Beispiel #1
0
int Stats::execute(int argc, char **argv)
{
    // Extract command line arguments.
    String inFile = "";
    String indexFile = "";
    bool basic = false;
    bool noeof = false;
    bool params = false;
    bool qual = false;
    bool phred = false;
    int maxNumReads = -1;
    bool unmapped = false;
    String pBaseQC = "";
    String cBaseQC = "";
    String regionList = "";
    int excludeFlags = 0;
    int requiredFlags = 0;
    bool withinRegion = false;
    int minMapQual = 0;
    String dbsnp = "";
    PosList *dbsnpListPtr = NULL;
    bool baseSum = false;
    int bufferSize = PileupHelper::DEFAULT_WINDOW_SIZE;

    ParameterList inputParameters;
    BEGIN_LONG_PARAMETERS(longParameterList)
        LONG_PARAMETER_GROUP("Required Parameters")
        LONG_STRINGPARAMETER("in", &inFile)
        LONG_PARAMETER_GROUP("Types of Statistics")
        LONG_PARAMETER("basic", &basic)
        LONG_PARAMETER("qual", &qual)
        LONG_PARAMETER("phred", &phred)
        LONG_STRINGPARAMETER("pBaseQC", &pBaseQC)
        LONG_STRINGPARAMETER("cBaseQC", &cBaseQC)
        LONG_PARAMETER_GROUP("Optional Parameters")
        LONG_INTPARAMETER("maxNumReads", &maxNumReads)
        LONG_PARAMETER("unmapped", &unmapped)
        LONG_STRINGPARAMETER("bamIndex", &indexFile)
        LONG_STRINGPARAMETER("regionList", &regionList)
        LONG_INTPARAMETER("excludeFlags", &excludeFlags)
        LONG_INTPARAMETER("requiredFlags", &requiredFlags)
        LONG_PARAMETER("noeof", &noeof)
        LONG_PARAMETER("params", &params)
        LONG_PARAMETER_GROUP("Optional phred/qual Only Parameters")
        LONG_PARAMETER("withinRegion", &withinRegion)
        LONG_PARAMETER_GROUP("Optional BaseQC Only Parameters")
        LONG_PARAMETER("baseSum", &baseSum)
        LONG_INTPARAMETER("bufferSize", &bufferSize)
        LONG_INTPARAMETER("minMapQual", &minMapQual)
        LONG_STRINGPARAMETER("dbsnp", &dbsnp)
        END_LONG_PARAMETERS();
   
    inputParameters.Add(new LongParameters ("Input Parameters", 
                                            longParameterList));

    inputParameters.Read(argc-1, &(argv[1]));

    // If no eof block is required for a bgzf file, set the bgzf file type to 
    // not look for it.
    if(noeof)
    {
        // Set that the eof block is not required.
        BgzfFileType::setRequireEofBlock(false);
    }

    // Check to see if the in file was specified, if not, report an error.
    if(inFile == "")
    {
        usage();
        inputParameters.Status();
        // In file was not specified but it is mandatory.
        std::cerr << "--in is a mandatory argument for stats, "
                  << "but was not specified" << std::endl;
        return(-1);
    }

    // Use the index file if unmapped or regionList is not empty.
    bool useIndex = (unmapped|| (!regionList.IsEmpty()));

    // IndexFile is required, so check to see if it has been set.
    if(useIndex && (indexFile == ""))
    {
        // In file was not specified, so set it to the in file
        // + ".bai"
        indexFile = inFile + ".bai";
    }
    ////////////////////////////////////////
    // Setup in case pileup is used.
    Pileup<PileupElementBaseQCStats> pileup(bufferSize);
    // Initialize start/end positions.
    myStartPos = 0;
    myEndPos = -1;
    
    // Open the output qc file if applicable.
    IFILE baseQCPtr = NULL;
    if(!pBaseQC.IsEmpty() && !cBaseQC.IsEmpty())
    {
        usage();
        inputParameters.Status();
        // Cannot specify both types of baseQC.
        std::cerr << "Cannot specify both --pBaseQC & --cBaseQC." << std::endl;
        return(-1);
    }
    else if(!pBaseQC.IsEmpty())
    {
        baseQCPtr = ifopen(pBaseQC, "w");
        PileupElementBaseQCStats::setPercentStats(true);
    }
    else if(!cBaseQC.IsEmpty())
    {
        baseQCPtr = ifopen(cBaseQC, "w");
        PileupElementBaseQCStats::setPercentStats(false);
    }

    if(baseQCPtr != NULL)
    {
        PileupElementBaseQCStats::setOutputFile(baseQCPtr);
        PileupElementBaseQCStats::printHeader();
    }
    if((baseQCPtr != NULL) || baseSum)
    {
        PileupElementBaseQCStats::setMapQualFilter(minMapQual);
        PileupElementBaseQCStats::setBaseSum(baseSum);
    }

    if(params)
    {
        inputParameters.Status();
    }

    // Open the file for reading.
    SamFile samIn;
    if(!samIn.OpenForRead(inFile))
    {
        fprintf(stderr, "%s\n", samIn.GetStatusMessage());
        return(samIn.GetStatus());
    }

    samIn.SetReadFlags(requiredFlags, excludeFlags);

    // Set whether or not basic statistics should be generated.
    samIn.GenerateStatistics(basic);

    // Read the sam header.
    SamFileHeader samHeader;
    if(!samIn.ReadHeader(samHeader))
    {
        fprintf(stderr, "%s\n", samIn.GetStatusMessage());
        return(samIn.GetStatus());
    }

    // Open the bam index file for reading if we are
    // doing unmapped reads (also set the read section).
    if(useIndex)
    {
        samIn.ReadBamIndex(indexFile);

        if(unmapped)
        {
            samIn.SetReadSection(-1);
        }

        if(!regionList.IsEmpty())
        {
            myRegionList = ifopen(regionList, "r");
        }
    }

    //////////////////////////
    // Read dbsnp if specified and doing baseQC
    if(((baseQCPtr != NULL) || baseSum) && (!dbsnp.IsEmpty()))
    {
        // Read the dbsnp file.
        IFILE fdbSnp;
        fdbSnp = ifopen(dbsnp,"r");
        // Determine how many entries.
        const SamReferenceInfo& refInfo = samHeader.getReferenceInfo();
        int maxRefLen = 0;
        for(int i = 0; i < refInfo.getNumEntries(); i++)
        {
            int refLen = refInfo.getReferenceLength(i);
            if(refLen >= maxRefLen)
            {
                maxRefLen = refLen + 1;
            }
        }
        
        dbsnpListPtr = new PosList(refInfo.getNumEntries(),maxRefLen);

        if(fdbSnp==NULL)
        {
            std::cerr << "Open dbSNP file " << dbsnp.c_str() << " failed!\n";
        }
        else if(dbsnpListPtr == NULL)
        {
            std::cerr << "Failed to init the memory allocation for the dbsnpList.\n";
        }
        else
        {
            // Read the dbsnp file.
            StringArray tokens;
            String buffer;
            int position = 0;
            int refID = 0;

            // Loop til the end of the file.
            while (!ifeof(fdbSnp))
            {
                // Read the next line.
                buffer.ReadLine(fdbSnp);
                // If it does not have at least 2 columns, 
                // continue to the next line.
                if (buffer.IsEmpty() || buffer[0] == '#') continue;
                tokens.AddTokens(buffer);
                if(tokens.Length() < 2) continue;

                if(!tokens[1].AsInteger(position))
                {
                    std::cerr << "Improperly formatted region line, start position "
                              << "(2nd column) is not an integer: "
                              << tokens[1]
                              << "; Skipping to the next line.\n";         
                    continue;
                }

                // Look up the reference name.
                refID = samHeader.getReferenceID(tokens[0]);
                if(refID != SamReferenceInfo::NO_REF_ID)
                {
                    // Reference id was found, so add it to the dbsnp
                    dbsnpListPtr->addPosition(refID, position);
                }
        
                tokens.Clear();
                buffer.Clear();
            }
        }
        ifclose(fdbSnp);
    }

    // Read the sam records.
    SamRecord samRecord;

    int numReads = 0;

    //////////////////////
    // Setup in case doing a quality count.
    // Quality histogram.
    const int MAX_QUAL = 126;
    const int START_QUAL = 33;
    uint64_t qualCount[MAX_QUAL+1];
    for(int i = 0; i <= MAX_QUAL; i++)
    {
        qualCount[i] = 0;
    }
    
    const int START_PHRED = 0;
    const int PHRED_DIFF = START_QUAL - START_PHRED;
    const int MAX_PHRED = MAX_QUAL - PHRED_DIFF;
    uint64_t phredCount[MAX_PHRED+1];
    for(int i = 0; i <= MAX_PHRED; i++)
    {
        phredCount[i] = 0;
    }
    
    int refPos = 0;
    Cigar* cigarPtr = NULL;
    char cigarChar = '?';
    // Exclude clips from the qual/phred counts if unmapped reads are excluded.
    bool qualExcludeClips = excludeFlags & SamFlag::UNMAPPED;

    //////////////////////////////////
    // When not reading by sections, getNextSection returns true
    // the first time, then false the next time.
    while(getNextSection(samIn))
    {
        // Keep reading records from the file until SamFile::ReadRecord
        // indicates to stop (returns false).
        while(((maxNumReads < 0) || (numReads < maxNumReads)) && samIn.ReadRecord(samHeader, samRecord))
        {
            // Another record was read, so increment the number of reads.
            ++numReads;
            // See if the quality histogram should be genereated.
            if(qual || phred)
            {
                // Get the quality.
                const char* qual = samRecord.getQuality();
                // Check for no quality ('*').
                if((qual[0] == '*') && (qual[1] == 0))
                {
                    // This record does not have a quality string, so no 
                    // quality processing is necessary.
                }
                else
                {
                    int index = 0;
                    cigarPtr = samRecord.getCigarInfo();
                    cigarChar = '?';
                    refPos = samRecord.get0BasedPosition();
                    if(!qualExcludeClips && (cigarPtr != NULL))
                    {
                        // Offset the reference position by any soft clips
                        // by subtracting the queryIndex of this start position.
                        // refPos is now the start position of the clips.
                        refPos -= cigarPtr->getQueryIndex(0);
                    }

                    while(qual[index] != 0)
                    {
                        // Skip this quality if it is clipped and we are skipping clips.
                        if(cigarPtr != NULL)
                        {
                            cigarChar = cigarPtr->getCigarCharOpFromQueryIndex(index);
                        }
                        if(qualExcludeClips && Cigar::isClip(cigarChar))
                        {
                            // Skip a clipped quality.
                            ++index;
                            // Increment the position.
                            continue;
                        }

                        if(withinRegion && (myEndPos != -1) && (refPos >= myEndPos))
                        {
                            // We have hit the end of the region, stop processing this
                            // quality string.
                            break;
                        }

                        if(withinRegion && (refPos < myStartPos))
                        {
                            // This position is not in the target.
                            ++index;
                            // Update the position if this is found in the reference or a clip.
                            if(Cigar::foundInReference(cigarChar) || Cigar::isClip(cigarChar))
                            {
                                ++refPos;
                            }
                            continue;
                        }

                        // Check for valid quality.
                        if((qual[index] < START_QUAL) || (qual[index] > MAX_QUAL))
                        {
                            if(qual)
                            {
                                std::cerr << "Invalid Quality found: " << qual[index] 
                                          << ".  Must be between "
                                          << START_QUAL << " and " << MAX_QUAL << ".\n";
                            }
                            if(phred)
                            {
                                std::cerr << "Invalid Phred Quality found: " << qual[index] - PHRED_DIFF
                                          << ".  Must be between "
                                          << START_QUAL << " and " << MAX_QUAL << ".\n";
                            }
                            // Skip an invalid quality.
                            ++index;
                            // Update the position if this is found in the reference or a clip.
                            if(Cigar::foundInReference(cigarChar) || Cigar::isClip(cigarChar))
                            {
                                ++refPos;
                            }
                            continue;
                        }
                        
                        // Increment the count for this quality.
                        ++(qualCount[(int)(qual[index])]);
                        ++(phredCount[(int)(qual[index]) - PHRED_DIFF]);
                        // Update the position if this is found in the reference or a clip.
                        if(Cigar::foundInReference(cigarChar) || Cigar::isClip(cigarChar))
                        {
                            ++refPos;
                        }
                        ++index;
                    }
                }
            }

            // Check the next thing to do for the read.
            if((baseQCPtr != NULL) || baseSum)
            {
                // Pileup the bases for this read.
                pileup.processAlignmentRegion(samRecord, myStartPos, myEndPos, dbsnpListPtr);
            }
        }

        // Done with a section, move on to the next one.

        // New section, so flush the pileup.
        pileup.flushPileup();
    }

    // Flush the rest of the pileup.
    if((baseQCPtr != NULL) || baseSum)
    {
        // Pileup the bases.
        pileup.processAlignmentRegion(samRecord, myStartPos, myEndPos, dbsnpListPtr);
        PileupElementBaseQCStats::printSummary();
        ifclose(baseQCPtr);
    }

    std::cerr << "Number of records read = " << 
        samIn.GetCurrentRecordCount() << std::endl;

    if(basic)
    {
        std::cerr << std::endl;
        samIn.PrintStatistics();
    }

    // Print the quality stats.
    if(qual)
    {
        std::cerr << std::endl;
        std::cerr << "Quality\tCount\n";
        for(int i = START_QUAL; i <= MAX_QUAL; i++)
        {
            std::cerr << i << "\t" << qualCount[i] << std::endl;
        }
    }
    // Print the phred quality stats.
    if(phred)
    {
        std::cerr << std::endl;
        std::cerr << "Phred\tCount\n";
        for(int i = START_PHRED; i <= MAX_PHRED; i++)
        {
            std::cerr << i << "\t" << phredCount[i] << std::endl;
        }
    }

    SamStatus::Status status = samIn.GetStatus();
    if(status == SamStatus::NO_MORE_RECS)
    {
        // A status of NO_MORE_RECS means that all reads were successful.
        status = SamStatus::SUCCESS;
    }

    return(status);
}
Beispiel #2
0
int Bam2FastQ::execute(int argc, char **argv)
{
    // Extract command line arguments.
    String inFile = "";
    bool readName = false;
    String refFile = "";
    String firstOut = "";
    String secondOut = "";
    String unpairedOut = "";

    bool interleave = false;
    bool noeof = false;
    bool gzip = false;
    bool params = false;

    myOutBase = "";
    myNumMateFailures = 0;
    myNumPairs = 0;
    myNumUnpaired = 0;
    mySplitRG = false;
    myQField = "";
    myNumQualTagErrors = 0;
    myReverseComp = true;
    myRNPlus = false;
    myFirstRNExt = DEFAULT_FIRST_EXT;
    mySecondRNExt = DEFAULT_SECOND_EXT;
    myCompression = InputFile::DEFAULT;

    ParameterList inputParameters;
    BEGIN_LONG_PARAMETERS(longParameterList)
        LONG_PARAMETER_GROUP("Required Parameters")
        LONG_STRINGPARAMETER("in", &inFile)
        LONG_PARAMETER_GROUP("Optional Parameters")
        LONG_PARAMETER("readName", &readName)
        LONG_PARAMETER("splitRG", &mySplitRG)
        LONG_STRINGPARAMETER("qualField", &myQField)
        LONG_PARAMETER("merge", &interleave)
        LONG_STRINGPARAMETER("refFile", &refFile)
        LONG_STRINGPARAMETER("firstRNExt", &myFirstRNExt)
        LONG_STRINGPARAMETER("secondRNExt", &mySecondRNExt)
        LONG_PARAMETER("rnPlus", &myRNPlus)
        LONG_PARAMETER("noReverseComp", &myReverseComp)
        LONG_PARAMETER("gzip", &gzip)
        LONG_PARAMETER("noeof", &noeof)
        LONG_PARAMETER("params", &params)
        LONG_PARAMETER_GROUP("Optional OutputFile Names")
        LONG_STRINGPARAMETER("outBase", &myOutBase)
        LONG_STRINGPARAMETER("firstOut", &firstOut)
        LONG_STRINGPARAMETER("secondOut", &secondOut)
        LONG_STRINGPARAMETER("unpairedOut", &unpairedOut)
        LONG_PHONEHOME(VERSION)
        END_LONG_PARAMETERS();
   
    inputParameters.Add(new LongParameters ("Input Parameters", 
                                            longParameterList));

    // parameters start at index 2 rather than 1.
    inputParameters.Read(argc, argv, 2);

    // If no eof block is required for a bgzf file, set the bgzf file type to 
    // not look for it.
    if(noeof)
    {
        // Set that the eof block is not required.
        BgzfFileType::setRequireEofBlock(false);
    }

    if(gzip)
    {
        myCompression = InputFile::GZIP;
    }

    // Check to see if the in file was specified, if not, report an error.
    if(inFile == "")
    {
        usage();
        inputParameters.Status();
        // In file was not specified but it is mandatory.
        std::cerr << "--in is a mandatory argument, "
                  << "but was not specified" << std::endl;
        return(-1);
    }

    // Cannot specify both interleaved & secondOut since secondOut would be N/A.
    if(interleave && !secondOut.IsEmpty())
    {
        usage();
        inputParameters.Status();
        std::cerr << "ERROR: Cannot specify --merge & --secondOut.\n";
        return(-1);
    }

    // Cannot specify both interleaved & secondOut since secondOut would be N/A.
    if(interleave && !secondOut.IsEmpty())
    {
        usage();
        inputParameters.Status();
        std::cerr << "ERROR: Cannot specify --merge & --secondOut.\n";
        return(-1);
    }

    // Cannot specify both splitRG & firstOut/secondOut/unpairedOut
    // since it needs a different file for each RG.
    if(mySplitRG && (!firstOut.IsEmpty() || 
                   !secondOut.IsEmpty() || !unpairedOut.IsEmpty()))
    {
        usage();
        inputParameters.Status();
        std::cerr << "ERROR: Cannot specify --splitRG & --firstOut/--secondOut/--unpairedOut.\n";
        std::cerr << "Use --outBase instead.\n";
        return(-1);
    }
    // Cannot specify splitRG & output to stdout.
    if(mySplitRG && (myOutBase[0] == '-'))
    {
        usage();
        inputParameters.Status();
        std::cerr << "ERROR: Cannot specify --splitRG & write to stdout.\n";
        return(-1);
    }

    // Check to see if the out file was specified, if not, generate it from
    // the input filename.
    if(myOutBase == "")
    {
        // Just remove the extension from the input filename.
        int extStart = inFile.FastFindLastChar('.');
        if(extStart <= 0)
        {
            myOutBase = inFile;
        }
        else
        {
            myOutBase = inFile.Left(extStart);
        }
    }

    if(mySplitRG)
    {
        std::string fqList = myOutBase.c_str();
        fqList += ".list";
        myFqList = ifopen(fqList.c_str(), "w");
        ifprintf(myFqList, "MERGE_NAME\tFASTQ1\tFASTQ2\tRG\n");
    }

    // Check to see if the first/second/single-ended were specified and
    // if not, set them.
    myFirstFileNameExt = "_1.fastq";
    mySecondFileNameExt = "_2.fastq";
    myUnpairedFileNameExt = ".fastq";
    if(interleave)
    {
        myFirstFileNameExt = "_interleaved.fastq";
        myFirstFileNameExt = "_interleaved.fastq";
    }
    getFileName(firstOut, myFirstFileNameExt);
    getFileName(secondOut, mySecondFileNameExt);
    getFileName(unpairedOut, myUnpairedFileNameExt);

    if(params)
    {
        inputParameters.Status();
    }

    // Open the files for reading/writing.
    // Open prior to opening the output files,
    // so if there is an error, the outputs don't get created.
    SamFile samIn;
    samIn.OpenForRead(inFile, &mySamHeader);
    // Skip non-primary reads.
    samIn.SetReadFlags(0, 0x0100);

    // Open the output files if not splitting RG
    if(!mySplitRG)
    {
        myUnpairedFile = ifopen(unpairedOut, "w", myCompression);

        // Only open the first file if it is different than an already opened file.
        if(firstOut != unpairedOut)
        {
            myFirstFile = ifopen(firstOut, "w", myCompression);
        }
        else
        {
            myFirstFile = myUnpairedFile;
        }

        // If it is interleaved or the 2nd file is not a new name, set it appropriately.
        if(interleave || secondOut == firstOut)
        {
            mySecondFile = myFirstFile;
        }
        else if(secondOut == unpairedOut)
        {
            mySecondFile = myUnpairedFile;
        }
        else
        {
            mySecondFile = ifopen(secondOut, "w", myCompression);
        }
    
        if(myUnpairedFile == NULL)
        {
            std::cerr << "Failed to open " << unpairedOut
                      << " so can't convert bam2FastQ.\n";
            return(-1);
        }
        if(myFirstFile == NULL)
        {
            std::cerr << "Failed to open " << firstOut
                      << " so can't convert bam2FastQ.\n";
            return(-1);
        }
        if(mySecondFile == NULL)
        {
            std::cerr << "Failed to open " << secondOut
                      << " so can't convert bam2FastQ.\n";
            return(-1);
        }
    }

    if((readName) || (strcmp(mySamHeader.getSortOrder(), "queryname") == 0))
    {
        readName = true;
    }
    else
    {
        // defaulting to coordinate sorted.
        samIn.setSortedValidation(SamFile::COORDINATE);
    }

    // Setup the '=' translation if the reference was specified.
    if(!refFile.IsEmpty())
    {
        GenomeSequence* refPtr = new GenomeSequence(refFile);
        samIn.SetReadSequenceTranslation(SamRecord::BASES);
        samIn.SetReference(refPtr);
    }

    SamRecord* recordPtr;
    int16_t samFlag;

    SamStatus::Status returnStatus = SamStatus::SUCCESS;
    while(returnStatus == SamStatus::SUCCESS)
    {
        recordPtr = myPool.getRecord();
        if(recordPtr == NULL)
        {
            // Failed to allocate a new record.
            throw(std::runtime_error("Failed to allocate a new SAM/BAM record"));
        }
        if(!samIn.ReadRecord(mySamHeader, *recordPtr))
        {
            // Failed to read a record.
            returnStatus = samIn.GetStatus();
            continue;
        }

        // Have a record.  Check to see if it is a pair or unpaired read.
        samFlag = recordPtr->getFlag();
        if(SamFlag::isPaired(samFlag))
        {
            if(readName)
            {
                handlePairedRN(*recordPtr);
            }
            else
            {
                handlePairedCoord(*recordPtr);
            }
        }
        else
        {
            ++myNumUnpaired;
            writeFastQ(*recordPtr, myUnpairedFile,
                       myUnpairedFileNameExt);
        }
    }

    // Flush All
    cleanUpMateMap(0, true);

    if(returnStatus == SamStatus::NO_MORE_RECS)
    {
        returnStatus = SamStatus::SUCCESS;
    }

    samIn.Close();
    closeFiles();
    
    // Output the results
    std::cerr << "\nFound " << myNumPairs << " read pairs.\n";
    std::cerr << "Found " << myNumUnpaired << " unpaired reads.\n";
    if(myNumMateFailures != 0)
    {
        std::cerr << "Failed to find mates for " << myNumMateFailures
                  << " reads, so they were written as unpaired\n"
                  << "  (not included in either of the above counts).\n";
    }
    if(myNumQualTagErrors != 0)
    {
        std::cerr << myNumQualTagErrors << " records did not have tag "
                  << myQField.c_str() << " or it was invalid, so the quality field was used for those records.\n";
    }

    return(returnStatus);
}
Beispiel #3
0
void testFlagRead(const char* fileName)
{
    SamFile inSam;
    SamFileHeader samHeader;
    SamRecord samRecord;

    ////////////////////////////////////////////////////////////
    // Required flag 0x48  (only flag 73 matches)
    // Exclude nothing
    assert(inSam.OpenForRead(fileName));
    assert(inSam.ReadHeader(samHeader));
    validateHeader(samHeader);
    inSam.SetReadFlags(0x48, 0x0);

    assert(inSam.ReadRecord(samHeader, samRecord) == true);
    validateRead1(samRecord);

    assert(inSam.ReadRecord(samHeader, samRecord) == false);

    inSam.Close();

    ////////////////////////////////////////////////////////////
    // No required flags.
    // Exclude 0x48.  This leaves just the one read with flag 133.
    assert(inSam.OpenForRead(fileName));
    assert(inSam.ReadHeader(samHeader));
    validateHeader(samHeader);
    inSam.SetReadFlags(0x0, 0x48);

    assert(inSam.ReadRecord(samHeader, samRecord) == true);
    validateRead2(samRecord);

    assert(inSam.ReadRecord(samHeader, samRecord) == false);

    inSam.Close();

    ////////////////////////////////////////////////////////////
    // Required flag 0x40 
    // Exclude 0x48.
    // This will not find any records since the exclude and required conflict.
    assert(inSam.OpenForRead(fileName));
    assert(inSam.ReadHeader(samHeader));
    validateHeader(samHeader);
    inSam.SetReadFlags(0x40, 0x48);

    assert(inSam.ReadRecord(samHeader, samRecord) == false);

    inSam.Close();

    ////////////////////////////////////////////////////////////
    // Required flag 0x4
    // Exclude 0x8.
    // Only finds flag 133.
    assert(inSam.OpenForRead(fileName));
    assert(inSam.ReadHeader(samHeader));
    validateHeader(samHeader);
    inSam.SetReadFlags(0x4, 0x8);

    assert(inSam.ReadRecord(samHeader, samRecord) == true);
    validateRead2(samRecord);

    assert(inSam.ReadRecord(samHeader, samRecord) == false);

    inSam.Close();

     ////////////////////////////////////////////////////////////
    // Required flag 0x4
    // Exclude nothing
    // Finds flags 133 & 141.
    assert(inSam.OpenForRead(fileName));
    assert(inSam.ReadHeader(samHeader));
    validateHeader(samHeader);
    inSam.SetReadFlags(0x4, 0x0);

    assert(inSam.ReadRecord(samHeader, samRecord) == true);
    validateRead2(samRecord);

    assert(inSam.ReadRecord(samHeader, samRecord) == true);
    validateRead8(samRecord);

    assert(inSam.ReadRecord(samHeader, samRecord) == true);
    validateRead10(samRecord);

    assert(inSam.ReadRecord(samHeader, samRecord) == false);

    inSam.Close();
}