view metagene_readthrough.py @ 17:c87c40e642af draft

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#!/usr/bin/env python2.7.3
#-*- coding: utf-8 -*-

'''
    Created on Dec. 2013
    @author: rachel legendre
    @copyright:  [e-mail]
    @license: GPL v3
'''

import os, sys, time, optparse, shutil, re, urllib, subprocess, tempfile
from urllib import unquote
from Bio import SeqIO
import csv
import pysam
import HTSeq
#from matplotlib import pyplot as pl
import matplotlib
matplotlib.use('Agg')
import matplotlib.pyplot as pl
from numpy import arange
from matplotlib import ticker as t
from PIL import Image
import ribo_functions
## suppress matplotlib warnings
import warnings



def stop_err( msg ):
    sys.stderr.write( "%s\n" % msg )
    sys.stderr.write( "Programme aborted at %s\n" % time.asctime(time.localtime(time.time())))
    sys.exit()
    
        
def compute_rpkm(length,count_gene,count_tot) :
    
    try : 
        rpkm = "{0:.4f}".format(count_gene*1000000.0/(count_tot*length))
    except ArithmeticError :
        stop_err( 'Illegal division by zero during computing RPKM')
    return float(rpkm)

 
def find_stop(seq) :
    '''
        Find stop codon in a sequence and return position of first nucleotide in stop
    '''
    stop_codon = ['TAA','TAG','TGA']
    for nt in range(0,len(seq)-3,3) :
        codon = seq[nt:nt+3]
        if codon in stop_codon :
            return nt
                
    return -1


def check_met(seq):
    '''
        Boolean function for testing presence or absence of methionine in 5 codons following stop codon 
    '''
    met = 'ATG'
    for pos in range(0,15,3) :
        codon = seq[pos:pos+3]
        if codon in met :
            return True
                
    return False


'''
    feature.iv is a GenomicInterval object :
    A GenomicInterval object has the following slots, some of which 
    are calculated from the other:
  
    chrom: The name of a sequence (i.e., chromosome, contig, or 
      the like). 
    start: The start of the interval. Even on the reverse strand,
      this is always the smaller of the two values 'start' and 'end'.
      Note that all positions should be given as 0-based value!
    end: The end of the interval. Following Python convention for 
      ranges, this in one more than the coordinate of the last base
      that is considered part of the sequence.
    strand: The strand, as a single character, '+' or '-'. '.' indicates
      that the strand is irrelevant. (Alternatively, pass a Strand object.)
    length: The length of the interval, i.e., end - start
    start_d: The "directional start" position. This is the position of the
     first base of the interval, taking the strand into account. Hence, 
     this is the same as 'start' except when strand == '-', in which 
     case it is end-1.
    end_d: The "directional end": Usually, the same as 'end', but for 
     strand=='-1', it is start-1.
  
'''
def check_overlapping(gff_reader,chrm,start,stop,strand, name):
    
    #### probleme avec les genes completement inclu...
    
    iv2 = HTSeq.GenomicInterval(chrm,start,stop,strand)
    for feature in gff_reader:
        if feature.type == "gene" :
            if feature.iv.overlaps(iv2) and feature.attr.get('gene_name') != name :
                ## if its a reverse gene, we replace start of extension by start of previous gene
                if strand == '-' :
                    return (feature.iv.end+3,stop)
                ## else we replace stop of extension by start of following gene
                else :
                    return (start,feature.iv.start-3)
    ## if no overlap are find, we return -1
    return (start,stop)


def pass_length(start,stop) :
    
    if (stop-start) > 25 :
        return True
    else :
        return False


def check_homo_coverage(gene,GFF,start,stop, aln) :
    
    chrom = GFF[gene]['chrom']
    ## compute number of nucleotides in CDS with a coverage equal to zero
    nt_cds_cov = 0
    nt_cds_num = 0
    for i in range(1,GFF[gene]['exon_number']+1) :
        for z in range(GFF[gene]['exon'][i]['start'],GFF[gene]['exon'][i]['stop']):
            nt_cds_num += 1
            if aln.count(chrom,z,z+1) == 0 :
                nt_cds_cov += 1
                    
    ## compute percent of CDS no covering
    if nt_cds_cov == 0 or nt_cds_num == 0:
        percent = 0
    else:
        percent = nt_cds_cov*100/nt_cds_num
    
    ## compute number of nucleotides with no coverage in extension
    nt_no_cover = 0
    for pos in range(start,stop,1) :
        if aln.count(chrom,pos,pos+1) == 0 :
            nt_no_cover += 1 
    #print gene, nt_cds_cov, percent, nt_no_cover
    #percent10 = (stop-start)*50/100
    if (nt_no_cover*100)/(stop-start) > percent :
        return False
    else :
        return True

def plot_gene ( aln, gene, start_extension, stop_extension, dirout ) :
    
    
    ### ignore matplotlib warnings for galaxy
    warnings.filterwarnings('ignore')
    try:
        strand = gene['strand']
        len_gene = gene['stop']-gene['start']
        if strand is "-" :
            len_ext = gene['stop']-start_extension
            ## coverage in all gene + extension
            start = start_extension-100
            stop = gene['stop']+100
            vector1 = [0]*(stop-start) 
            #for pileupcolumn in aln.pileup( gene['chrom'], start, stop):
            #    vector.append(pileupcolumn.n)
            for read in aln.fetch(gene['chrom'], start, stop):
                if read.is_reverse :
                    ## for get footprint in P-site (estimate) we take 3 nt in middle of read
                    #pos = (read.pos+13)-start
                    pos = (read.pos-start) + (read.rlen/2)-1
                    for z in range(pos,(pos+3)) :
                        ## le fetch contient des reads qui chevauchent les 30 nt de la fin du gene mais dont le site P
                        ## se trouve avant notre vector, le z devient negatif et la couverture augmente en fin de vecteur (ce qui est faux)
                        if z > 0 :
                            try :
                                vector1[z] += 1
                            except IndexError :
                                pass
            vector1.reverse()
            mean_read = float(sum(vector1))/float(len(vector1))
            cov = [(x/mean_read) for x in vector1]
            idx_tot = arange(len(cov))
            ## coverage in extension
            start_ext = start_extension-40
            stop_ext = stop_extension+30
            vector2 = [0]*(stop_ext-start_ext)
            #for pileupcolumn in aln.pileup( gene['chrom'], start, stop):
            #    vector.append(pileupcolumn.n)
            for read in aln.fetch(gene['chrom'], start_extension, stop_ext):
                if read.is_reverse :
                    ## get footprint in P-site
                    #pos = (read.pos+13)-start_ext
                    pos = (read.pos-start_ext) + (read.rlen/2)-1
                    for z in range(pos,(pos+3)) :
                        if z > 0 :
                            try :
                                vector2[z] += 1
                            except IndexError :
                                pass
            vector2.reverse()
            #mean_read = float(sum(vector))/float(len(vector))
            cov_ext = [(x/mean_read) for x in vector2]
            _max = max(cov_ext[30::])
            idx_ext = arange(len(cov_ext))
            
        else :
            len_ext = stop_extension-gene['start']
            start = gene['start']-100
            stop = stop_extension+100
            vector = [0]*(stop-start) 
            #for pileupcolumn in aln.pileup( gene['chrom'], start, stop):
                #vector.append(pileupcolumn.n)    
            for read in aln.fetch(gene['chrom'], start, stop):
                if not read.is_reverse :
                    ## get footprint in P-site
                    #pos = (read.pos+12)-start
                    pos = (read.pos-start) + (read.rlen/2)-1
                    for z in range(pos,(pos+3)) :
                        if z > 0 :
                            try :
                                vector[z] += 1
                            except IndexError :
                                pass
            mean_read = float(sum(vector))/float(len(vector))
            cov = [(x/mean_read) for x in vector]
            idx_tot = arange(len(cov))
            
            ## coverage in extension
            start_ext = gene['stop']-30
            stop_ext = stop_extension+40
            vector = [0]*(stop-start_ext) 
            for read in aln.fetch(gene['chrom'], start_ext, stop_extension):
                if not read.is_reverse :
                    ## get footprint in P-site
                    pos = (read.pos-start_ext) + (read.rlen/2)-1
                    for z in range(pos,(pos+3)) :
                        if z > 0 :
                            try :
                                vector[z] += 1
                            except IndexError :
                                pass
                    
            cov_ext = [(x/mean_read) for x in vector]
            idx_ext = arange(len(cov_ext))
            _max = max(cov_ext[30::])
            
        #### PLOT FIGURE ####
        
        font = {'family' : 'serif','color': 'grey','weight' : 'normal','size' : 16 }
        
        
        fig = pl.figure(num=1)
        ## create a big subplot for common y axis on two last subplot  
        ax = fig.add_subplot(2,1,2)
        ## hide all spines 
        ax.spines['top'].set_visible(False)
        ax.spines['bottom'].set_visible(False)
        ax.spines['left'].set_visible(False)
        ax.spines['right'].set_visible(False)
        ax.tick_params(labelcolor='w', top='off', bottom='off', left='off', right='off')
            
        ## plot gene structure
        ax1 = fig.add_subplot(3,1,1)
        ax1.set_title(gene['name'])
        ## hide all spines
        ax1.spines['right'].set_color('none')
        ax1.spines['top'].set_color('none')
        ax1.spines['left'].set_color('none')
        ax1.spines['bottom'].set_color('none')
        ax1.set_ylim(0,5)
        #set xlim as second plot with 100nt switch
        ax1.set_xlim(-100,len(idx_tot)-100)
        ## hide ticks
        pl.yticks([])
        pl.xticks([])
        ## if it's a "simple" gene 
        if gene['exon_number'] == 1 :
            exon = arange(len_gene)
            x = [3]*len_gene
            ax1.plot(exon,x,color='#9B9E9C')
            ax1.fill_between(exon,2,x,color='#9B9E9C')
        ## if it's a gene with introns    
        else :
            ## plot a line representing intron
            intron = arange(len_gene)
            y = [2.5]*len_gene
            ax1.plot(intron,y,color='#9B9E9C')
            
            ## plotting each intron
            start = gene['start']
            if strand == '+' :
                for exon in range(1,gene['exon_number']+1,1) :
                    len_exon = gene['exon'][exon]['stop']-gene['exon'][exon]['start']
                    idx = gene['exon'][exon]['start'] - start 
                    exo = arange(idx,idx+len_exon)
                    x = [3]*len_exon
                    ax1.plot(exo,x,color='#9B9E9C')
                    ax1.fill_between(exo,2,x,color='#9B9E9C')
            else :
                ## if it's a reverse gene we must reverse exon's position
                start = gene['start']
                tabF = [2.5]*len_gene
                tabR = [2.5]*len_gene
                for exon in range(1,gene['exon_number']+1,1) :
                    len_exon = gene['exon'][exon]['stop']-gene['exon'][exon]['start']
                    idx = gene['exon'][exon]['start'] - start 
                    exo = arange(idx,idx+len_exon)
                    for z in exo :
                        tabF[z] = 3
                        tabR[z] = 2
                tabF.reverse()
                tabR.reverse()
                #pl.ylim(0,5)
                ax1.plot(tabF,color='#9B9E9C')
                ax1.plot(tabR,color='#9B9E9C')
                x = arange(len(tabR))
                ax1.fill_between(x,tabF,tabR,color='#9B9E9C')
                
        ## insert arrows (as genome browser representation)
        yloc = 2.5
        narrows = 20
        exonwidth = .8
        spread = .4 * len_gene / narrows
        for i in range(narrows):
            loc = (float(i) * len_gene / narrows)+ (len(idx_tot)/100)*2
            if strand == '+' :
                x = [loc - spread, loc, loc - spread]
            else:
                x = [loc - spread, loc, loc - spread]
            y = [yloc - exonwidth / 5, yloc, yloc + exonwidth / 5]
            ax1.plot(x, y, lw=1.4, color='#676B69')
       
        
        # plot coverage in all gene + extension
        ax2 = fig.add_subplot(3,1,2)
        ## fixe limit to length of coverage for x axis
        ax2.set_xlim(0,len(idx_tot))
        ## fixe 4 ticks and associated labels for y axis
        ax2.set_yticklabels(arange(0,int(max(cov)+20),int((max(cov)+20)/4)))
        ax2.set_yticks(arange(0,int(max(cov)+20),int((max(cov)+20)/4)))
        ### add start and stop of gene in axe in place of number
        ax2.set_xticks([100,(100+len_gene),(100+len_ext)])
        ax2.set_xticklabels(["{:,}".format(gene['start']),"{:,}".format(gene['stop']),""])
        ## hide spines and any axis
        ax2.spines['right'].set_visible(False)
        ax2.spines['top'].set_visible(False)
        ax2.yaxis.set_ticks_position('left')
        ax2.xaxis.set_ticks_position('bottom')
        ## plot and fill
        ax2.plot(idx_tot, cov, color="#CC0011")
        ax2.fill_between(idx_tot, 0,cov,color="#CC0011")
        ax2.set_title("Genomic coordinates (%s,%s)"% (gene['strand'],gene['chrom']) ,fontdict={'fontsize':'small'})
        
        # plot zoom coverage in extension
        ax3 = fig.add_subplot(3,1,3)
        ## fixe limit to length of coverage for x axis
        ax3.set_ylim(0,int(_max))
        # we hide spines
        ax3.spines['right'].set_visible(False)
        ax3.spines['top'].set_visible(False)
        ax3.yaxis.set_ticks_position('left')
        ax3.xaxis.set_ticks_position('bottom')
        ## add stop and in-frame stop in x axis
        pl.xticks([30,( stop_extension-start_extension)+30],["stop codon","next in-frame stop"])
        ## fixe good position for labels      
        (ax3.get_xticklabels()[0]).set_horizontalalignment('center')
        (ax3.get_xticklabels()[1]).set_horizontalalignment('left')
        #if max coverage is lower than 2, we have a illegal division by zero
        if _max > 2 :
            ax3.set_yticklabels(arange(0,int(_max+1),int((_max+1)/3)))
            ax3.set_yticks(arange(0,int(_max+1),int((_max+1)/3)))
        else :
            ## 
            ax3.set_ylim(0,_max)
            ax3.ticklabel_format(style='sci', scilimits=(-2,2), axis='y',useOffset=False)
     
        ax3.plot(idx_ext, cov_ext, color="#CC0011")
        ax3.fill_between(idx_ext, 0,cov_ext,color="#CC0011")
        
        
        ## get scale of subplot 3
        #ax3.text(ax3.get_xlim()[1]-50,ax3.get_ylim()[1]-1, r'50 nt', fontdict=font)
        #pl.arrow( ax3.get_xlim()[1]-50, ax3.get_ylim()[1]-2, ax3.get_xlim()[1]-50, 0, fc="grey", ec="grey",lw=2)
        
        ## set common y label
        ax.set_ylabel('Normalized footprint counts',labelpad=20)
        
        ## draw and save plot
        pl.draw()
        #pl.show()
        pl.savefig(dirout+".png",format='png', dpi=300)
        pl.clf()
        
            
        ## Make thumbnail for html page
        infile = dirout+'.png'
        size = 128, 128
        im = Image.open(infile)
        im.thumbnail(size, Image.ANTIALIAS)
        im.save(dirout+"_thumbnail.png", "PNG")
        warnings.resetwarnings()
                
    except Exception, e:
        stop_err( 'Error during gene plotting : ' + str( e ) )

    
def compute_analysis(bam, GFF, fasta, gff, dirout, extend) :
    
    try:
        background_file = dirout+"/background_sequence.fasta"
        file_back = open(background_file, 'w')
        file_context = open(dirout+"/stop_context.fasta", 'w')
        file_extension = open(dirout+"/extensions.fasta", 'w')
        ## Opening Bam file with pysam librarie
        pysam.index(bam)
        aln = pysam.Samfile(bam, "rb",header=True, check_header = True)
        ## Opening fasta file in a dict with BioPython
        SeqDict = SeqIO.to_dict(SeqIO.parse(open(fasta,"r"),"fasta"))
            
        ## count total read in bam file
        cmd = "samtools view -c %s " % (bam)
        proc = subprocess.Popen( args=cmd, shell=True, stdout = subprocess.PIPE)
        count_tot = int(proc.stdout.read().rstrip())
        returncode = proc.wait()
        
        ## opening a GFF reader for check overlapping
        gff_reader = HTSeq.GFF_Reader(gff)

        with open(dirout+"/readthrough_result.csv","w") as out :
            myheader = ['Gene','Name', 'FAIL', 'Stop context','chrom','start extension','stop extension','length extension','RPKM CDS', 'RPKM extension','ratio','Annotation','sequence']
            writer = csv.writer(out,delimiter='\t')
            writer.writerow(myheader)    
            for gene in GFF['order'] :
                #print GFF[gene]
                ## maybe no start position in GTF file so we must to check and replace
                exon_number = GFF[gene]['exon_number']
                try : GFF[gene]['start'] 
                except :
                    if GFF[gene]['strand'] == '+' :
                        GFF[gene]['start'] = GFF[gene]['exon'][1]['start']
                    else :
                        GFF[gene]['start'] = GFF[gene]['exon'][exon_number]['stop']
                ## also for stop coordinates
                try : GFF[gene]['stop'] 
                except :
                    if GFF[gene]['strand'] == '+' :
                        GFF[gene]['stop'] = GFF[gene]['exon'][exon_number]['stop']
    
                    else :
                        GFF[gene]['stop'] = GFF[gene]['exon'][1]['start']
                
                               
                indic = ""
                # compute rpkm of CDS :
                len_cds = GFF[gene]['stop']-GFF[gene]['start']
                count_cds = 0
                rpkm_cds = 0
                count_cds = 0
                try :
                    ### count method of pysam doesn't strand information
                    if GFF[gene]['strand'] == '+' :
                        for track in aln.fetch(GFF[gene]['chrom'],GFF[gene]['start']+12,GFF[gene]['stop']-15) :
                            if not track.is_reverse :
                                count_cds += 1
                    else :
                        for track in aln.fetch(GFF[gene]['chrom'],GFF[gene]['start']+15,GFF[gene]['stop']-12) :
                            if track.is_reverse :
                                count_cds += 1
                    rpkm_cds = compute_rpkm(len_cds,count_cds,count_tot)
                except ValueError:
                    ## genere warning about gtf coordinates
                    #warnings.warn("Please check coordinates in GFF : maybe stop or start codon are missing" )
                    pass
                ## Only if gene is translate :
                if rpkm_cds > 0 and count_cds > 128:  
                    ## search footprint in UTR3
                    count = 0
                    try :
                        if GFF[gene]['strand'] == '+' :
                            contexte_stop = str(SeqDict[GFF[gene]['chrom']].seq[GFF[gene]['stop']-6:GFF[gene]['stop']+6])
                            #print gene, contexte_stop
                            start_extension = GFF[gene]['stop']
                            stop_extension = GFF[gene]['stop']+90
                            for track in aln.fetch(GFF[gene]['chrom'],start_extension+15,stop_extension) :
                                if not track.is_reverse :
                                    count += 1
                                    
                            ## get sequence of extension
                            seq = str(SeqDict[GFF[gene]['chrom']].seq[start_extension:stop_extension])
                                
                        else :
                            contexte_stop = str(SeqDict[GFF[gene]['chrom']].seq[GFF[gene]['start']-7:GFF[gene]['start']+5].reverse_complement())
                            #print gene, contexte_stop
                            start_extension = GFF[gene]['start']-90
                            stop_extension = GFF[gene]['start']
                            for track in aln.fetch(GFF[gene]['chrom'],start_extension,stop_extension-15) :
                                if track.is_reverse :
                                    count += 1
                            ## get sequence of extension
                            seq = str(SeqDict[GFF[gene]['chrom']].seq[start_extension:stop_extension-1].reverse_complement())
                             
                       
                        ## if we have coverage after cds stop codon    
                        if count > 10 :    
                            res = find_stop(seq)
                            if res == -1 :
                                '''
                                    Write results with no stop but RPF in extension
                                '''
                                ## check if next in-frame codon is far than 90 nt extension :
                                if GFF[gene]['strand'] == '+' :
                                    pos = check_overlapping(gff_reader,GFF[gene]['chrom'],GFF[gene]['stop']+1,GFF[gene]['stop']+extend,'+',GFF[gene]['name'])
                                    start_extension = pos[0]-1
                                    stop_extension = pos[1]
                                    #start_extension = GFF[gene]['stop']
                                    #stop_extension = GFF[gene]['stop']+extend
                                    seq = str(SeqDict[GFF[gene]['chrom']].seq[start_extension:stop_extension])
                                    
                                    #print gene,count,pos,'\n',seq
                                    
                                    if (seq):
                                        res = find_stop(seq)
                                        if res == -1 :
                                            mylist = [gene,GFF[gene]['name'],'no stop',contexte_stop, GFF[gene]['chrom'], start_extension, stop_extension,stop_extension-start_extension,rpkm_cds,'-','-',GFF[gene]['note'],seq]
                                            writer.writerow(mylist)
                                        else :
                                            indic = 'ok'
                                            #print res
                                            #stop_extension = start_extension + res +3
                                else :
                                    pos = check_overlapping(gff_reader,GFF[gene]['chrom'],GFF[gene]['start']-extend,GFF[gene]['start']-1,'-',GFF[gene]['name'])
                                    start_extension = pos[0]
                                    stop_extension = pos[1]+1
                                    #start_extension = GFF[gene]['start']-extend
                                    #stop_extension = GFF[gene]['start']
                                    seq = str(SeqDict[GFF[gene]['chrom']].seq[start_extension:stop_extension-1].reverse_complement())
                                    
                                    #print gene,count,pos,'\n',seq
                                    
                                    if (seq):
                                        res = find_stop(seq)
                                        if res == -1 :
                                            mylist = [gene,GFF[gene]['name'],'no stop',contexte_stop, GFF[gene]['chrom'], start_extension, stop_extension,stop_extension-start_extension,rpkm_cds,'-','-',GFF[gene]['note'],seq]
                                            writer.writerow(mylist)
                                        else :
                                            indic = 'ok'
                                            #print res
                                            #start_extension = stop_extension - res -3
                            else :
                                indic = 'ok'
                                
                                
                            if indic == 'ok' :        
                                ## We save new coordinates
                                if GFF[gene]['strand'] == '+' :
                                    stop_extension = start_extension + res +3
                                    #print gene, count
                                    #print gene,start_extension,stop_extension
                                    seq = str(SeqDict[GFF[gene]['chrom']].seq[start_extension:stop_extension])
                                    #print seq
                                    count_stop = aln.count(GFF[gene]['chrom'],stop_extension-2,stop_extension+2)
                                    if pass_length(start_extension,stop_extension) :
                                        count_ext = aln.count(GFF[gene]['chrom'],start_extension+9,stop_extension-15)
                                    if stop_extension > GFF[gene]['stop']+9 :
                                        stop_ok = 1
                                    else :
                                        stop_ok = 0
                                else :
                                    start_extension = stop_extension - res - 3
                                    #print gene, count
                                    #print gene,start_extension,stop_extension
                                    seq = str(SeqDict[GFF[gene]['chrom']].seq[start_extension-1:stop_extension-1].reverse_complement())
                                    #print seq
                                    count_stop = aln.count(GFF[gene]['chrom'],start_extension-2,start_extension+2)
                                    if pass_length(start_extension,stop_extension) :
                                        count_ext = aln.count(GFF[gene]['chrom'],start_extension+15,stop_extension-9)
                                    if start_extension < GFF[gene]['start']-9 :
                                        stop_ok = 1
                                    else :
                                        stop_ok = 0
                                ## if we are no methionine in 5 codons following stop of CDS
                                if (not check_met(seq) ):
                                    ## if we have footprint in stop codon extension and stop extension is further than cds_stop+9
                                    if count_stop > 2 and stop_ok == 1 :
                                        homo_cov = check_homo_coverage(gene,GFF,start_extension,stop_extension,aln)
                                        if (homo_cov) :
                                            '''
                                                write result witch corresponding to readthrough
                                            '''
                                            ##if length of extension upper than 25 we can compute rpkm 
                                            if (not pass_length(start_extension,stop_extension)) :
                                                len_ext = stop_extension-start_extension
                                                rpkm_ext = 'nan'
                                                ratio = 'nan'
                                            else :
                                                len_ext = stop_extension-start_extension
                                                rpkm_ext = compute_rpkm(len_ext,count_ext,count_tot)
                                                ## compute ratio between extension coverage and cds coverage (rpkm)
                                                ratio = rpkm_ext/rpkm_cds
                                                #print gene,ratio
                                                #print start_extension,stop_extension
                                            mylist = [gene,GFF[gene]['name'],'-',contexte_stop,GFF[gene]['chrom'], start_extension, stop_extension,stop_extension-start_extension,rpkm_cds,rpkm_ext,ratio,GFF[gene]['note'],seq]
                                            writer.writerow(mylist)
                                            file_context.write('>'+gene+'\n'+contexte_stop+'\n')
                                            file_extension.write('>'+gene+'\n'+seq+'\n')
                                        else :
                                            '''
                                                write result witch corresponding to readthrough but with no homogeneous coverage
                                            '''
                                            if (not pass_length(start_extension,stop_extension)) :
                                                len_ext = stop_extension-start_extension
                                                rpkm_ext = 'nan'
                                                ratio = 'nan'
                                            else :
                                                len_ext = stop_extension-start_extension
                                                rpkm_ext = compute_rpkm(len_ext,count_ext,count_tot)
                                                ## compute ratio between extension coverage and cds coverage (rpkm)
                                                ratio = rpkm_ext/rpkm_cds
                                            mylist = [gene,GFF[gene]['name'],'hetero cov',contexte_stop, GFF[gene]['chrom'], start_extension, stop_extension,stop_extension-start_extension,rpkm_cds,rpkm_ext,ratio,GFF[gene]['note'],seq]
                                            writer.writerow(mylist)
                                            file_context.write('>'+gene+'\n'+contexte_stop+'\n')
                                            file_extension.write('>'+gene+'\n'+seq+'\n')
                                            #print ">"+gene+"\n"+contexte_stop
                                        
                                        ## plot gene :
                                        plot_gene(aln, GFF[gene], start_extension, stop_extension, dirout+"/"+gene)
                                        
                                       
                                        
                                    else :
                                        '''
                                            write result with no footprint in stop codon of extension
                                        '''
                                        mylist = [gene,GFF[gene]['name'],'no RPF in stop',contexte_stop, GFF[gene]['chrom'], start_extension, stop_extension,stop_extension-start_extension,rpkm_cds,'-','-',GFF[gene]['note'],seq]
                                        writer.writerow(mylist)
                                        file_context.write('>'+gene+'\n'+contexte_stop+'\n')
                                        file_extension.write('>'+gene+'\n'+seq+'\n')
                                        #print ">"+gene+"\n"+contexte_stop
                                else :
                                    '''
                                        write result with RPF maybe result of reinitiation on a start codon
                                    '''
                                    if pass_length(start_extension,stop_extension) :
                                        mylist = [gene,GFF[gene]['name'],'Met after stop', contexte_stop, GFF[gene]['chrom'], start_extension, stop_extension,stop_extension-start_extension,rpkm_cds,'-','-',GFF[gene]['note'],seq]
                                        writer.writerow(mylist)
                                        file_context.write('>'+gene+'\n'+contexte_stop+'\n')
                                        file_extension.write('>'+gene+'\n'+seq+'\n')
                                        #print ">"+gene+"\n"+contexte_stop
                        else :
                            ## if its not a interesting case, we get stop context of genes without readthrough
                            if GFF[gene]['strand'] == '+' :
                                contexte_stop = str(SeqDict[GFF[gene]['chrom']].seq[GFF[gene]['stop']-6:GFF[gene]['stop']+6])
                                file_back.write(contexte_stop+'\n')
                            else :
                                contexte_stop = str(SeqDict[GFF[gene]['chrom']].seq[GFF[gene]['start']-7:GFF[gene]['start']+5].reverse_complement())
                                file_back.write(contexte_stop+'\n')
                            
                    ## excluded UT with incorrect positions        
                    except ValueError:
                        pass
                            
              
        file_context.close()
        file_back.close()
        file_extension.close()
    except Exception,e:
        stop_err( 'Error during computing analysis : ' + str( e ) ) 


    
def write_html_page(html,subfolder) :
        
        
    try :

        gene_table = ''
        gene_table += '<table>'
        gene_table += '<thead><tr><th data-sort="string">Gene</th><th>Plot</th><th data-sort="string">Name</th><th>Stop context</th><th>Coordinates</th><th>RPKM CDS</th><th>RPKM extension</th><th data-sort="float">ratio</th><th>Extension</th></tr></thead><tbody>'

        with open(os.path.join(subfolder,'readthrough_result.csv'), 'rb') as csvfile:
            spamreader = csv.reader(csvfile, delimiter='\t')
            ## skip the header
            next(spamreader, None)
            ##test if file is empty or not
            if next(spamreader, None):
                for row in spamreader:
                    if row[2] == '-' :
                        gene_table += '<tr><td>%s</td><td><a href="%s.png" data-lightbox="%s"><img src="%s_thumbnail.png" /></a></td><td><a title="%s">%s</a></td><td>%s</td><td>%s:%s-%s</td><td>%s</td><td>%s</td><td>%s</td><td>%s</td></tr>' %(row[0], row[0], row[0], row[0], row[11], row[1], row[3], row[4], row[5], row[6], row[8], row[9], row[10], row[12])
                    
                gene_table += '</tbody></table>' 
            else :
                gene_table = 'Sorry, there are no stop codon readthrough genes in your data\n'
     
 
        html_str = """  
<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN"
  "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
    
<html xmlns="http://www.w3.org/1999/xhtml">
    <head>
        <link rel="stylesheet" href="http://code.jquery.com/ui/1.10.3/themes/smoothness/jquery-ui.css" />
        <script src="http://code.jquery.com/jquery-1.10.2.min.js"></script>
        <script src="http://ajax.googleapis.com/ajax/libs/jquery/1.9.1/jquery.min.js"></script>
        <script src="http://code.jquery.com/ui/1.10.3/jquery-ui.js"></script>
        <script src="lightbox/js/jquery-1.10.2.min.js"></script>
        <script src="lightbox/js/lightbox-2.6.min.js"></script>
        <link href="lightbox/css/lightbox.css" rel="stylesheet" />
        <meta http-equiv="Content-Type" content="text/html; charset=utf-8" />
        <title>Dual coding result file</title>
        <link rel="stylesheet" href="http://code.jquery.com/ui/1.10.3/themes/smoothness/jquery-ui.css" />
        <script>
          (function(d){d.fn.stupidtable=function(b){return this.each(function(){var a=d(this);b=b||{};b=d.extend({},d.fn.stupidtable.default_sort_fns,b);var n=function(a,b){for(var f=[],c=0,e=a.slice(0).sort(b),h=0;h<a.length;h++){for(c=d.inArray(a[h],e);-1!=d.inArray(c,f);)c++;f.push(c)}return f},q=function(a,b){for(var d=a.slice(0),c=0,e=0;e<b.length;e++)c=b[e],d[c]=a[e];return d};a.on("click","th",function(){var m=a.children("tbody").children("tr"),g=d(this),f=0,c=d.fn.stupidtable.dir;a.find("th").slice(0, g.index()).each(function(){var a=d(this).attr("colspan")||1;f+=parseInt(a,10)});var e=g.data("sort-default")||c.ASC;g.data("sort-dir")&&(e=g.data("sort-dir")===c.ASC?c.DESC:c.ASC);var h=g.data("sort")||null;null!==h&&(a.trigger("beforetablesort",{column:f,direction:e}),a.css("display"),setTimeout(function(){var l=[],p=b[h];m.each(function(a,b){var c=d(b).children().eq(f),e=c.data("sort-value"),c="undefined"!==typeof e?e:c.text();l.push(c)});var k;k=e==c.ASC?n(l,p):n(l,function(a,b){return-p(a,b)}); a.find("th").data("sort-dir",null).removeClass("sorting-desc sorting-asc");g.data("sort-dir",e).addClass("sorting-"+e);k=d(q(m,k));a.children("tbody").remove();a.append("<tbody />").append(k);a.trigger("aftertablesort",{column:f,direction:e});a.css("display")},10))})})};d.fn.stupidtable.dir={ASC:"asc",DESC:"desc"};d.fn.stupidtable.default_sort_fns={"int":function(b,a){return parseInt(b,10)-parseInt(a,10)},"float":function(b,a){return parseFloat(b)-parseFloat(a)},string:function(b,a){return b<a?-1: b>a?1:0},"string-ins":function(b,a){b=b.toLowerCase();a=a.toLowerCase();return b<a?-1:b>a?1:0}}})(jQuery);
          (function($) {

            $.fn.stupidtable = function(sortFns) {
              return this.each(function() {
                var $table = $(this);
                sortFns = sortFns || {};
          
                // ==================================================== //
                //                  Utility functions                   //
                // ==================================================== //
          
                // Merge sort functions with some default sort functions.
                sortFns = $.extend({}, $.fn.stupidtable.default_sort_fns, sortFns);
          
                // Return the resulting indexes of a sort so we can apply
                // this result elsewhere. This returns an array of index numbers.
                // return[0] = x means "arr's 0th element is now at x"
                var sort_map = function(arr, sort_function) {
                  var map = [];
                  var index = 0;
                  var sorted = arr.slice(0).sort(sort_function);
                  for (var i=0; i<arr.length; i++) {
                      index = $.inArray(arr[i], sorted);
          
                      // If this index is already in the map, look for the next index.
                      // This handles the case of duplicate entries.
                      while ($.inArray(index, map) != -1) {
                          index++;
                      }
                      map.push(index);
                  }
          
                  return map;
                };
          
                // Apply a sort map to the array.
                var apply_sort_map = function(arr, map) {
                  var clone = arr.slice(0),
                      newIndex = 0;
                  for (var i=0; i<map.length; i++) {
                    newIndex = map[i];
                    clone[newIndex] = arr[i];
                  }
                  return clone;
                };
          
                // ==================================================== //
                //                  Begin execution!                    //
                // ==================================================== //
          
                // Do sorting when THs are clicked
                $table.on("click", "th", function() {
                  var trs = $table.children("tbody").children("tr");
                  var $this = $(this);
                  var th_index = 0;
                  var dir = $.fn.stupidtable.dir;
          
                  $table.find("th").slice(0, $this.index()).each(function() {
                    var cols = $(this).attr("colspan") || 1;
                    th_index += parseInt(cols,10);
                  });
          
                  // Determine (and/or reverse) sorting direction, default `asc`
                  var sort_dir = $this.data("sort-default") || dir.ASC;
                  if ($this.data("sort-dir"))
                     sort_dir = $this.data("sort-dir") === dir.ASC ? dir.DESC : dir.ASC;
          
                  // Choose appropriate sorting function. 
                  var type = $this.data("sort") || null;
          
                  // Prevent sorting if no type defined
                  if (type === null) {
                    return;
                  }
          
                  // Trigger `beforetablesort` event that calling scripts can hook into;
                  // pass parameters for sorted column index and sorting direction
                  $table.trigger("beforetablesort", {column: th_index, direction: sort_dir});
                  // More reliable method of forcing a redraw
                  $table.css("display");
          
                  // Run sorting asynchronously on a timout to force browser redraw after
                  // `beforetablesort` callback. Also avoids locking up the browser too much.
                  setTimeout(function() {
                    // Gather the elements for this column
                    var column = [];
                    var sortMethod = sortFns[type];
          
                    // Push either the value of the `data-order-by` attribute if specified
                    // or just the text() value in this column to column[] for comparison.
                    trs.each(function(index,tr) {
                      var $e = $(tr).children().eq(th_index);
                      var sort_val = $e.data("sort-value");
                      var order_by = typeof(sort_val) !== "undefined" ? sort_val : $e.text();
                      column.push(order_by);
                    });
          
                    // Create the sort map. This column having a sort-dir implies it was
                    // the last column sorted. As long as no data-sort-desc is specified,
                    // we're free to just reverse the column.
                    var theMap;
                    if (sort_dir == dir.ASC)
                      theMap = sort_map(column, sortMethod);
                    else
                      theMap = sort_map(column, function(a, b) { return -sortMethod(a, b); });
          
                    // Reset siblings
                    $table.find("th").data("sort-dir", null).removeClass("sorting-desc sorting-asc");
                    $this.data("sort-dir", sort_dir).addClass("sorting-"+sort_dir);
          
                    var sortedTRs = $(apply_sort_map(trs, theMap));
                    $table.children("tbody").remove();
                    $table.append("<tbody />").append(sortedTRs);
          
                    // Trigger `aftertablesort` event. Similar to `beforetablesort`
                    $table.trigger("aftertablesort", {column: th_index, direction: sort_dir});
                    // More reliable method of forcing a redraw
                    $table.css("display");
                  }, 10);
                });
              });
            };
          
            // Enum containing sorting directions
            $.fn.stupidtable.dir = {ASC: "asc", DESC: "desc"};
          
            $.fn.stupidtable.default_sort_fns = {
              "int": function(a, b) {
                return parseInt(a, 10) - parseInt(b, 10);
              },
              "float": function(a, b) {
                return parseFloat(a) - parseFloat(b);
              },
              "string": function(a, b) {
                if (a < b) return -1;
                if (a > b) return +1;
                return 0;
              },
              "string-ins": function(a, b) {
                a = a.toLowerCase();
                b = b.toLowerCase();
                if (a < b) return -1;
                if (a > b) return +1;
                return 0;
              }
            };
          
          })(jQuery);
          $(function(){
            var table = $("table").stupidtable();
          
            table.on("beforetablesort", function (event, data) {
              // data.column - the index of the column sorted after a click
              // data.direction - the sorting direction (either asc or desc)
              $("#msg").text("Sorting index " + data.column)
            });
            table.on("aftertablesort", function (event, data) {
              var th = $(this).find("th");
              th.find(".arrow").remove();
              var dir = $.fn.stupidtable.dir;
              var arrow = data.direction === dir.ASC ? "&uarr;" : "&darr;";
              th.eq(data.column).append('<span class="arrow">' + arrow +'</span>');
            });
          });
        </script>
        <style type="text/css">
            label {
              display: inline-block;
              width: 5em;
            }
            table {
              border-collapse: collapse;
            }
            th, td {
              padding: 5px 10px;
              border: 1px solid #999;
            }
            th {
              background-color: #a7d3ff;
            }
            th[data-sort]{
              cursor:pointer;
            }
            a[data-lightbox]{
              cursor:zoom-in;
            }
            #msg {
              color: green;
            }
        </style>
    </head>
    <body>
        <h1>Readthrough analyse results</h1>
            %s
    </body>
</html> """ % (gene_table)
         
        html_file = open(html,"w")
        html_file.write(html_str)
        html_file.close()    
        
        
    except Exception, e :
        stop_err('Error during html page creation : ' + str( e ) )
        
        
def __main__():

    #Parse command line options
    parser = optparse.OptionParser()
    parser.add_option("-g", "--gff", dest="gff", type= "string",
                  help="GFF annotation file", metavar="FILE")
                    
    parser.add_option("-f", "--fasta", dest="fasta", type= "string",
                  help="Fasta file ", metavar="FILE")
            
    parser.add_option("-b", "--bam", dest="bamfile", type= "string",
                  help="Bam Ribo-Seq alignments ", metavar="FILE")
                    
    parser.add_option("-d", "--dirout", dest="dirout", type= "string",
                  help="write report in this html file and in associated directory", metavar="STR,STR")   
    
    parser.add_option("-e", "--extend", dest="extend", type= "int",default = 300 ,
                  help="Lenght of extension after stop in number of base pairs (depends on your organisme)", metavar="INT")     
                    
    parser.add_option("-q", "--quiet",
                  action="store_false", dest="verbose", default=True,
                  help="don't print status messages to stdout")
                    
    (options, args) = parser.parse_args()
    sys.stdout.write("Begin readthrough analysis at %s\n" % time.asctime( time.localtime(time.time())))

    try:
        (html_file, subfolder ) = options.dirout.split(",")
        if not os.path.exists(subfolder):
            try:
                os.mkdir(subfolder)
            except Exception, e :
                stop_err('Error running make directory : ' + str(e)) 
        ## identify GFF or GTF format from 9th column
        with open (options.gff,"r") as gffile :
            for line in gffile :
                if '#' in line :
                    ## skip header
                    gffile.next()
                elif 'gene_id' in line :
                    ## launch gtf reader :
                    GFF = ribo_functions.store_gtf(options.gff)
                    break
                elif 'ID=' in line :
                    ## launch gff reader 
                    GFF = ribo_functions.store_gff(options.gff)
                    break
                else :
                    stop_err( 'Please check your annotation file is in correct format, GFF or GTF' )
            
        #GFF = store_gff(options.gff)
        #GFF = ribo_functions.store_gtf(options.gff)
        ## check gff reading
        if not GFF['order'] :
            stop_err( 'Incorrect GFF file' )
        clean_file = ribo_functions.cleaning_bam(options.bamfile)
        compute_analysis(clean_file, GFF, options.fasta, options.gff, subfolder, options.extend)
        if os.path.exists( clean_file ):
            os.remove( clean_file )
        
        write_html_page(html_file,subfolder)
        ##paste jquery script in result directory :
        jq_src = os.path.join(os.path.dirname(__file__),'lightbox')
        shutil.copytree(jq_src,os.path.join(subfolder,'lightbox'))
        
        
        sys.stdout.write("Finish readthrough analysis at %s\n" % time.asctime( time.localtime(time.time())))
    except Exception, e:
        stop_err( 'Error running metagene readthrough analysis : ' + str( e ) )
    

if __name__=="__main__":
    __main__()