 'ACT': 'T', 'ACC': 'T', 'ACA': 'T', 'ACG': 'T', 'AAT': 'N', 'AAC': 'N', 'AAA': 'K', 'AAG': 'K', 'AGT': 'S', 'AGC': 'S', 'AGA': 'R', 'AGG': 'R', 'GTT': 'V', 'GTC': 'V', 'GTA': 'V', 'GTG': 'V', 'GCT': 'A', 'GCC': 'A', 'GCA': 'A', 'GCG': 'A', 'GAT': 'D', 'GAC': 'D', 'GAA': 'E', 'GAG': 'E', 'GGT': 'G', 'GGC': 'G', 'GGA': 'G', 'GGG': 'G'}
#mycoplasma genetic code
geneticcode_4={'TTT':'F', 'TTC':'F', 'TTA':'L', 'TTG':'L', 'TCT': 'S', 'TCC': 'S','TCA': 'S','TCG': 'S', 'TAT': 'Y','TAC': 'Y', 'TAA': '*', 'TAG': '*', 'TGT': 'C', 'TGC': 'C', 'TGA': 'W', 'TGG': 'W', 'CTT': 'L','CTC': 'L','CTA': 'L','CTG': 'L', 'CCT': 'P', 'CCC': 'P', 'CCA': 'P', 'CCG': 'P', 'CAT': 'H', 'CAC': 'H', 'CAA': 'Q', 'CAG': 'Q', 'CGT': 'R', 'CGC': 'R', 'CGA': 'R', 'CGG': 'R', 'ATT': 'I', 'ATC': 'I', 'ATA': 'I', 'ATG': 'M', 'ACT': 'T', 'ACC': 'T', 'ACA': 'T', 'ACG': 'T', 'AAT': 'N', 'AAC': 'N', 'AAA': 'K', 'AAG': 'K', 'AGT': 'S', 'AGC': 'S', 'AGA': 'R', 'AGG': 'R', 'GTT': 'V', 'GTC': 'V', 'GTA': 'V', 'GTG': 'V', 'GCT': 'A', 'GCC': 'A', 'GCA': 'A', 'GCG': 'A', 'GAT': 'D', 'GAC': 'D', 'GAA': 'E', 'GAG': 'E', 'GGT': 'G', 'GGC': 'G', 'GGA': 'G', 'GGG': 'G'}
geneticcodes=[geneticcode_1,geneticcode_1,geneticcode_1,geneticcode_4]
geneticcode=geneticcodes[genetic_code_number-1]
codonsynonyms={}
for codon in geneticcode.keys():
thiscodon={}
codonsynonyms[codon]=0.0
for x in range(3):
numsyn=0.0
numnotstop=3
for y in ['A', 'C', 'G', 'T']:
if codon[x]!=y:
newcodon=codon[:x]+y+codon[x+1:]
if geneticcode[newcodon]==geneticcode[codon]:
numsyn=numsyn+1
elif geneticcode[newcodon]=='*':
numnotstop=numnotstop-1
codonsynonyms[codon]=codonsynonyms[codon]+(numsyn/numnotstop)
S=0.0
N=0.0
S1=0.0
N1=0.0
S2=0.0
N2=0.0
Sd=0.0
Nd=0.0
pS=0.0
pN=0.0
gapcount=0
numcodons=0
varianceS=0.0
varianceN=0.0
z=0.0
dN=0.0
dS=0.0
SNPtype={}
AAfromtype={}
AAtotype={}
Fisher=-0
#if len(CDS)!=len(SNPseq):
# print "Error: sequences must be the same length to calculate dN/dS!"
# [N, S, dN, dS, pN, pS, varianceS, varianceN, z, (len(CDS)-gapcount), Nd, Sd, Fisher], SNPtype, AAfromtype, AAtotype
for x in range(0,len(CDS),3):
if len(CDS)<x+3:
continue
numcodons=numcodons+1
codon=CDS[x:x+3]
SNPcodon=SNPseq[x:x+3]
codonposn=[CDSbasenumbers[x], CDSbasenumbers[x+1], CDSbasenumbers[x+2]]
if codon!=SNPcodon:
for y,z in enumerate(codon):
if SNPcodon[y]!=z:
newSNPcodon=codon[:y]+SNPcodon[y]+codon[y+1:]
if 'N' in codon or 'N' in newSNPcodon or '-' in codon or '-' in newSNPcodon:
SNPtype[codonposn[y]]='-'
elif geneticcode[newSNPcodon]=='*':
SNPtype[codonposn[y]]='2'
elif geneticcode[newSNPcodon]=='*':
SNPtype[codonposn[y]]='3'
elif geneticcode[newSNPcodon] == geneticcode[codon]:
SNPtype[codonposn[y]]='S'
else:
SNPtype[codonposn[y]]='N'
if not 'N' in codon and not 'N' in SNPcodon and geneticcode[codon]!=geneticcode[SNPcodon]:
AAfromtype[codonposn[y]]=geneticcode[codon]
AAtotype[codonposn[y]]=geneticcode[SNPcodon]
if 'N' in codon or 'N' in SNPcodon or '-' in codon or '-' in SNPcodon:
gapcount=gapcount+3
continue
# if geneticcode[codon]=='*' or geneticcode[SNPcodon]=='*':
# continue
# print codon, x
#s=float(codonsynonyms[codon])/3
#n=float(3-s)
S1=S1+(float(codonsynonyms[codon]))
S2=S2+(float(codonsynonyms[SNPcodon]))
sd=0.0
nd=0.0
pathcount=0
if codon!=SNPcodon:
sd, nd, pathcount=countcodonchanges(codon, SNPcodon, geneticcode, sd, nd)
#if nochanges==2:
# sd=float(sd)/2
# nd=float(nd)/2
#elif nochanges==3:
# sd=float(sd)/6
# nd=float(nd)/6
if pathcount>0:
sd=float(sd)/pathcount
nd=float(nd)/pathcount
#elif codon!= SNPcodon:
# print codon, SNPcodon, "All paths lead to early stop codon in SNP sequence."
#return
Sd=Sd+sd
Nd=Nd+nd
#pS=pS+(sd/s)
#pN=pN+(nd/n)
#print codon, SNPcodon
S=(S1+S2)/2
N=len(CDS)-gapcount-S
#pNb=pN/numcodons
#pSb=pS/numcodons
if N!=0:
pN=Nd/N
if S!=0:
pS=Sd/S
if pS==0:
#print "No sites are synonymous."
return [N, S, dN, dS, pN, pS, varianceS, varianceN, z, (len(CDS)-gapcount), Nd, Sd, Fisher], SNPtype, AAfromtype, AAtotype
if pS<0.75 and pN<0.75:
dS=(-3*(math.log(1-((pS*4)/3))))/4
dN=(-3*(math.log(1-((pN*4)/3))))/4
varianceS=(9 * pS * (1 -pS))/(((3 - 4 *pS) **2) * (len(CDS)-gapcount));
varianceN=(9 * pN * (1 -pN))/(((3 - 4 *pN) **2) * (len(CDS)-gapcount));
z=(dN - dS) / math.sqrt(varianceS + varianceN)
#Fisher exact test of S and N
#oddsratio, Fisher = stats.fisher_exact([[N, S], [Nd, Sd]])
Fisher=fisher.pvalue(N,S,Nd,Sd).two_tail
else:
#print "Too divergent for JC! Using pN/pS instead."
dS=pS
dN=pN
#print dN, dS, S, N, S+N, Sd, Nd, pS, pN#, pSb, pNb, dS, dN, pN/pS, dN/dS
#print "N =", N
#print "S =", S
#print "dN/dS =", dN/dS
#print [N, S, dN, dS, pN, pS, varianceS, varianceN, z, (len(CDS)-gapcount), Nd, Sd, Fisher], SNPtype, AAfromtype, AAtotype
return [N, S, dN, dS, pN, pS, varianceS, varianceN, z, (len(CDS)-gapcount), Nd, Sd, Fisher], SNPtype, AAfromtype, AAtotype
#########################################################
# Function to concatenate CDSs for one of the sequences #
#########################################################
def concatenate_CDS_sequences(record, sequence, ref):
def add_feature_seq(feature):
#start=ref_pos_to_aln_pos[feature.location.start.position]
#end=ref_pos_to_aln_pos[feature.location.end.position-1]
#my_seq= Seq(sequences[sequence][start : end+1])
#if feature.strand==-1:
# my_seq=my_seq.reverse_complement()
#print len(my_seq),
#my_seq=str(feature.extract(sequences[sequence]))
#my_ref= Seq(sequences[ref][start : end+1])
#if feature.strand==-1:
# my_ref=my_ref.reverse_complement()
# #print len(my_ref)
#my_ref=str(my_ref)
my_ref=str(feature.extract(sequences[ref]))
#for base in xrange(len(my_ref)):
# if my_ref[base]=="-" and my_seq[base]=="-":
# continue
# elif my_ref[base]=="-" or my_seq[base]=="-":
# return
#
# if len(str(my_seq.replace("-",""))) % 3 or len(str(my_ref.replace("-",""))) % 3:
# return
#if len(str(my_seq)) % 3 or len(str(my_ref)) % 3:
# return
x=0
#for y in feature:
# if my_ref[x]!="-":
# CDSbasenumbers.append(ref_pos_to_aln_pos[y])
# x+=1
my_seq=[]
my_ref=[]
tCDSbasenumbers=[]
for part in feature.location.parts:
start=ref_pos_to_aln_pos[part.start]
end=ref_pos_to_aln_pos[part.end]
tmy_seq= Seq(sequences[sequence][start : end])
if feature.strand==-1:
tmy_seq=tmy_seq.reverse_complement()
my_seq.append(str(tmy_seq))
tref_seq= Seq(sequences[ref][start : end])
if feature.strand==-1:
tref_seq=tref_seq.reverse_complement()
my_ref.append(str(tref_seq))
rmed=[]
if part.strand==-1:
for x, y in enumerate(xrange(end-1, start-1, -1)):
if str(tref_seq)[x]!="-":
tCDSbasenumbers.append(y)
else:
rmed.append([x,y,str(tref_seq)[x], my_ref[-1][x], my_seq[-1][x]])
else:
for x, y in enumerate(xrange(start, end)):
if str(tref_seq)[x]!="-":
tCDSbasenumbers.append(y)
else:
rmed.append([x,y,str(tref_seq)[x], my_ref[-1][x], my_seq[-1][x]])
#print start, end, tCDSbasenumbers, part.strand
#if part.strand==-1:
# sys.exit()
toprint=[len(my_seq)]
my_seq=''.join(my_seq)
my_ref=''.join(my_ref)
toprint+=[str(tref_seq), rmed, len(my_seq), len(my_ref), len(tCDSbasenumbers), feature.strand, start, end, end-start, x]
#print feature.id, feature.strand, len(str(my_seq.replace("-",""))) % 3, len(str(my_ref.replace("-",""))) % 3
for base in xrange(len(my_ref)):
if my_ref[base]=="-" and my_seq[base]=="-":
continue
elif my_ref[base]=="-" or my_seq[base]=="-":
return "", "", []
if len(str(my_seq.replace("-",""))) % 3 or len(str(my_ref.replace("-",""))) % 3:
return "", "", []
my_ref=my_ref.replace("-","")
my_seq=my_seq.replace("-","")
if len(my_seq)!=len(tCDSbasenumbers):
print ' '.join(map(str,toprint))
print len(my_seq), len(my_ref), len(tCDSbasenumbers), feature.strand
print my_seq
print my_ref
sys.exit()
return my_seq, my_ref, tCDSbasenumbers
concatenated_sequence=[]
concatenated_ref=[]
CDSbasenumbers=[]
for feature in record.features:
if feature.type=="CDS" and not "pseudo" in feature.qualifiers:
ms, mr, tc=add_feature_seq(feature)
#print len(ms), len(concatenated_sequence)
concatenated_sequence.append(ms)
concatenated_ref.append(mr)
CDSbasenumbers=CDSbasenumbers+tc
#print len(ms), len(concatenated_sequence)
#print len(''.join(concatenated_sequence)), len(''.join(concatenated_ref)), len(CDSbasenumbers)
#print CDSbasenumbers
return ''.join(concatenated_sequence), ''.join(concatenated_ref), CDSbasenumbers
########################################
# Function to get a name for a feature #
########################################
def get_best_feature_name(feature):
name_types=["gene", "primary_name", "systematic_id", "locus_tag"]
for name in name_types:
if feature.qualifiers.has_key(name):
return feature.qualifiers[name][0]
return ""
###########################################
# Function to get a product for a feature #
###########################################
def get_feature_product(feature):
if feature.qualifiers.has_key("product"):
return feature.qualifiers["product"][0]
return ""
class SNPanalysis:
def __init__(self, fastq='', directory='', mapped={}, runmaq='n', CDSseq=''):
self.fastq=fastq
self.directory=directory
self.mapped=mapped
self.runmaq=runmaq
self.CDSseq=CDSseq
self.N=0.0
self.S=0.0
self.dN=0.0
self.dS=0.0
self.pN=0.0
self.pS=0.0
self.varianceN=0.0
self.varianceS=0.0
self.z=0.0
self.Nd=0.0
self.Ns=0.0
self.goodlen=0
self.CDSseq=''
self.snpsummary={}
self.nummapped=0
self.percentmapped=0.0
self.intragenic=0
self.intergenic=0
self.SNPs={}
if __name__ == "__main__":
#argv=sys.argv[1:]
#ref, inputfile, outfile, tabfile, align, embl, raxml, graphs, bootstrap, model, chisquare, recomb=getOptions(argv)
(options, args)=get_user_options()
check_input_validity(options, args)
snps={}
refbases={}
bases={}#0=A 1=C 2=G 3=T
nostates=0
converter={}
convertback={}
snpbases={}
snpstructs=[]
count=0
runpileups='n'
print '\nReading input alignment...',
sys.stdout.flush()
#else:
lines=[]
count=-1
sequences={}
names=set([])
curseqlist=[]
append=curseqlist.append
for linea in open(options.inputfile, "rU"):
linea=linea.strip()
if len(linea)==0:
continue
if linea[0]==">":
if count>-1:
sequence=''.join(curseqlist)
sequences[name]=sequence
count=count+1
curseqlist=[]
append=curseqlist.append
name=linea.split()[0][1:]
lines.append(linea.split()[0][1:]+'\n')
else:
append(linea)
if count>-1:
sequence=''.join(curseqlist)
sequences[name]=sequence
#for line in lines:
# words=line.strip().split('\n')
# sequences[words[0].split()[0]]=''.join(words[1:])
snpstructs.append(SNPanalysis())
ref=options.ref
reflen=len(sequences[sequences.keys()[0]])
for sequence in sequences.keys():
if len(sequences[sequence])!=reflen:
print "\nERROR!: sequences are not all of the same length!!!\n"
sys.exit()
# if sequence!=ref:
# sequences[sequence]=sequences[sequence].upper().replace('N','-')
# else:
# sequences[sequence]=sequences[sequence].upper()
if not options.gaps:
sequences[sequence]=sequences[sequence].upper().replace('-','N')
else:
sequences[sequence]=sequences[sequence].upper()
#replace uncertainties in each sequence
for x in ["R", "S", "B", "Y", "W", "D", "K", "H", "M", "V"]:
sequences[sequence]=sequences[sequence].replace(x,"N")
#print sequences.keys()
print "Found", len(sequences.keys()), "sequences of length", reflen
sys.stdout.flush()
if not ref in sequences.keys():
print "Error!!!: Reference ("+ref+") not in alignment"
sys.exit()
count=0
if ref!='':
aln_pos_to_ref_pos={}
ref_pos_to_aln_pos={}
#print reflen
refseq=sequences[ref].replace('-','')
reflennogaps=len(refseq)
for x, y in enumerate(sequences[ref]):
if y!='-':
aln_pos_to_ref_pos[x]=count#use aln_pos_to_ref_pos to convert alignment positions to positions in ref sequence
ref_pos_to_aln_pos[count]=x#opposite to aln_pos_to_ref_pos. Use this to convert reference positions to alignment positions
count=count+1
snplocations=[]
snpbases=[]
basecounts=[]
###################
# Open embl files #
###################
if options.embl!='':
print "\nReading EMBL file(s)...",
try:
record = open_annotation(options.embl)
except StandardError:
print "\nCannot open annotation file"
sys.exit()
print "Done"
if len(record.seq)!=reflennogaps:
DoError("embl file ("+str(len(record.seq))+") and alignment ("+str(reflennogaps)+") have different reference lengths")
sys.exit()
#Identify snps in the alignment
print "\nIdentifying SNPs...",
sys.stdout.flush()
constants={"A":0, "C":0, "G":0, "T":0}
for x in range(reflen):
numbases=0
foundbases={}
if sequences[ref][x].upper()=="N":
continue
for key in sequences.keys():
base=sequences[key][x].upper()
if base not in foundbases.keys() and base not in ['-', "N"]:
foundbases[base]=1
numbases=numbases+1
elif base not in ['-', "N"]:
foundbases[base]+=1
if numbases>1:
snplocations.append(x)
snpbases.append(foundbases)
basecounts.append(numbases)
elif numbases==1:
constants[foundbases.keys()[0]]+=1
print "Done"
print "Found", len(snplocations), "sites with a SNP"
print "Constant bases:"
for base in ["A","C","G","T"]:
print base+":", constants[base]
print
sys.stdout.flush()
seqsort=sequences.keys()
seqsort.sort()
allsnps={}
refbases={}
alssnpsummary={}
tempsnps={}
tempsnpsummary={'A':{'C':0, 'G':0, 'T':0}, 'C':{'A':0, 'G':0, 'T':0}, 'G':{'C':0, 'A':0, 'T':0}, 'T':{'C':0, 'G':0, 'A':0}}
olddone=-1
if options.embl!='' and ref!='':
def add_subfeature_positions(feature, pseudo, featurenum):
if feature.type in ['CDS', 'rRNA', 'tRNA']:
#print feature.id, feature.strand, feature.type, feature.location, len(embldata)
for part in feature.location.parts:
#print part.start, part.end
start=ref_pos_to_aln_pos[part.start]
end=ref_pos_to_aln_pos[part.end]
for x in xrange(start,end):
embldata[x]=featurenum
print "\nCalculating dN/dS values...",# NEEDS FIXING!!!
sys.stdout.flush()
#dndsout=open('dNdS_by_CDS.out','w')
sys.stdout.flush()
dnbydsstats={}
comp={'A':'T','T':'A','G':'C','C':'G', 'N':'N', '-':'-'}
embldata=[]
for i in range(0,reflen):
embldata.append(-1)
for y, feature in enumerate(record.features):
if feature.type in ['CDS', 'rRNA', 'tRNA']:
if "pseudo" in feature.qualifiers:
pseudo=True
else:
pseudo=False
add_subfeature_positions(feature, pseudo, y)
#print embldata
#refCDSseq=concatenate_CDS_sequences(record, ref)
print 'Done'
sys.stdout.flush()
tmpseqs={}
snptypes={}
AAfromtypes={}
AAtotypes={}
for sequence in seqsort:
if sequence==ref:
continue
print sequence+'...',
sys.stdout.flush()
tmpCDSseq, refCDSseq, CDSbasenumbers=concatenate_CDS_sequences(record, sequence, ref)
#print len(tmpCDSseq), len(refCDSseq), len(CDSbasenumbers)
dnbydsstats[sequence], snptypes[sequence], AAfromtypes[sequence], AAtotypes[sequence]=dnbyds(refCDSseq, tmpCDSseq, CDSbasenumbers, genetic_code_number=options.genetic_code_number)
#print dnbydsstats[sequence]
#N, S, dN, dS, pN, pS, varianceS, varianceN, z, (len(CDS)-gapcount), Nd, Sd
if dnbydsstats[sequence][3]!=0:
print "dNdS = %.2f, p-value = %.2f" % (dnbydsstats[sequence][2]/dnbydsstats[sequence][3], dnbydsstats[sequence][12])
#print >> dndsout, '\t'+str(dnbydsstats[sequence][2]/dnbydsstats[sequence][3]),
sys.stdout.flush()
else:
print "-"
#dndsout.close()
#print summary file and alignment file
print "\nWriting output file(s)...",
sys.stdout.flush()
output=open(options.outfile+'.out','w')
#if len(allsnps.keys())>1:
# print >> output, 'Ref_sequence\tPosition_in_ref',
#else:
print >> output, 'Position_in_alignment',
if ref!='':
print >> output, '\tPosition_in_'+ref,
if options.embl!='':
print >> output, '\tCDS/rRNA/tRNA/Intergenic\tstrand\tCDS_name\tproduct\tSynonymous/Non-synonymous',
print >> output, '\tRef_base\tSNP_base\tTotal',
for name in seqsort:
if name!=ref:
print >> output, '\t'+name,
print >> output, '\n',
#
if options.tabfile==True:
tabout=open(options.outfile+'_snps.tab','w')
#covgraph=open(outfile+'_cov.txt','w')
#covcountgraph=open(outfile+'_covcount.txt','w')
#minquality=10
#minqualityperdepth=4
snpsequence={}
for name in seqsort:
snpsequence[name]=''
refsequence=''
if ref=='':
ref=sequences.keys()[0]
if options.tabfile==True:
print >> tabout, 'ID SNP'
poscount=1
#for key in sequences.keys():
#snpsort=allsnps[key].keys()
#snpsort.sort()
strainsnpsummary={}
for name in seqsort:
if name!=ref:
strainsnpsummary[name]={}
strainsnpsummary[name]['count']=0
for x in ['A', 'C', 'G', 'T']:
strainsnpsummary[name][x]={}
for y in ['A', 'C', 'G', 'T']:
strainsnpsummary[name][x][y]=0
for x, j in enumerate(snplocations):
outstring=''
tabstring=''
#if len(allsnps.keys())>1:
# outstring=outstring+key+'\t'+str(j)
#else:
outstring=outstring+str(j+1)
snpcolour='1'
if sequences[ref][j]!='-':
outstring=outstring+'\t'+str(aln_pos_to_ref_pos[j]+1)
if options.embl!='':
# if embldata[aln_pos_to_ref_pos[j]]<0:
# outstring=outstring+'\tIntergenic\t-\t-\t'
# else:
# outstring=outstring+'\t'+record.features[embldata[aln_pos_to_ref_pos[j]]].type+'\t'+str(record.features[embldata[aln_pos_to_ref_pos[j]]].strand)+'\t'+get_best_feature_name(record.features[embldata[aln_pos_to_ref_pos[j]]])+'\t'+get_feature_product(record.features[embldata[aln_pos_to_ref_pos[j]]])
# snptype='-'
if embldata[j]<0:
outstring=outstring+'\tIntergenic\t-\t-\t-'
else:
outstring=outstring+'\t'+record.features[embldata[j]].type+'\t'+str(record.features[embldata[j]].strand)+'\t'+get_best_feature_name(record.features[embldata[j]])+'\t'+get_feature_product(record.features[embldata[j]])
snptype='-'
for name in seqsort:
if name==ref:
continue
# if snptypes[name].has_key(aln_pos_to_ref_pos[j]):
# if snptypes[name][aln_pos_to_ref_pos[j]]=='S':
# snpcolour='3'
# elif snptypes[name][aln_pos_to_ref_pos[j]]=='N':
# snpcolour='2'
# elif snptypes[name][aln_pos_to_ref_pos[j]]=='2':
# snpcolour='4'
# elif snptypes[name][aln_pos_to_ref_pos[j]]=='3':
# snpcolour='4'
# if snptype=='-' and snptypes[name][aln_pos_to_ref_pos[j]]!='-':
# snptype=snptypes[name][aln_pos_to_ref_pos[j]]
# elif snptypes[name][aln_pos_to_ref_pos[j]] not in snptype and snptypes[name][aln_pos_to_ref_pos[j]]!='-':