int main(int argc, char* argv[]) {

	string basFileName;

	if (argc < 2) {
		cout << "usage: bas2  file.bas.h5 " << endl;
		cout << "bas2 = bas ls" << endl;
		cout << " -t --titleindices  Print the titles of reads and their indices in the file." << endl;
	}
	basFileName = argv[1];
	int argi = 1;
	bool printSummary      = true;
	bool printTitleIndices = false;
	while (argi < argc) {
		if (strcmp(argv[argi], "-t") == 0 or
				strcmp(argv[argi], "--titleindices") == 0) {
			printTitleIndices = true;
			printSummary = false;
		}
		++argi;
	}
	HDFBasReader reader;
	reader.Initialize(basFileName);
	int numReads = reader.GetNumReads();
	cout << "Num reads: " << numReads << endl;
	vector<int> readLengths;
	reader.GetAllReadLengths(readLengths);
	DNALength totalLength = 0;
	VectorIndex i;
	int numAbove100 = 0;
	for (i = 0; i< readLengths.size(); i++ ){
		totalLength += readLengths[i];
		if (readLengths[i] > 100) {
			++numAbove100;
		}
	}

	if (printSummary) {
		if (numReads > 0 ){
			cout << "Average read length: " << totalLength / (1.0*numReads) << endl;
		}
		cout << "A total of " << numAbove100  << " have length > 100" << endl;

		return 0;
	}
	if (printTitleIndices) {
		FASTQSequence seq;
		int seqIndex = 0;
		while (reader.GetNext(seq)) {
			cout << seqIndex << " " << seq.title << " " << seq.length << endl;
			++seqIndex;
		}
	}
}
Exemple #2
0
int main(int argc, char* argv[]) {
	
	string inFileName, outFileName;

	if (argc < 3) {
		cout << "usage: filterHDFPls in out idx1 [idx2 idx3]..." << endl;
		exit(1);
	}
	inFileName  = argv[1];
	outFileName = argv[2];
	
	vector<int> readIndices;
	int argi = 3;
	int minLength = 0;
	int minAvgQual = 0;
	while (argi < argc) {
		if (strcmp(argv[argi], "-minLength") == 0) {
			minLength = atoi(argv[++argi]);
		}
		else if (strcmp(argv[argi], "-minAvgQual") == 0) {
			minAvgQual = atoi(argv[++argi]);
		}
		++argi;
	}
	
	std::sort(readIndices.begin(), readIndices.end());
	HDFBasReader reader;
	HDFBasWriter writer;

	reader.Initialize(inFileName);
	writer.Initialize(outFileName, reader.GetMovieName(), reader.GetRunCode());
	
	int ri;
	int curReadIndex = 0;
	FASTQSequence seq;
	for (ri = 0; ri < readIndices.size(); ri++, curReadIndex++ ){
		reader.GetNext(seq);
		bool skipRead = false;
		if (seq.length < minLength) { skipRead = true;}
		if (seq.GetAverageQuality() < minAvgQual) { skipRead = true; }
		if (skipRead) { continue; }
		// all ok, write read out.
		writer.Write(seq);
	}


}
TEST(SubreadsTest, EndToEnd_Multiple)
{
    // setup
    const string movieName = "m140905_042212_sidney_c100564852550000001823085912221377_s1_X0";

    vector<string> baxFilenames;
    baxFilenames.push_back(tests::Data_Dir + "/" + movieName + ".1.bax.h5");

    const string generatedBam = movieName + ".subreads.bam";
    const string scrapBam = movieName + ".scraps.bam";

    // run conversion
    const int result = RunBax2Bam(baxFilenames, "--subread");
    EXPECT_EQ(0, result);

    // open BAX reader on original data
    HDFBasReader baxReader;
    baxReader.IncludeField("Basecall");
    baxReader.IncludeField("DeletionQV");
    baxReader.IncludeField("DeletionTag");
    baxReader.IncludeField("InsertionQV");
    baxReader.IncludeField("PreBaseFrames");
    baxReader.IncludeField("MergeQV");
    baxReader.IncludeField("SubstitutionQV");
    baxReader.IncludeField("HQRegionSNR");
    // not using SubTag or PulseWidth here

    string baxBasecallerVersion;
    string baxBindingKit;
    string baxSequencingKit;

    const int initOk = baxReader.Initialize(baxFilenames.front());
    EXPECT_EQ(1, initOk);
    if (initOk == 1) {

        if (baxReader.scanDataReader.fileHasScanData && baxReader.scanDataReader.initializedRunInfoGroup) {

            if (baxReader.scanDataReader.runInfoGroup.ContainsAttribute("BindingKit")) {
                HDFAtom<std::string> bkAtom;
                if (bkAtom.Initialize(baxReader.scanDataReader.runInfoGroup, "BindingKit")) {
                    bkAtom.Read(baxBindingKit);
                    bkAtom.dataspace.close();
                }
            }

            if (baxReader.scanDataReader.runInfoGroup.ContainsAttribute("SequencingKit")) {
                HDFAtom<std::string> skAtom;
                if (skAtom.Initialize(baxReader.scanDataReader.runInfoGroup, "SequencingKit")) {
                    skAtom.Read(baxSequencingKit);
                    skAtom.dataspace.close();
                }
            }
        }

        baxReader.GetChangeListID(baxBasecallerVersion);
    }

    // read region table info
    boost::scoped_ptr<HDFRegionTableReader> regionTableReader(new HDFRegionTableReader);
    RegionTable regionTable;
    std::string fn = baxFilenames.front();
    EXPECT_TRUE(regionTableReader->Initialize(fn) != 0);
    regionTable.Reset();
    regionTableReader->ReadTable(regionTable);
    regionTableReader->Close();

    EXPECT_NO_THROW(
    {
        // open BAM file
        BamFile bamFile(generatedBam);

        // check BAM header information
        const BamHeader& header = bamFile.Header();
        EXPECT_EQ(string("1.5"),     header.Version());
        EXPECT_EQ(string("unknown"), header.SortOrder());
        EXPECT_EQ(string("3.0.1"),   header.PacBioBamVersion());
        EXPECT_TRUE(header.Sequences().empty());
        EXPECT_TRUE(header.Comments().empty());
        ASSERT_FALSE(header.Programs().empty());

        const vector<string> readGroupIds = header.ReadGroupIds();
        ASSERT_FALSE(readGroupIds.empty());
        const ReadGroupInfo& rg = header.ReadGroup(readGroupIds.front());

        string rawId = movieName + "//SUBREAD";
        string md5Id;
        MakeMD5(rawId, md5Id, 8);
        EXPECT_EQ(md5Id, rg.Id());

        EXPECT_EQ(string("PACBIO"), rg.Platform());
        EXPECT_EQ(movieName, rg.MovieName());

        EXPECT_TRUE(rg.SequencingCenter().empty());
        EXPECT_TRUE(rg.Date().empty());
        EXPECT_TRUE(rg.FlowOrder().empty());
        EXPECT_TRUE(rg.KeySequence().empty());
        EXPECT_TRUE(rg.Library().empty());
        EXPECT_TRUE(rg.Programs().empty());
        EXPECT_TRUE(rg.PredictedInsertSize().empty());
        EXPECT_TRUE(rg.Sample().empty());

        EXPECT_EQ("SUBREAD", rg.ReadType());
        EXPECT_EQ(baxBasecallerVersion, rg.BasecallerVersion());
        EXPECT_EQ(baxBindingKit, rg.BindingKit());
        EXPECT_EQ(baxSequencingKit, rg.SequencingKit());
        EXPECT_EQ(75, std::stod(rg.FrameRateHz()));
        EXPECT_EQ("dq", rg.BaseFeatureTag(BaseFeature::DELETION_QV));
        EXPECT_EQ("dt", rg.BaseFeatureTag(BaseFeature::DELETION_TAG));
        EXPECT_EQ("iq", rg.BaseFeatureTag(BaseFeature::INSERTION_QV));
        EXPECT_EQ("ip", rg.BaseFeatureTag(BaseFeature::IPD));
        EXPECT_EQ("mq", rg.BaseFeatureTag(BaseFeature::MERGE_QV));
        EXPECT_EQ("sq", rg.BaseFeatureTag(BaseFeature::SUBSTITUTION_QV));
        EXPECT_FALSE(rg.HasBaseFeature(BaseFeature::SUBSTITUTION_TAG));
        EXPECT_EQ(FrameCodec::V1, rg.IpdCodec());

        // compare 1st record from each file
        SMRTSequence baxRecord;
        UInt holeNumber = 0;
        vector<float> hqSnr;

        size_t intervalIdx = 0;
        vector<SubreadInterval> subreadIntervals;

        size_t numTested = 0;
        EntireFileQuery entireFile(bamFile);
        for (BamRecord& bamRecord : entireFile) {
            if (intervalIdx >= subreadIntervals.size())
            {
                while (baxReader.GetNext(baxRecord))
                {
                    holeNumber  = baxRecord.zmwData.holeNumber;

                    ComputeSubreadIntervals(&subreadIntervals, regionTable, holeNumber);

                    /* this is for debugging subread interval problems
                    int hqStart = 0;
                    int hqEnd = 0;
                    int hqScore = 0;
                    LookupHQRegion(holeNumber,
                                   regionTable,
                                   hqStart,
                                   hqEnd,
                                   hqScore);

                    vector<ReadInterval> subreadIntervals_;
                    CollectSubreadIntervals(baxRecord, &regionTable, subreadIntervals_);

                    for (int i = subreadIntervals_.size() - 1; i >= 0; --i)
                    {
                        auto& in = subreadIntervals_[i];
                        int inStart = max(hqStart, in.start);
                        int inEnd   = min(hqEnd,   in.end);
                        if (inEnd <= inStart)
                            subreadIntervals_.erase(subreadIntervals_.begin() + i);
                    }

                    cerr << "hqRegion: " << hqStart << ", " << hqEnd << endl;
                    cerr << "subreadRegions:" << endl;
                    for (const auto& in : subreadIntervals_)
                        cerr << "  l, r: " << in.start << ", " << in.end << endl;

                    cerr << "adapterDerived:" << endl;
                    for (const auto& in : subreadIntervals)
                        cerr << "  l, r: " << in.Start << ", " << in.End << endl;

                    cerr << endl;
                    // */

                    if (subreadIntervals.empty())
                        continue;

                    intervalIdx = 0;

                    hqSnr.clear();
                    hqSnr.push_back(baxRecord.HQRegionSnr('A'));
                    hqSnr.push_back(baxRecord.HQRegionSnr('C'));
                    hqSnr.push_back(baxRecord.HQRegionSnr('G'));
                    hqSnr.push_back(baxRecord.HQRegionSnr('T'));

                    EXPECT_GT(hqSnr[0], 0);
                    EXPECT_GT(hqSnr[1], 0);
                    EXPECT_GT(hqSnr[2], 0);
                    EXPECT_GT(hqSnr[3], 0);

                    goto compare;
                }

                goto cleanup;
            }

compare:
            const BamRecordImpl& bamRecordImpl = bamRecord.Impl();
            EXPECT_EQ(4680,bamRecordImpl.Bin());
            EXPECT_EQ(0,   bamRecordImpl.InsertSize());
            EXPECT_EQ(255, bamRecordImpl.MapQuality());
            EXPECT_EQ(-1,  bamRecordImpl.MatePosition());
            EXPECT_EQ(-1,  bamRecordImpl.MateReferenceId());
            EXPECT_EQ(-1,  bamRecordImpl.Position());
            EXPECT_EQ(-1,  bamRecordImpl.ReferenceId());
            EXPECT_FALSE(bamRecordImpl.IsMapped());

            const int subreadStart = subreadIntervals[intervalIdx].Start;
            const int subreadEnd   = subreadIntervals[intervalIdx].End;

            const string expectedName = movieName + "/" +
                    to_string(holeNumber)   + "/" +
                    to_string(subreadStart) + "_" +
                    to_string(subreadEnd);
            EXPECT_EQ(expectedName, bamRecordImpl.Name());

            using PacBio::BAM::QualityValue;
            using PacBio::BAM::QualityValues;

            const DNALength length = subreadEnd - subreadStart;

            string expectedSequence;
            expectedSequence.assign((const char*)baxRecord.seq + subreadStart, length);

            const string bamSequence = bamRecord.Sequence();
            const QualityValues bamQualities = bamRecord.Qualities();
            EXPECT_EQ(expectedSequence, bamSequence);
            EXPECT_TRUE(bamQualities.empty());

            const QualityValues bamDeletionQVs = bamRecord.DeletionQV();
            const QualityValues bamInsertionQVs = bamRecord.InsertionQV();
            const QualityValues bamMergeQVs = bamRecord.MergeQV();
            const QualityValues bamSubstitutionQVs = bamRecord.SubstitutionQV();

            for (size_t i = 0; i < length; ++i) {
                const size_t pos = subreadStart + i;

                EXPECT_EQ((QualityValue)baxRecord.GetDeletionQV(pos),     bamDeletionQVs.at(i));
                EXPECT_EQ((QualityValue)baxRecord.GetInsertionQV(pos),    bamInsertionQVs.at(i));
                EXPECT_EQ((QualityValue)baxRecord.GetMergeQV(pos),        bamMergeQVs.at(i));
                EXPECT_EQ((QualityValue)baxRecord.GetSubstitutionQV(pos), bamSubstitutionQVs.at(i));
            }

            if (baxRecord.deletionTag)
            {
                string expectedDeletionTags;
                expectedDeletionTags.assign((char*)baxRecord.deletionTag + subreadStart,
                                            (char*)baxRecord.deletionTag + subreadStart + length);
                const string& bamDeletionTags = bamRecord.DeletionTag();
                EXPECT_EQ(expectedDeletionTags, bamDeletionTags);
            }

            if (baxRecord.substitutionTag)
            {
                string expectedSubstitutionTags;
                expectedSubstitutionTags.assign((char*)baxRecord.substitutionTag + subreadStart,
                                            (char*)baxRecord.substitutionTag + subreadStart + length);
                const string& bamSubstitutionTags = bamRecord.SubstitutionTag();
                EXPECT_EQ(expectedSubstitutionTags, bamSubstitutionTags);
            }

            // TODO: IPDs
            const LocalContextFlags ctxFlags = subreadIntervals[intervalIdx].LocalContextFlags;

            EXPECT_EQ(md5Id,        bamRecord.ReadGroupId());
            EXPECT_EQ(movieName,    bamRecord.MovieName());
            EXPECT_EQ(1,            bamRecord.NumPasses());
            EXPECT_EQ(holeNumber,   bamRecord.HoleNumber());
            EXPECT_EQ(subreadStart, bamRecord.QueryStart());
            EXPECT_EQ(subreadEnd,   bamRecord.QueryEnd());
            EXPECT_EQ(hqSnr,        bamRecord.SignalToNoise());
            EXPECT_EQ(ctxFlags,     bamRecord.LocalContextFlags());

            numTested++;
            intervalIdx++;
        }

cleanup:
        EXPECT_GT(numTested, 1);

        // cleanup
        baxReader.Close();
        RemoveFile(generatedBam);
        RemoveFile(scrapBam);

    }); // EXPECT_NO_THROW
Exemple #4
0
int main(int argc, char* argv[]) {
    string program = "pls2fasta";
    string versionString = VERSION;
    AppendPerforceChangelist(PERFORCE_VERSION_STRING, versionString);

	string plsFileName, fastaOutName;
	vector<string> plsFileNames;
	bool trimByRegion, maskByRegion;
	trimByRegion = false;
	maskByRegion = false;
	int argi = 3;
	RegionTable regionTable;
	string regionsFOFNName = "";
	vector<string> regionFileNames;
	bool splitSubreads = true;
	int minSubreadLength = 0;
	bool addSimulatedData = false;
	bool printSimulatedCoordinate = false;
	bool printSimulatedSequenceIndex = false;
  bool printFastq = false;
  bool printCcs   = false;
  int  lineLength = 50;
  int minReadScore = 0;
  vector<int> holeNumbers;
  CommandLineParser clp;
  bool printOnlyBest = false;

  clp.SetProgramName(program);
  clp.SetVersion(versionString);
  clp.RegisterStringOption("in.pls.h5", &plsFileName, "Input pls.h5/bax.h5/fofn file.", true);
  clp.RegisterStringOption("out.fasta", &fastaOutName, "Output fasta/fastq file.", true);
  clp.RegisterPreviousFlagsAsHidden();
  clp.RegisterFlagOption("trimByRegion", &trimByRegion, "Trim away low quality regions.");
  clp.RegisterFlagOption("maskByRegion", &maskByRegion, "Mask low quality regions with 'N'.");
  clp.RegisterStringOption("regionTable", &regionsFOFNName, "Optional HDF file with a /PulseData/Regions dataset.");
  clp.RegisterIntOption("minSubreadLength", &minSubreadLength, "Do not write subreads less than the specified length.", CommandLineParser::PositiveInteger);
  clp.RegisterFlagOption("noSplitSubreads", &splitSubreads, "Do not split reads on adapter sequences.");
  clp.RegisterIntListOption("holeNumber", &holeNumbers, "Only print this hole number (or list of numbers).");
  clp.RegisterFlagOption("fastq", &printFastq, "Print in FASTQ format with quality.");
  clp.RegisterFlagOption("ccs", &printCcs, "Print de novo CCS sequences");
  clp.RegisterIntOption("lineLength", &lineLength, "Specify fasta/fastq line length", CommandLineParser::PositiveInteger);
  clp.RegisterIntOption("minReadScore", &minReadScore, "Minimum read score to print a read.  The score is "
                        "a number between 0 and 1000 and represents the expected accuracy percentage * 10. "
                        "A typical value would be between 750 and 800.  This does not apply to ccs reads.", CommandLineParser::NonNegativeInteger);
  clp.RegisterFlagOption("best", &printOnlyBest, "If a CCS sequence exists, print this.  Otherwise, print the longest"
                         "subread.  This does not support fastq.");
  string description = ("Converts pls.h5/bax.h5/fofn files to fasta or fastq files. Although fasta files are provided"
  " with every run, they are not trimmed nor split into subreads. This program takes "
  "additional annotation information, such as the subread coordinates and high quality regions "
  "and uses them to create fasta sequences that are substrings of all bases called. Most of the time "
  "you will want to trim low quality reads, so you should specify -trimByRegion.");
  clp.SetProgramSummary(description);
                        
  clp.ParseCommandLine(argc, argv);

    cerr << "[INFO] " << GetTimestamp() << " [" << program << "] started."  << endl;
	if (trimByRegion and maskByRegion) {
		cout << "ERROR! You cannot both trim and mask regions. Use one or the other." << endl;
		exit(1);
	}
		 
  if (printFastq) {
    // Setting lineLength to 0 flags to print on one line.
    lineLength = 0;
  }

	if (FileOfFileNames::IsFOFN(plsFileName)) {
		FileOfFileNames::FOFNToList(plsFileName, plsFileNames);
	}
	else {
		plsFileNames.push_back(plsFileName);
	}
	if (regionsFOFNName == "") {
		regionFileNames = plsFileNames;
	}
	else {
		if (FileOfFileNames::IsFOFN(regionsFOFNName)) {
			FileOfFileNames::FOFNToList(regionsFOFNName, regionFileNames);
		}
		else {
			regionFileNames.push_back(regionsFOFNName);
		}
	}



	ofstream fastaOut;
	CrucialOpen(fastaOutName, fastaOut);
	int plsFileIndex;
	HDFRegionTableReader hdfRegionReader;
  sort(holeNumbers.begin(), holeNumbers.end());
	for (plsFileIndex = 0; plsFileIndex < plsFileNames.size(); plsFileIndex++) {
		if (trimByRegion or maskByRegion or splitSubreads) {
			hdfRegionReader.Initialize(regionFileNames[plsFileIndex]);
			hdfRegionReader.ReadTable(regionTable);
			regionTable.SortTableByHoleNumber();
		}
		
		ReaderAgglomerate reader;
    HDFBasReader ccsReader;

    if (printOnlyBest) {
      ccsReader.SetReadBasesFromCCS();
      ccsReader.Initialize(plsFileNames[plsFileIndex]);
    }
    if (printCcs == false) {
  		reader.IgnoreCCS();
    }
    else {
      reader.hdfBasReader.SetReadBasesFromCCS();
    }
		if (addSimulatedData) {
			reader.hdfBasReader.IncludeField("SimulatedCoordinate");
			reader.hdfBasReader.IncludeField("SimulatedSequenceIndex");
		}

        if (reader.SetReadFileName(plsFileNames[plsFileIndex]) == 0) {
          cout << "ERROR, could not determine file type."
               << plsFileNames[plsFileIndex] << endl;
          exit(1);
        }
        if (reader.Initialize() == 0) {
          cout << "ERROR, could not initialize file "
               << plsFileNames[plsFileIndex] << endl;
          exit(1);
        }

		DNALength simulatedCoordinate;
		DNALength simulatedSequenceIndex;
		reader.SkipReadQuality();
		SMRTSequence seq;
		vector<ReadInterval> subreadIntervals;;
    SMRTSequence ccsSeq;
		while (reader.GetNext(seq)) {
      if (printOnlyBest) {
        ccsReader.GetNext(ccsSeq);
      }

      if (holeNumbers.size() != 0 and 
          binary_search(holeNumbers.begin(), holeNumbers.end(), seq.zmwData.holeNumber) == false) {
        continue;
      }

      if (seq.length == 0) {
        continue;
      }

			if (addSimulatedData) {
				reader.hdfBasReader.simulatedCoordinateArray.Read(reader.hdfBasReader.curRead-1, reader.hdfBasReader.curRead, &simulatedCoordinate);
				reader.hdfBasReader.simulatedSequenceIndexArray.Read(reader.hdfBasReader.curRead-1, reader.hdfBasReader.curRead, &simulatedSequenceIndex);
			}

		  if (printCcs == true) {
        if (printFastq == false) {
          seq.PrintSeq(fastaOut);
        }
        else {
          seq.PrintFastq(fastaOut, lineLength);
        }
        continue;
      }	

      //
      // Determine the high quality boundaries of the read.  This is
      // the full read is no hq regions exist, or it is stated to
      // ignore regions.
      //
      DNALength hqReadStart, hqReadEnd;
      int hqRegionScore;
      if (GetReadTrimCoordinates(seq, seq.zmwData, regionTable, hqReadStart, hqReadEnd, hqRegionScore) == false or 
          (trimByRegion == false and maskByRegion == false)) {
        hqReadStart = 0;
        hqReadEnd   = seq.length;
      }
      
      //
      // Mask off the low quality portions of the reads.
      //
			if (maskByRegion) {
        if (hqReadStart > 0) {
          fill(&seq.seq[0], &seq.seq[hqReadStart], 'N');
        }
        if (hqReadEnd != seq.length) {
          fill(&seq.seq[hqReadEnd], &seq.seq[seq.length], 'N');
        }
			}
      


      //
      // Now possibly print the full read with masking.  This could be handled by making a 
      // 
			if (splitSubreads == false) {
        ReadInterval wholeRead(0, seq.length);
        // The set of subread intervals is just the entire read.
        subreadIntervals.clear();
        subreadIntervals.push_back(wholeRead);
			}
			else {
				//
				// Print subread coordinates no matter whether or not reads have subreads.
				//
				subreadIntervals.clear(); // clear old, new intervals are appended.
				CollectSubreadIntervals(seq, &regionTable, subreadIntervals);
      }
      //
      // Output all subreads as separate sequences.
      //
      int intvIndex;
      SMRTSequence bestSubreadSequence;
      int bestSubreadScore = -1;
      int bestSubreadIndex = 0;
      int bestSubreadStart = 0, bestSubreadEnd = 0;
      SMRTSequence bestSubread;
      for (intvIndex = 0; intvIndex < subreadIntervals.size(); intvIndex++) {
        SMRTSequence subreadSequence, subreadSequenceRC;
					
        subreadSequence.subreadStart = subreadIntervals[intvIndex].start;
        subreadSequence.subreadEnd   = subreadIntervals[intvIndex].end;
          
        // 
        // When trimming by region, only output the parts of the
        // subread that overlap the hq region.
        //
        if (trimByRegion == true) {
          subreadSequence.subreadStart = max((DNALength) subreadIntervals[intvIndex].start, hqReadStart);
          subreadSequence.subreadEnd   = min((DNALength) subreadIntervals[intvIndex].end, hqReadEnd);
        }

        if (subreadSequence.subreadStart >= subreadSequence.subreadEnd or 
            subreadSequence.subreadEnd - subreadSequence.subreadStart <= minSubreadLength) {
          //
          // There is no high qualty portion of this subread. Skip it.
          //
          continue;
        }

        if (hqRegionScore < minReadScore) {
          continue;
        }

        //
        // Print the subread, adding the coordinates as part of the title.
        //
        subreadSequence.ReferenceSubstring(seq, subreadSequence.subreadStart, 
                                           subreadSequence.subreadEnd - subreadSequence.subreadStart);
        stringstream titleStream;
        titleStream << seq.title;
        if (splitSubreads) {
          //
          // Add the subread coordinates if splitting on subread.
          //
          titleStream << "/" 
                      << subreadSequence.subreadStart
                      << "_" << subreadSequence.subreadEnd;
        }
          
        // 
        // If running on simulated data, add where the values were simulated from.
        //
        if (addSimulatedData) {
          titleStream << ((FASTASequence*)&seq)->title << "/chrIndex_" 
                      << simulatedSequenceIndex << "/position_"<< simulatedCoordinate;
          ((FASTASequence*)&seq)->CopyTitle(titleStream.str());
        }

        subreadSequence.CopyTitle(titleStream.str());

        //
        // Eventually replace with WriterAgglomerate.
        //
        if (printOnlyBest == false) {
          if (subreadSequence.length > 0) {
            if (printFastq == false) {
              ((FASTASequence*)&subreadSequence)->PrintSeq(fastaOut);
            }
            else {
              subreadSequence.PrintFastq(fastaOut, lineLength);
            }
          }
          delete[] subreadSequence.title;
        }
        else {
          int subreadWeightedScore = subreadSequence.length * hqRegionScore;
          if (subreadWeightedScore > bestSubreadScore) {
            bestSubreadIndex = intvIndex;
            bestSubread = subreadSequence;
            bestSubreadScore = subreadWeightedScore;
          }
        }
      }

      if (printOnlyBest) {
        if (ccsSeq.length > 0) {
          if (printFastq == false) {
            ccsSeq.PrintSeq(fastaOut);
          }
          else {
            ccsSeq.PrintFastq(fastaOut, ccsSeq.length);
          }
        }
        else {
          if (bestSubreadScore >= 0) {
            if (printFastq == false) {
              bestSubread.PrintSeq(fastaOut);
            }
            else {
              bestSubread.PrintFastq(fastaOut, bestSubread.length);
            }
            bestSubread.Free();
          }
        }
        ccsSeq.Free();
      }
      seq.Free();
    }
    reader.Close();
    hdfRegionReader.Close();
  }
  cerr << "[INFO] " << GetTimestamp() << " [" << program << "] ended."  << endl;
}