""" MakeRdsFromBam Created on Jun 3, 2010 @author: sau """ try: import psyco psyco.full() except: pass import sys, string, optparse, re import pysam from commoncode import readDataset, writeLog def main(argv=None): if not argv: argv = sys.argv verstring = "%s: version 1.0" % sys.argv[0] print verstring usage = "usage: %prog label samfile outrdsfile [propertyName::propertyValue] [options]\ \ninput reads must be sorted to properly record multireads" parser = optparse.OptionParser(usage=usage) parser.add_option("--append", action="store_false", dest="init", help="append to existing rds file [default: create new]") parser.add_option("--RNA", action="store_true", dest="rnaDataType", help="set data type to RNA [default: DNA]") parser.add_option("-S", "--sam", action="store_true", dest="useSamFile", help="input file is in sam format") parser.add_option("--index", action="store_true", dest="doIndex", help="index the output rds file") parser.add_option("--cache", type="int", dest="cachePages", help="number of cache pages to use [default: 100000") parser.add_option("-m", "--multiCount", type="int", dest="maxMultiReadCount", help="multi counts over this value are discarded [default: 10]") parser.add_option("--rawreadID", action="store_false", dest="trimReadID", help="use the raw read names") parser.set_defaults(init=True, doIndex=False, useSamFile=False, cachePages=100000, maxMultiReadCount=10, rnaDataType=False, trimReadID=True) (options, args) = parser.parse_args(argv[1:]) try: label = args[0] except IndexError: print "no label specified - see --help for usage" sys.exit(1) if options.useSamFile: fileMode = "r" else: fileMode = "rb" try: samfile = pysam.Samfile(args[1], fileMode) except IndexError: print "no samfile specified - see --help for usage" sys.exit(1) except ValueError: print "samfile index not found" sys.exit(1) if options.rnaDataType: dataType = "RNA" else: dataType = "DNA" try: outDbName = args[2] except IndexError: print "no outrdsfile specified - see --help for usage" sys.exit(1) writeLog(outDbName + '.log', verstring, string.join(sys.argv[1:])) rds = readDataset(outDbName, options.init, dataType, verbose=True) if not options.init and options.doIndex: try: if rds.hasIndex(): rds.dropIndex() except: pass if 'sam_mapped' not in rds.getMetadata(): rds.insertMetadata([('sam_mapped', 'True')]) defaultCacheSize = rds.getDefaultCacheSize() if options.cachePages > defaultCacheSize: if options.init: rds.setDBcache(options.cachePages, default=True) else: rds.setDBcache(options.cachePages) propertyList = [] for arg in sys.argv: if '::' in arg: (pname, pvalue) = arg.strip().split('::') propertyList.append((pname, pvalue)) if len(propertyList) > 0: rds.insertMetadata(propertyList) countReads = {"unmapped": 0, "total": 0, "unique": 0, "multi": 0, "multiDiscard": 0, "splice": 0 } readsize = 0 insertSize = 100000 uniqueInsertList = [] multiInsertList = [] spliceInsertList = [] processedEntryDict = {} uniqueReadDict = {} multiReadDict = {} spliceReadDict = {} samFileIterator = samfile.fetch(until_eof=True) for read in samFileIterator: if read.is_unmapped: countReads["unmapped"] += 1 continue if readsize == 0: take = (0, 2, 3) # CIGAR operation (M/match, D/del, N/ref_skip) readsize = sum([length for op,length in read.cigar if op in take]) if options.init: rds.insertMetadata([('readsize', readsize)]) #Build the read dictionaries try: readSequence = read.seq except KeyError: readSequence = "" pairReadSuffix = getPairedReadNumberSuffix(read) readName = "%s%s%s" % (read.qname, readSequence, pairReadSuffix) if options.trimReadID: rdsEntryName = "%s:%s:%d%s" % (label, read.qname, countReads["total"], pairReadSuffix) else: rdsEntryName = read.qname if processedEntryDict.has_key(readName): if isSpliceEntry(read.cigar): if spliceReadDict.has_key(readName): del spliceReadDict[readName] else: if uniqueReadDict.has_key(readName): del uniqueReadDict[readName] if multiReadDict.has_key(readName): (read, priorCount, rdsEntryName) = multiReadDict[readName] count = priorCount + 1 multiReadDict[readName] = (read, count, rdsEntryName) else: multiReadDict[readName] = (read, 1, rdsEntryName) else: processedEntryDict[readName] = "" if isSpliceEntry(read.cigar): spliceReadDict[readName] = (read,rdsEntryName) else: uniqueReadDict[readName] = (read, rdsEntryName) if countReads["total"] % insertSize == 0: for entry in uniqueReadDict.keys(): (readData, rdsEntryName) = uniqueReadDict[entry] chrom = samfile.getrname(readData.rname) uniqueInsertList.append(getRDSEntry(readData, rdsEntryName, chrom, readsize)) countReads["unique"] += 1 for entry in spliceReadDict.keys(): (readData, rdsEntryName) = spliceReadDict[entry] chrom = samfile.getrname(readData.rname) spliceInsertList.append(getRDSSpliceEntry(readData, rdsEntryName, chrom, readsize)) countReads["splice"] += 1 for entry in multiReadDict.keys(): (readData, count, rdsEntryName) = multiReadDict[entry] chrom = samfile.getrname(readData.rname) if count > options.maxMultiReadCount: countReads["multiDiscard"] += 1 else: multiInsertList.append(getRDSEntry(readData, rdsEntryName, chrom, readsize, weight=count)) countReads["multi"] += 1 rds.insertUniqs(uniqueInsertList) rds.insertMulti(multiInsertList) uniqueInsertList = [] uniqueReadDict = {} multiInsertList = [] multiReadDict = {} if dataType == 'RNA': rds.insertSplices(spliceInsertList) spliceInsertList = [] spliceReadDict = {} print '.', sys.stdout.flush() processedEntryDict = {} countReads["total"] += 1 if len(uniqueReadDict.keys()) > 0: for entry in uniqueReadDict.keys(): (readData, rdsEntryName) = uniqueReadDict[entry] chrom = samfile.getrname(readData.rname) uniqueInsertList.append(getRDSEntry(readData, rdsEntryName, chrom, readsize)) countReads["unique"] += 1 rds.insertUniqs(uniqueInsertList) if len(multiReadDict.keys()) > 0: for entry in multiReadDict.keys(): (readData, count, rdsEntryName) = multiReadDict[entry] chrom = samfile.getrname(readData.rname) if count > options.maxMultiReadCount: countReads["multiDiscard"] += 1 else: multiInsertList.append(getRDSEntry(readData, rdsEntryName, chrom, readsize, weight=count)) countReads["multi"] += 1 countReads["multi"] += len(multiInsertList) if len(spliceReadDict.keys()) > 0 and dataType == "RNA": for entry in spliceReadDict.keys(): (readData, rdsEntryName) = spliceReadDict[entry] chrom = samfile.getrname(readData.rname) spliceInsertList.append(getRDSSpliceEntry(readData, rdsEntryName, chrom, readsize)) countReads["splice"] += 1 rds.insertSplices(spliceInsertList) countString = "\n%d unmapped reads discarded" % countReads["unmapped"] countString += "\t%d unique reads" % countReads["unique"] countString += "\t%d multi reads" % countReads["multi"] countString += "\t%d multi reads count > %d discarded" % (countReads["multiDiscard"], options.maxMultiReadCount) if dataType == 'RNA': countString += "\t%d spliced reads" % countReads["splice"] print countString.replace('\t', '\n') writeLog(outDbName + '.log', verstring, countString) if options.doIndex: print "building index...." if options.cachePages > defaultCacheSize: rds.setDBcache(options.cachePages) rds.buildIndex(options.cachePages) else: rds.buildIndex(defaultCacheSize) def getRDSEntry(alignedRead, readName, chrom, readSize, weight=1): start = int(alignedRead.pos) stop = int(start+readSize) sense = getReadSense(alignedRead.is_reverse) try: mismatchTag = alignedRead.opt("MD") mismatches = getMismatches(mismatchTag, alignedRead.seq, sense) except KeyError: mismatches = "" return (readName, chrom, start, stop, sense, 1.0/weight, '', mismatches) def getRDSSpliceEntry(alignedRead, readName, chrom, readSize): (readName, chrom, start, stop, sense, weight, flag, mismatches) = getRDSEntry(alignedRead, readName, chrom, readSize) startL, startR, stopL, stopR = getSpliceBounds(start, readSize, alignedRead.cigar) return (readName, chrom, startL, stopL, startR, stopR, sense, 1.0, "", mismatches) def getPairedReadNumberSuffix(read): readSuffix = "" if not isPairedRead(read): return "" if read.is_read1: readSuffix = "/1" elif read.is_read2: readSuffix = "/2" return readSuffix def isPairedRead(read): return read.is_proper_pair and (read.is_read1 or read.is_read2) def isSpliceEntry(cigarTupleList): isSplice = False for operation,length in cigarTupleList: if operation == 3: isSplice = True break return isSplice def getReadSense(reverse): if reverse: sense = "-" else: sense = "+" return sense def getMismatches(mismatchTag, querySequence="", sense="+", logErrors=False): output = [] deletionMarker = "^" position = 0 lengths = re.findall("\d+", mismatchTag) mismatchSequences = re.findall("\d+([ACGTN]|\\^[ACGTN]+)", mismatchTag) for mismatchEntry in range(len(mismatchSequences)): mismatch = mismatchSequences[mismatchEntry] position = position + int(lengths[mismatchEntry]) if string.find(mismatch, deletionMarker) == 0: continue try: if querySequence: genomicNucleotide = querySequence[position] else: genomicNucleotide = "N" if sense == '-': mismatch = getComplementNucleotide(mismatch) genomicNucleotide = getComplementNucleotide(genomicNucleotide) elandCompatiblePosition = int(position + 1) output.append('%s%d%s' % (mismatch, elandCompatiblePosition, genomicNucleotide)) position += 1 except IndexError: if logErrors: errorMessage = "getMismatch IndexError; tag: %s, seq: %s, pos: %d" % (mismatchTag, querySequence, position) writeLog("MakeRdsFromBamError.log", "1.0", errorMessage) return "" return string.join(output, ",") def getComplementNucleotide(nucleotide): complement = {"A":"T", "T":"A", "C":"G", "G":"C", "N":"N" } return complement[nucleotide] def getSpliceBounds(start, readsize, cigarTupleList): stopR = int(start + readsize) offset = 0 for operation,length in cigarTupleList: if operation == 3: stopL = int(start + offset) startR = int(stopL + length) return start, startR, stopL, stopR else: offset += length if __name__ == "__main__": main(sys.argv)