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ROSE2_main.py
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ROSE2_main.py
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#!/usr/bin/env python
'''
PROGRAM TO STITCH TOGETHER REGIONS TO FORM ENHANCERS, MAP READ DENSITY TO STITCHED REGIONS,
AND RANK ENHANCERS BY READ DENSITY TO DISCOVER SUPER-ENHANCERS
May2, 2014
VERSION 0.2
CONTACT: [email protected]
'''
from __future__ import absolute_import # , division, print_function, unicode_literals
import utils
import sys
# import ROSE_utils
import time
import copy
import os
import numpy
import subprocess
from collections import defaultdict
#==================================================================
#=====================HELPER FUNCTIONS=============================
#==================================================================
def getBamChromList(bamFileList):
'''
gets the consensus list of chromosomes mapped by the bams
'''
#start w/ the first bam
cmd = 'samtools idxstats %s' % (bamFileList[0])
idxStats = subprocess.Popen(cmd,stdout=subprocess.PIPE,shell=True)
idxStats= idxStats.communicate()
finalChromList = [line.split('\t')[0] for line in idxStats[0].split('\n')[0:-2]]
#now go through each additional bam
for bamFile in bamFileList:
cmd = 'samtools idxstats %s' % (bamFile)
idxStats = subprocess.Popen(cmd,stdout=subprocess.PIPE,shell=True)
idxStats= idxStats.communicate()
chromList = [line.split('\t')[0] for line in idxStats[0].split('\n')[0:-2]]
finalChromList = [chrom for chrom in finalChromList if chromList.count(chrom) != 0]
return utils.uniquify(finalChromList)
def checkRefCollection(referenceCollection):
'''
makes sure the names of all loci in the reference collection are unique
'''
namesList = [locus.ID() for locus in referenceCollection.getLoci()]
if len(namesList) != len(utils.uniquify(namesList)):
print("ERROR: REGIONS HAVE NON-UNIQUE IDENTIFIERS")
sys.exit()
else:
print("REFERENCE COLLECTION PASSES QC")
return
def filterGFF(gffFile,chromList):
'''
takes in a gff and filters out all lines that don't belong to a chrom in the chromList
'''
gff = utils.parseTable(gffFile,'\t')
filteredGFF = []
excludeList=[]
for line in gff:
if chromList.count(line[0]) ==1:
filteredGFF.append(line)
else:
excludeList.append(line[0])
excludeList = utils.uniquify(excludeList)
if len(excludeList) > 0:
print("EXCLUDED GFF REGIONS FROM THE FALLING CHROMS: %s" % (','.join(excludeList)))
return filteredGFF
#==================================================================
#=====================REGION STITCHING=============================
#==================================================================
def optimizeStitching(locusCollection, name, outFolder, stepSize=500):
'''
takes a locus collection and starts writing out stitching stats at step sized intervals
'''
maxStitch = 15000 # set a hard wired match stitching parameter
stitchTable = [['STEP', 'NUM_REGIONS', 'TOTAL_CONSTIT', 'TOTAL_REGION', 'MEAN_CONSTIT', 'MEDIAN_CONSTIT', 'MEAN_REGION', 'MEDIAN_REGION', 'MEAN_STITCH_FRACTION', 'MEDIAN_STITCH_FRACTION']]
# first consolidate the collection
locusCollection = locusCollection.stitchCollection(stitchWindow=0)
total_constit = sum([locus.len() for locus in locusCollection.getLoci()])
step = 0
while step <= maxStitch:
print("Getting stitch stats for %s (bp)" % (step))
stitchCollection = locusCollection.stitchCollection(stitchWindow=step)
num_regions = len(stitchCollection)
stitchLoci = stitchCollection.getLoci()
regionLengths = [locus.len() for locus in stitchLoci]
total_region = sum(regionLengths)
constitLengths = []
for locus in stitchLoci:
constitLoci = locusCollection.getOverlap(locus)
constitLengths.append(sum([locus.len() for locus in constitLoci]))
meanConstit = round(numpy.mean(constitLengths), 2)
medianConstit = round(numpy.median(constitLengths), 2)
meanRegion = round(numpy.mean(regionLengths), 2)
medianRegion = round(numpy.median(regionLengths), 2)
stitchFractions = [float(constitLengths[i]) / float(regionLengths[i]) for i in range(len(regionLengths))]
meanStitchFraction = round(numpy.mean(stitchFractions), 2)
medianStitchFraction = round(numpy.median(stitchFractions), 2)
newLine = [step, num_regions, total_constit, total_region, meanConstit, medianConstit, meanRegion, medianRegion, meanStitchFraction, medianStitchFraction]
stitchTable.append(newLine)
step += stepSize
# write the stitch table to disk
stitchParamFile = '%s%s_stitch_params.tmp' % (outFolder, name)
utils.unParseTable(stitchTable, stitchParamFile, '\t')
# call the rscript
rCmd = 'Rscript ./ROSE2_stitchOpt.R %s %s %s' % (stitchParamFile, outFolder, name)
print(rCmd)
# get back the stitch parameter
rOutput = subprocess.Popen(rCmd, stdout=subprocess.PIPE, shell=True)
rOutputTest = rOutput.communicate()
print(rOutputTest)
stitchParam = rOutputTest[0].split('\n')[2]
try:
stitchParam = int(stitchParam)
except ValueError:
print("INVALID STITCHING PARAMETER. STITCHING OPTIMIZATION FAILED")
sys.exit()
# delete? the table
# os.system('rm -f %s' % (stitchParamFile))
return stitchParam
def regionStitching(referenceCollection, name, outFolder, stitchWindow, tssWindow, annotFile, removeTSS=True):
print('PERFORMING REGION STITCHING')
# first have to turn bound region file into a locus collection
# need to make sure this names correctly... each region should have a unique name
#referenceCollection
debugOutput = []
# filter out all bound regions that overlap the TSS of an ACTIVE GENE
if removeTSS:
print('REMOVING TSS FROM REGIONS USING AN EXCLUSION WINDOW OF %sBP' % (tssWindow))
# first make a locus collection of TSS
startDict = utils.makeStartDict(annotFile)
# now makeTSS loci for active genes
removeTicker = 0
# this loop makes a locus centered around +/- tssWindow of transcribed genes
# then adds it to the list tssLoci
tssLoci = []
for geneID in startDict.keys():
tssLoci.append(utils.makeTSSLocus(geneID, startDict, tssWindow, tssWindow))
# this turns the tssLoci list into a LocusCollection
# 50 is the internal parameter for LocusCollection and doesn't really matter
tssCollection = utils.LocusCollection(tssLoci, 50)
# gives all the loci in referenceCollection
boundLoci = referenceCollection.getLoci()
# this loop will check if each bound region is contained by the TSS exclusion zone
# this will drop out a lot of the promoter only regions that are tiny
# typical exclusion window is around 2kb
for locus in boundLoci:
if len(tssCollection.getContainers(locus, 'both')) > 0:
# if true, the bound locus overlaps an active gene
referenceCollection.remove(locus)
debugOutput.append([locus.__str__(), locus.ID(), 'CONTAINED'])
removeTicker += 1
print('REMOVED %s LOCI BECAUSE THEY WERE CONTAINED BY A TSS' % (removeTicker))
# referenceCollection is now all enriched region loci that don't overlap an active TSS
if stitchWindow == '':
print('DETERMINING OPTIMUM STITCHING PARAMTER')
optCollection = copy.deepcopy(referenceCollection)
stitchWindow = optimizeStitching(optCollection, name, outFolder, stepSize=500)
print('USING A STITCHING PARAMETER OF %s' % stitchWindow)
stitchedCollection = referenceCollection.stitchCollection(stitchWindow, 'both')
if removeTSS:
# now replace any stitched region that overlap 2 distinct genes
# with the original loci that were there
fixedLoci = []
tssLoci = []
for geneID in startDict.keys():
tssLoci.append(utils.makeTSSLocus(geneID, startDict, 50, 50))
# this turns the tssLoci list into a LocusCollection
# 50 is the internal parameter for LocusCollection and doesn't really matter
tssCollection = utils.LocusCollection(tssLoci, 50)
removeTicker = 0
originalTicker = 0
for stitchedLocus in stitchedCollection.getLoci():
overlappingTSSLoci = tssCollection.getOverlap(stitchedLocus, 'both')
tssNames = [startDict[tssLocus.ID()]['name'] for tssLocus in overlappingTSSLoci]
tssNames = utils.uniquify(tssNames)
if len(tssNames) > 2:
# stitchedCollection.remove(stitchedLocus)
originalLoci = referenceCollection.getOverlap(stitchedLocus, 'both')
originalTicker += len(originalLoci)
fixedLoci += originalLoci
debugOutput.append([stitchedLocus.__str__(), stitchedLocus.ID(), 'MULTIPLE_TSS'])
removeTicker += 1
else:
fixedLoci.append(stitchedLocus)
print('REMOVED %s STITCHED LOCI BECAUSE THEY OVERLAPPED MULTIPLE TSSs' % (removeTicker))
print('ADDED BACK %s ORIGINAL LOCI' % (originalTicker))
fixedCollection = utils.LocusCollection(fixedLoci, 50)
return fixedCollection, debugOutput, stitchWindow
else:
return stitchedCollection, debugOutput, stitchWindow
#==================================================================
#=====================REGION LINKING MAPPING=======================
#==================================================================
def mapCollection(stitchedCollection, referenceCollection, bamFileList, mappedFolder, output, refName):
'''
makes a table of factor density in a stitched locus and ranks table by number of loci stitched together
'''
print('FORMATTING TABLE')
loci = stitchedCollection.getLoci()
locusTable = [['REGION_ID', 'CHROM', 'START', 'STOP', 'NUM_LOCI', 'CONSTITUENT_SIZE']]
lociLenList = []
# strip out any that are in chrY
for locus in list(loci):
if locus.chr() == 'chrY':
loci.remove(locus)
for locus in loci:
# numLociList.append(int(stitchLocus.ID().split('_')[1]))
lociLenList.append(locus.len())
# numOrder = order(numLociList,decreasing=True)
lenOrder = utils.order(lociLenList, decreasing=True)
ticker = 0
for i in lenOrder:
ticker += 1
if ticker % 1000 == 0:
print(ticker)
locus = loci[i]
# First get the size of the enriched regions within the stitched locus
refEnrichSize = 0
refOverlappingLoci = referenceCollection.getOverlap(locus, 'both')
for refLocus in refOverlappingLoci:
refEnrichSize += refLocus.len()
try:
stitchCount = int(locus.ID().split('_')[0])
except ValueError:
stitchCount = 1
coords = [int(x) for x in locus.coords()]
locusTable.append([locus.ID(), locus.chr(), min(coords), max(coords), stitchCount, refEnrichSize])
print('GETTING MAPPED DATA')
print("USING A BAMFILE LIST:")
print(bamFileList)
for bamFile in bamFileList:
bamFileName = bamFile.split('/')[-1]
print('GETTING MAPPING DATA FOR %s' % bamFile)
# assumes standard convention for naming enriched region gffs
# opening up the mapped GFF
print('OPENING %s%s_%s_MAPPED/matrix.txt' % (mappedFolder, refName, bamFileName))
mappedGFF = utils.parseTable('%s%s_%s_MAPPED/matrix.txt' % (mappedFolder, refName, bamFileName), '\t')
signalDict = defaultdict(float)
print('MAKING SIGNAL DICT FOR %s' % (bamFile))
mappedLoci = []
for line in mappedGFF[1:]:
chrom = line[1].split('(')[0]
start = int(line[1].split(':')[-1].split('-')[0])
end = int(line[1].split(':')[-1].split('-')[1])
mappedLoci.append(utils.Locus(chrom, start, end, '.', line[0]))
try:
signalDict[line[0]] = float(line[2]) * (abs(end - start))
except ValueError:
print('WARNING NO SIGNAL FOR LINE:')
print(line)
continue
mappedCollection = utils.LocusCollection(mappedLoci, 500)
locusTable[0].append(bamFileName)
for i in range(1, len(locusTable)):
signal = 0.0
line = locusTable[i]
lineLocus = utils.Locus(line[1], line[2], line[3], '.')
overlappingRegions = mappedCollection.getOverlap(lineLocus, sense='both')
for region in overlappingRegions:
signal += signalDict[region.ID()]
locusTable[i].append(signal)
utils.unParseTable(locusTable, output, '\t')
#==================================================================
#=========================MAIN METHOD==============================
#==================================================================
def main():
'''
main run call
'''
debug = False
from optparse import OptionParser
usage = "usage: %prog [options] -g [GENOME] -i [INPUT_REGION_GFF] -r [RANKBY_BAM_FILE] -o [OUTPUT_FOLDER] [OPTIONAL_FLAGS]"
parser = OptionParser(usage=usage)
# required flags
parser.add_option("-i", "--i", dest="input", nargs=1, default=None,
help="Enter a .gff or .bed file of binding sites used to make enhancers")
parser.add_option("-r", "--rankby", dest="rankby", nargs=1, default=None,
help="bamfile to rank enhancer by")
parser.add_option("-o", "--out", dest="out", nargs=1, default=None,
help="Enter an output folder")
parser.add_option("-g", "--genome", dest="genome", nargs=1, default=None,
help="Enter the genome build (MM9,MM8,HG18,HG19)")
# optional flags
parser.add_option("-b", "--bams", dest="bams", nargs=1, default=None,
help="Enter a comma separated list of additional bam files to map to")
parser.add_option("-c", "--control", dest="control", nargs=1, default=None,
help="bamfile to rank enhancer by")
parser.add_option("-s", "--stitch", dest="stitch", nargs=1, default='',
help="Enter a max linking distance for stitching. Default will determine optimal stitching parameter")
parser.add_option("-t", "--tss", dest="tss", nargs=1, default=0,
help="Enter a distance from TSS to exclude. 0 = no TSS exclusion")
parser.add_option("--mask", dest="mask", nargs=1, default=None,
help="Mask a set of regions from analysis. Provide a .bed or .gff of masking regions")
# RETRIEVING FLAGS
(options, args) = parser.parse_args()
if not options.input or not options.rankby or not options.out or not options.genome:
print('hi there')
parser.print_help()
exit()
# making the out folder if it doesn't exist
outFolder = utils.formatFolder(options.out, True)
# figuring out folder schema
gffFolder = utils.formatFolder(outFolder + 'gff/', True)
mappedFolder = utils.formatFolder(outFolder + 'mappedGFF/', True)
# GETTING INPUT FILE
if options.input.split('.')[-1] == 'bed':
# CONVERTING A BED TO GFF
inputGFFName = options.input.split('/')[-1][0:-4]
inputGFFFile = '%s%s.gff' % (gffFolder, inputGFFName)
utils.bedToGFF(options.input, inputGFFFile)
elif options.input.split('.')[-1] == 'gff':
# COPY THE INPUT GFF TO THE GFF FOLDER
inputGFFFile = options.input
os.system('cp %s %s' % (inputGFFFile, gffFolder))
else:
print('WARNING: INPUT FILE DOES NOT END IN .gff or .bed. ASSUMING .gff FILE FORMAT')
# COPY THE INPUT GFF TO THE GFF FOLDER
inputGFFFile = options.input
os.system('cp %s %s' % (inputGFFFile, gffFolder))
# GETTING THE LIST OF BAMFILES TO PROCESS
if options.control:
bamFileList = [options.rankby, options.control]
else:
bamFileList = [options.rankby]
if options.bams:
bamFileList += options.bams.split(',')
#bamFileList = utils.uniquify(bamFileList) # makes sad when you have the same control bam over and over again
# optional args
# Stitch parameter
if options.stitch == '':
stitchWindow = ''
else:
stitchWindow = int(options.stitch)
# tss options
tssWindow = int(options.tss)
if tssWindow != 0:
removeTSS = True
else:
removeTSS = False
# GETTING THE BOUND REGION FILE USED TO DEFINE ENHANCERS
print('USING %s AS THE INPUT GFF' % (inputGFFFile))
inputName = inputGFFFile.split('/')[-1].split('.')[0]
# GETTING THE GENOME
genome = options.genome
print('USING %s AS THE GENOME' % genome)
# GETTING THE CORRECT ANNOT FILE
cwd = os.getcwd()
genomeDict = {
'HG18': '%s/annotation/hg18_refseq.ucsc' % (cwd),
'MM9': '%s/annotation/mm9_refseq.ucsc' % (cwd),
'HG19': '%s/annotation/hg19_refseq.ucsc' % (cwd),
'MM8': '%s/annotation/mm8_refseq.ucsc' % (cwd),
'MM10': '%s/annotation/mm10_refseq.ucsc' % (cwd),
'RN4': '%s/annotation/rn4_refseq.ucsc' % (cwd),
}
annotFile = genomeDict[genome.upper()]
# MAKING THE START DICT
print('MAKING START DICT')
startDict = utils.makeStartDict(annotFile)
#GET CHROMS FOUND IN THE BAMS
print('GETTING CHROMS IN BAMFILES')
bamChromList = getBamChromList(bamFileList)
print("USING THE FOLLOWING CHROMS")
print(bamChromList)
#LOADING IN THE GFF AND FILTERING BY CHROM
print('LOADING AND FILTERING THE GFF')
inputGFF = filterGFF(inputGFFFile,bamChromList)
# LOADING IN THE BOUND REGION REFERENCE COLLECTION
print('LOADING IN GFF REGIONS')
referenceCollection = utils.gffToLocusCollection(inputGFF)
print('CHECKING REFERENCE COLLECTION:')
checkRefCollection(referenceCollection)
# MASKING REFERENCE COLLECTION
# see if there's a mask
if options.mask:
maskFile = options.mask
# if it's a bed file
if maskFile.split('.')[-1].upper() == 'BED':
maskGFF = utils.bedToGFF(maskFile)
elif maskFile.split('.')[-1].upper() == 'GFF':
maskGFF = utils.parseTable(maskFile, '\t')
else:
print("MASK MUST BE A .gff or .bed FILE")
sys.exit()
maskCollection = utils.gffToLocusCollection(maskGFF)
# now mask the reference loci
referenceLoci = referenceCollection.getLoci()
filteredLoci = [locus for locus in referenceLoci if len(maskCollection.getOverlap(locus, 'both')) == 0]
print("FILTERED OUT %s LOCI THAT WERE MASKED IN %s" % (len(referenceLoci) - len(filteredLoci), maskFile))
referenceCollection = utils.LocusCollection(filteredLoci, 50)
# NOW STITCH REGIONS
print('STITCHING REGIONS TOGETHER')
stitchedCollection, debugOutput, stitchWindow = regionStitching(referenceCollection, inputName, outFolder, stitchWindow, tssWindow, annotFile, removeTSS)
# NOW MAKE A STITCHED COLLECTION GFF
print('MAKING GFF FROM STITCHED COLLECTION')
stitchedGFF = utils.locusCollectionToGFF(stitchedCollection)
# making sure start/stop ordering are correct
for i in range(len(stitchedGFF)):
line = stitchedGFF[i]
start = int(line[3])
stop = int(line[4])
if start > stop:
line[3] = stop
line[4] = start
print(stitchWindow)
print(type(stitchWindow))
if not removeTSS:
stitchedGFFFile = '%s%s_%sKB_STITCHED.gff' % (gffFolder, inputName, str(stitchWindow / 1000))
stitchedGFFName = '%s_%sKB_STITCHED' % (inputName, str(stitchWindow / 1000))
debugOutFile = '%s%s_%sKB_STITCHED.debug' % (gffFolder, inputName, str(stitchWindow / 1000))
else:
stitchedGFFFile = '%s%s_%sKB_STITCHED_TSS_DISTAL.gff' % (gffFolder, inputName, str(stitchWindow / 1000))
stitchedGFFName = '%s_%sKB_STITCHED_TSS_DISTAL' % (inputName, str(stitchWindow / 1000))
debugOutFile = '%s%s_%sKB_STITCHED_TSS_DISTAL.debug' % (gffFolder, inputName, str(stitchWindow / 1000))
# WRITING DEBUG OUTPUT TO DISK
if debug:
print('WRITING DEBUG OUTPUT TO DISK AS %s' % (debugOutFile))
utils.unParseTable(debugOutput, debugOutFile, '\t')
# WRITE THE GFF TO DISK
print('WRITING STITCHED GFF TO DISK AS %s' % (stitchedGFFFile))
utils.unParseTable(stitchedGFF, stitchedGFFFile, '\t')
# SETTING UP THE OVERALL OUTPUT FILE
outputFile1 = outFolder + stitchedGFFName + '_ENHANCER_REGION_MAP.txt'
print('OUTPUT WILL BE WRITTEN TO %s' % (outputFile1))
# MAPPING TO THE NON STITCHED (ORIGINAL GFF)
# MAPPING TO THE STITCHED GFF
# Try to use the bamliquidatior_path.py script on cluster, otherwise, failover to local (in path), otherwise fail.
bamliquidator_path = 'bamliquidator_batch'
bamFileListUnique = list(bamFileList)
bamFileListUnique = utils.uniquify(bamFileListUnique)
#prevent redundant mapping
print("MAPPING TO THE FOLLOWING BAMS:")
print(bamFileListUnique)
for bamFile in bamFileListUnique:
bamFileName = bamFile.split('/')[-1]
# MAPPING TO THE STITCHED GFF
mappedOut1Folder = '%s%s_%s_MAPPED' % (mappedFolder, stitchedGFFName, bamFileName)
mappedOut1File = '%s%s_%s_MAPPED/matrix.txt' % (mappedFolder, stitchedGFFName, bamFileName)
if utils.checkOutput(mappedOut1File, 0.2, 0.2):
print("FOUND %s MAPPING DATA FOR BAM: %s" % (stitchedGFFFile, mappedOut1File))
else:
cmd1 = bamliquidator_path + " --sense . -e 200 --match_bamToGFF -r %s -o %s %s" % (stitchedGFFFile, mappedOut1Folder, bamFile)
print(cmd1)
os.system(cmd1)
if utils.checkOutput(mappedOut1File,0.2,5):
print("SUCCESSFULLY MAPPED TO %s FROM BAM: %s" % (stitchedGFFFile, bamFileName))
else:
print("ERROR: FAILED TO MAP %s FROM BAM: %s" % (stitchedGFFFile, bamFileName))
sys.exit()
print('BAM MAPPING COMPLETED NOW MAPPING DATA TO REGIONS')
# CALCULATE DENSITY BY REGION
# NEED TO FIX THIS FUNCTION TO ACCOUNT FOR DIFFERENT OUTPUTS OF LIQUIDATOR
mapCollection(stitchedCollection, referenceCollection, bamFileList, mappedFolder, outputFile1, refName=stitchedGFFName)
print('CALLING AND PLOTTING SUPER-ENHANCERS')
if options.control:
rankbyName = options.rankby.split('/')[-1]
controlName = options.control.split('/')[-1]
cmd = 'R --no-save %s %s %s %s < ROSE2_callSuper.R' % (outFolder, outputFile1, inputName, controlName)
else:
rankbyName = options.rankby.split('/')[-1]
controlName = 'NONE'
cmd = 'R --no-save %s %s %s %s < ROSE2_callSuper.R' % (outFolder, outputFile1, inputName, controlName)
print(cmd)
os.system(cmd)
# calling the gene mapper
time.sleep(20)
superTableFile = "%s_SuperEnhancers.table.txt" % (inputName)
if options.control:
cmd = "python ROSE2_geneMapper.py -g %s -r %s -c %s -i %s%s &" % (genome, options.rankby, options.control, outFolder, superTableFile)
else:
cmd = "python ROSE2_geneMapper.py -g %s -r %s -i %s%s &" % (genome, options.rankby, outFolder, superTableFile)
os.system(cmd)
stretchTableFile = "%s_StretchEnhancers.table.txt" % (inputName)
if options.control:
cmd = "python ROSE2_geneMapper.py -g %s -r %s -c %s -i %s%s &" % (genome, options.rankby, options.control, outFolder, stretchTableFile)
else:
cmd = "python ROSE2_geneMapper.py -g %s -r %s -i %s%s &" % (genome, options.rankby, outFolder, stretchTableFile)
os.system(cmd)
superStretchTableFile = "%s_SuperStretchEnhancers.table.txt" % (inputName)
if options.control:
cmd = "python ROSE2_geneMapper.py -g %s -r %s -c %s -i %s%s &" % (genome, options.rankby, options.control, outFolder, superStretchTableFile)
else:
cmd = "python ROSE2_geneMapper.py -g %s -r %s -i %s%s &" % (genome, options.rankby, outFolder, superStretchTableFile)
os.system(cmd)
if __name__ == "__main__":
main()