BamReader.cpp

// ***************************************************************************
// BamReader.cpp (c) 2009 Derek Barnett, Michael Str�mberg
// Marth Lab, Department of Biology, Boston College
// All rights reserved.
// ---------------------------------------------------------------------------
// Last modified: 13 October 2010 (DB)
// ---------------------------------------------------------------------------
// Uses BGZF routines were adapted from the bgzf.c code developed at the Broad
// Institute.
// ---------------------------------------------------------------------------
// Provides the basic functionality for reading BAM files
// ***************************************************************************

// C++ includes
#include <algorithm>
#include <iterator>
#include <string>
#include <vector>
#include <iostream>
#include "BGZF.h"
#include "BamReader.h"
using namespace BamTools;
using namespace std;

struct BamReader::BamReaderPrivate {

    // -------------------------------
    // structs, enums, typedefs
    // -------------------------------
    enum RegionState { BEFORE_REGION = 0
                     , WITHIN_REGION
                     , AFTER_REGION
                     };

    // -------------------------------
    // data members
    // -------------------------------

    // general file data
    BgzfData  mBGZF;
    string    HeaderText;
    BamIndex* Index;
    RefVector References;
    bool      HasIndex;
    int64_t   AlignmentsBeginOffset;
    string    Filename;
    string    IndexFilename;
    
    // index caching mode
    BamIndex::BamIndexCacheMode IndexCacheMode;
    
    // system data
    bool IsBigEndian;

    // user-specified region values
    BamRegion Region;
    bool HasAlignmentsInRegion;

    // parent BamReader
    BamReader* Parent;
    
    // BAM character constants
    const char* DNA_LOOKUP;
    const char* CIGAR_LOOKUP;

    // constructor & destructor
    BamReaderPrivate(BamReader* parent);
    ~BamReaderPrivate(void);

    // -------------------------------
    // "public" interface

    // file operations
    void Close(void);
    bool Open(const std::string& filename, 
              const std::string& indexFilename, 
              const bool lookForIndex, 
              const bool preferStandardIndex);
    bool Rewind(void);
    bool SetRegion(const BamRegion& region);

    // access alignment data
    bool GetNextAlignment(BamAlignment& bAlignment);
    bool GetNextAlignmentCore(BamAlignment& bAlignment);

    // access auxiliary data
    int GetReferenceID(const string& refName) const;

    // index operations
    bool CreateIndex(bool useStandardIndex);
    void SetIndexCacheMode(const BamIndex::BamIndexCacheMode mode);


    // internal methods
    private:

        // ---------------------------------------
        // reading alignments and auxiliary data

        // adjusts requested region if necessary (depending on where data actually begins)
        void AdjustRegion(BamRegion& region);
        // fills out character data for BamAlignment data
        bool BuildCharData(BamAlignment& bAlignment);
        // checks to see if alignment overlaps current region
        RegionState IsOverlap(BamAlignment& bAlignment);
        // retrieves header text from BAM file
        void LoadHeaderData(void);
        // retrieves BAM alignment under file pointer
        bool LoadNextAlignment(BamAlignment& bAlignment);
        // builds reference data structure from BAM file
        void LoadReferenceData(void);
        // mark references with 'HasAlignments' status
        void MarkReferences(void);

        // ---------------------------------
        // index file handling

        // clear out inernal index data structure
        void ClearIndex(void);
        // loads index from BAM index file
        bool LoadIndex(const bool lookForIndex, const bool preferStandardIndex);
};

// -----------------------------------------------------
// BamReader implementation (wrapper around BRPrivate)
// -----------------------------------------------------
// constructor
BamReader::BamReader(void) {
    d = new BamReaderPrivate(this);
}

// destructor
BamReader::~BamReader(void) {
    delete d;
    d = 0;
}

// file operations
void BamReader::Close(void) { d->Close(); }
bool BamReader::HasIndex(void) const { return d->HasIndex; }
bool BamReader::IsIndexLoaded(void) const { return HasIndex(); }
bool BamReader::IsOpen(void) const { return d->mBGZF.IsOpen; }
bool BamReader::Jump(int refID, int position)  { return d->SetRegion( BamRegion(refID, position) ); }
bool BamReader::Open(const std::string& filename, 
                     const std::string& indexFilename, 
                     const bool lookForIndex, 
                     const bool preferStandardIndex) 
{ 
    return d->Open(filename, indexFilename, lookForIndex, preferStandardIndex); 
}
bool BamReader::Rewind(void) { return d->Rewind(); }
bool BamReader::SetRegion(const BamRegion& region) { return d->SetRegion(region); }
bool BamReader::SetRegion(const int& leftRefID, const int& leftBound, const int& rightRefID, const int& rightBound) {
    return d->SetRegion( BamRegion(leftRefID, leftBound, rightRefID, rightBound) );
}

// access alignment data
bool BamReader::GetNextAlignment(BamAlignment& bAlignment) { return d->GetNextAlignment(bAlignment); }
bool BamReader::GetNextAlignmentCore(BamAlignment& bAlignment) { return d->GetNextAlignmentCore(bAlignment); }

// access auxiliary data
const string BamReader::GetHeaderText(void) const { return d->HeaderText; }
int BamReader::GetReferenceCount(void) const { return d->References.size(); }
const RefVector& BamReader::GetReferenceData(void) const { return d->References; }
int BamReader::GetReferenceID(const string& refName) const { return d->GetReferenceID(refName); }
const std::string BamReader::GetFilename(void) const { return d->Filename; }

// index operations
bool BamReader::CreateIndex(bool useStandardIndex) { return d->CreateIndex(useStandardIndex); }
void BamReader::SetIndexCacheMode(const BamIndex::BamIndexCacheMode mode) { d->SetIndexCacheMode(mode); }

// -----------------------------------------------------
// BamReaderPrivate implementation
// -----------------------------------------------------

// constructor
BamReader::BamReaderPrivate::BamReaderPrivate(BamReader* parent)
    : Index(0)
    , HasIndex(false)
    , AlignmentsBeginOffset(0)
    , IndexCacheMode(BamIndex::LimitedIndexCaching)
    , HasAlignmentsInRegion(true)
    , Parent(parent)
    , DNA_LOOKUP("=ACMGRSVTWYHKDBN")
    , CIGAR_LOOKUP("MIDNSHP")
{ 
    IsBigEndian = SystemIsBigEndian();
}

// destructor
BamReader::BamReaderPrivate::~BamReaderPrivate(void) {
    Close();
}

// adjusts requested region if necessary (depending on where data actually begins)
void BamReader::BamReaderPrivate::AdjustRegion(BamRegion& region) {
  
    // check for valid index first
    if ( Index == 0 ) return;
  
    // see if any references in region have alignments
    HasAlignmentsInRegion = false;
    int currentId = region.LeftRefID;
    while ( currentId <= region.RightRefID ) { 
	HasAlignmentsInRegion = Index->HasAlignments(currentId);
	if ( HasAlignmentsInRegion ) break;
	++currentId;
    }
    
    // if no data found on any reference in region
    if ( !HasAlignmentsInRegion ) return;

    // if left bound of desired region had no data, use first reference that had data
    // otherwise, leave requested region as-is
    if ( currentId != region.LeftRefID ) {
	region.LeftRefID = currentId;
	region.LeftPosition = 0;
    }
}

// fills out character data for BamAlignment data
bool BamReader::BamReaderPrivate::BuildCharData(BamAlignment& bAlignment) {
  
    // calculate character lengths/offsets
    const unsigned int dataLength     = bAlignment.SupportData.BlockLength - BAM_CORE_SIZE;
    const unsigned int seqDataOffset  = bAlignment.SupportData.QueryNameLength + (bAlignment.SupportData.NumCigarOperations * 4);
    const unsigned int qualDataOffset = seqDataOffset + (bAlignment.SupportData.QuerySequenceLength+1)/2;
    const unsigned int tagDataOffset  = qualDataOffset + bAlignment.SupportData.QuerySequenceLength;
    const unsigned int tagDataLength  = dataLength - tagDataOffset;
      
    // set up char buffers
    const char* allCharData = bAlignment.SupportData.AllCharData.data();
    const char* seqData     = ((const char*)allCharData) + seqDataOffset;
    const char* qualData    = ((const char*)allCharData) + qualDataOffset;
          char* tagData     = ((char*)allCharData) + tagDataOffset;
  
    // store alignment name (relies on null char in name as terminator)
    bAlignment.Name.assign((const char*)(allCharData));    

    // save query sequence
    bAlignment.QueryBases.clear();
    bAlignment.QueryBases.reserve(bAlignment.SupportData.QuerySequenceLength);
    for (unsigned int i = 0; i < bAlignment.SupportData.QuerySequenceLength; ++i) {
        char singleBase = DNA_LOOKUP[ ( (seqData[(i/2)] >> (4*(1-(i%2)))) & 0xf ) ];
        bAlignment.QueryBases.append(1, singleBase);
    }
  
    // save qualities, converting from numeric QV to 'FASTQ-style' ASCII character
    bAlignment.Qualities.clear();
    bAlignment.Qualities.reserve(bAlignment.SupportData.QuerySequenceLength);
    for (unsigned int i = 0; i < bAlignment.SupportData.QuerySequenceLength; ++i) {
        char singleQuality = (char)(qualData[i]+33);
        bAlignment.Qualities.append(1, singleQuality);
    }
    
    // if QueryBases is empty (and this is a allowed case)
    if ( bAlignment.QueryBases.empty() ) 
        bAlignment.AlignedBases = bAlignment.QueryBases;
    
    // if QueryBases contains data, then build AlignedBases using CIGAR data
    else {
    
        // resize AlignedBases
        bAlignment.AlignedBases.clear();
        bAlignment.AlignedBases.reserve(bAlignment.SupportData.QuerySequenceLength);
      
        // iterate over CigarOps
        int k = 0;
        vector<CigarOp>::const_iterator cigarIter = bAlignment.CigarData.begin();
        vector<CigarOp>::const_iterator cigarEnd  = bAlignment.CigarData.end();
        for ( ; cigarIter != cigarEnd; ++cigarIter ) {
            
            const CigarOp& op = (*cigarIter);
            switch(op.Type) {
              
                case ('M') :
                case ('I') :
                    bAlignment.AlignedBases.append(bAlignment.QueryBases.substr(k, op.Length)); // for 'M', 'I' - write bases
                    // fall through
                
                case ('S') :
                    k += op.Length;                                     // for 'S' - soft clip, skip over query bases
                    break;
                    
                case ('D') :
                    bAlignment.AlignedBases.append(op.Length, '-');     // for 'D' - write gap character
                    break;
                    
                case ('P') :
                    bAlignment.AlignedBases.append( op.Length, '*' );   // for 'P' - write padding character
                    break;
                    
                case ('N') :
                    bAlignment.AlignedBases.append( op.Length, 'N' );  // for 'N' - write N's, skip bases in original query sequence
                    break;
                    
                case ('H') :
                    break;  // for 'H' - hard clip, do nothing to AlignedBases, move to next op
                    
                default:
                    fprintf(stderr, "ERROR: Invalid Cigar op type\n"); // shouldn't get here
                    exit(1);
            }
        }
    }
 
    // -----------------------
    // Added: 3-25-2010 DB
    // Fixed: endian-correctness for tag data
    // -----------------------
    if ( IsBigEndian ) {
        int i = 0;
        while ( (unsigned int)i < tagDataLength ) {
          
            i += 2; // skip tag type (e.g. "RG", "NM", etc)
            uint8_t type = toupper(tagData[i]);     // lower & upper case letters have same meaning 
            ++i;                                    // skip value type
    
            switch (type) {
                
                case('A') :
                case('C') : 
                    ++i;
                    break;

                case('S') : 
                    SwapEndian_16p(&tagData[i]); 
                    i += sizeof(uint16_t);
                    break;
                    
                case('F') :
                case('I') : 
                    SwapEndian_32p(&tagData[i]);
                    i += sizeof(uint32_t);
                    break;
                
                case('D') : 
                    SwapEndian_64p(&tagData[i]);
                    i += sizeof(uint64_t);
                    break;
                
                case('H') :
                case('Z') : 
                    while (tagData[i]) { ++i; }
                    ++i; // increment one more for null terminator
                    break;
                
                default : 
                    fprintf(stderr, "ERROR: Invalid tag value type\n"); // shouldn't get here
                    exit(1);
            }
        }
    }
    
    // store TagData
    bAlignment.TagData.clear();
    bAlignment.TagData.resize(tagDataLength);
    memcpy((char*)bAlignment.TagData.data(), tagData, tagDataLength);
    
    // clear the core-only flag
    bAlignment.SupportData.HasCoreOnly = false;
    
    // return success
    return true;
}

// clear index data structure
void BamReader::BamReaderPrivate::ClearIndex(void) {
    delete Index;
    Index = 0;
    HasIndex = false;
}

// closes the BAM file
void BamReader::BamReaderPrivate::Close(void) {
    
    // close BGZF file stream
    mBGZF.Close();
    
    // clear out index data
    ClearIndex();
    
    // clear out header data
    HeaderText.clear();
    
    // clear out region flags
    Region.clear();
}

// creates index for BAM file, saves to file
// default behavior is to create the BAM standard index (".bai")
// set flag to false to create the BamTools-specific index (".bti")
bool BamReader::BamReaderPrivate::CreateIndex(bool useStandardIndex) {

    // clear out prior index data
    ClearIndex();
    
    // create index based on type requested
    if ( useStandardIndex ) 
        Index = new BamStandardIndex(&mBGZF, Parent);
    else
        Index = new BamToolsIndex(&mBGZF, Parent);
    
    // set index cache mode to full for writing
    Index->SetCacheMode(BamIndex::FullIndexCaching);
    
    // build new index
    bool ok = true;
    ok &= Index->Build();
    HasIndex = ok;
    
    // mark empty references
    MarkReferences();
    
    // attempt to save index data to file
    ok &= Index->Write(Filename); 
    
    // set client's desired index cache mode 
    Index->SetCacheMode(IndexCacheMode);
    
    // return success/fail of both building & writing index
    return ok;
}

// get next alignment (from specified region, if given)
bool BamReader::BamReaderPrivate::GetNextAlignment(BamAlignment& bAlignment) {

    // if valid alignment found, attempt to parse char data, and return success/failure
    if ( GetNextAlignmentCore(bAlignment) )
        return BuildCharData(bAlignment);
    
    // no valid alignment found
    else return false;
}

// retrieves next available alignment core data (returns success/fail)
// ** DOES NOT parse any character data (read name, bases, qualities, tag data)
//    these can be accessed, if necessary, from the supportData 
// useful for operations requiring ONLY positional or other alignment-related information
bool BamReader::BamReaderPrivate::GetNextAlignmentCore(BamAlignment& bAlignment) {

    // if region is set but has no alignments
    if ( !Region.isNull() && !HasAlignmentsInRegion )
        return false;
  
    // if valid alignment available
    if ( LoadNextAlignment(bAlignment) ) {

        // set core-only flag
        bAlignment.SupportData.HasCoreOnly = true;
      
	// if region not specified with at least a left boundary, return success
	if ( !Region.isLeftBoundSpecified() ) return true;
	
        // determine region state (before, within, after)
        BamReader::BamReaderPrivate::RegionState state = IsOverlap(bAlignment);

        // if alignment lies after region, return false
        if ( state == AFTER_REGION ) return false;

        while ( state != WITHIN_REGION ) {
            // if no valid alignment available (likely EOF) return failure
            if ( !LoadNextAlignment(bAlignment) ) return false;
            // if alignment lies after region, return false (no available read within region)
            state = IsOverlap(bAlignment);
            if ( state == AFTER_REGION ) return false;
        }

        // return success (alignment found that overlaps region)
        return true;
    }

    // no valid alignment
    else return false;
}

// returns RefID for given RefName (returns References.size() if not found)
int BamReader::BamReaderPrivate::GetReferenceID(const string& refName) const {

    // retrieve names from reference data
    vector<string> refNames;
    RefVector::const_iterator refIter = References.begin();
    RefVector::const_iterator refEnd  = References.end();
    for ( ; refIter != refEnd; ++refIter) 
        refNames.push_back( (*refIter).RefName );

    // return 'index-of' refName ( if not found, returns refNames.size() )
    return distance(refNames.begin(), find(refNames.begin(), refNames.end(), refName));
}

// returns region state - whether alignment ends before, overlaps, or starts after currently specified region
// this *internal* method should ONLY called when (at least) IsLeftBoundSpecified == true
BamReader::BamReaderPrivate::RegionState BamReader::BamReaderPrivate::IsOverlap(BamAlignment& bAlignment) {
    
    // if alignment is on any reference sequence before left bound
    if ( bAlignment.RefID < Region.LeftRefID ) return BEFORE_REGION;
  
    // if alignment starts on left bound reference 
    else if ( bAlignment.RefID == Region.LeftRefID ) {
      
	// if alignment starts at or after left boundary
	if ( bAlignment.Position >= Region.LeftPosition) {
	  
	    // if right boundary is specified AND 
	    // left/right boundaries are on same reference AND 
	    // alignment starts past right boundary
	    if ( Region.isRightBoundSpecified() && 
	         Region.LeftRefID == Region.RightRefID && 
	         bAlignment.Position > Region.RightPosition )
		return AFTER_REGION;
	    
	    // otherwise, alignment is within region
	    return WITHIN_REGION;
	}
	
	// alignment starts before left boundary
	else {
	    // check if alignment overlaps left boundary
	    if ( bAlignment.GetEndPosition() >= Region.LeftPosition ) return WITHIN_REGION;
	    else return BEFORE_REGION;
	}
    }
  
    // alignment starts on a reference after the left bound
    else {
      
	// if region has a right boundary
	if ( Region.isRightBoundSpecified() ) {
	  
	    // alignment is on reference between boundaries
	    if ( bAlignment.RefID < Region.RightRefID ) return WITHIN_REGION;
	  
	    // alignment is on reference after right boundary
	    else if ( bAlignment.RefID > Region.RightRefID ) return AFTER_REGION;
	  
	    // alignment is on right bound reference
	    else {
		// check if alignment starts before or at right boundary
		if ( bAlignment.Position <= Region.RightPosition ) return WITHIN_REGION;		
		else return AFTER_REGION;
	    }
	}
      
	// otherwise, alignment is after left bound reference, but there is no right boundary
	else return WITHIN_REGION;
    }
}

// load BAM header data
void BamReader::BamReaderPrivate::LoadHeaderData(void) {

    // check to see if proper BAM header
    char buffer[4];
    if (mBGZF.Read(buffer, 4) != 4) {
        fprintf(stderr, "Could not read header type\n");
        exit(1);
    }

    if (strncmp(buffer, "BAM\001", 4)) {
        fprintf(stderr, "wrong header type!\n");
        exit(1);
    }

    // get BAM header text length
    mBGZF.Read(buffer, 4);
    unsigned int headerTextLength = BgzfData::UnpackUnsignedInt(buffer);
    if ( IsBigEndian ) SwapEndian_32(headerTextLength); 
    
    // get BAM header text
    char* headerText = (char*)calloc(headerTextLength + 1, 1);
    mBGZF.Read(headerText, headerTextLength);
    HeaderText = (string)((const char*)headerText);

    // clean up calloc-ed temp variable
    free(headerText);
}

// load existing index data from BAM index file (".bti" OR ".bai"), return success/fail
bool BamReader::BamReaderPrivate::LoadIndex(const bool lookForIndex, const bool preferStandardIndex) {

    // clear out any existing index data
    ClearIndex();

    // if no index filename provided, so we need to look for available index files
    if ( IndexFilename.empty() ) {
      
        // attempt to load BamIndex based on current Filename provided & preferStandardIndex flag
        const BamIndex::PreferredIndexType type = (preferStandardIndex ? BamIndex::STANDARD : BamIndex::BAMTOOLS);
        Index = BamIndex::FromBamFilename(Filename, &mBGZF, Parent, type);
        
        // if null, return failure
        if ( Index == 0 ) return false;
        
        // generate proper IndexFilename based on type of index created
        IndexFilename = Filename + Index->Extension();
    }
    
    else {
      
        // attempt to load BamIndex based on IndexFilename provided by client
        Index = BamIndex::FromIndexFilename(IndexFilename, &mBGZF, Parent);
        
        // if null, return failure
        if ( Index == 0 ) return false;
    }

    // set cache mode for BamIndex
    Index->SetCacheMode(IndexCacheMode);

    // loading the index data from file
    HasIndex = Index->Load(IndexFilename);
    
    // mark empty references
    MarkReferences();
    
    // return index status
    return HasIndex;
}

// populates BamAlignment with alignment data under file pointer, returns success/fail
bool BamReader::BamReaderPrivate::LoadNextAlignment(BamAlignment& bAlignment) {

    // read in the 'block length' value, make sure it's not zero
    char buffer[4];
    mBGZF.Read(buffer, 4);
    bAlignment.SupportData.BlockLength = BgzfData::UnpackUnsignedInt(buffer);
    if ( IsBigEndian ) { SwapEndian_32(bAlignment.SupportData.BlockLength); }
    if ( bAlignment.SupportData.BlockLength == 0 ) return false;

    // read in core alignment data, make sure the right size of data was read
    char x[BAM_CORE_SIZE];
    if ( mBGZF.Read(x, BAM_CORE_SIZE) != BAM_CORE_SIZE ) return false; 

    if ( IsBigEndian ) {
        for ( int i = 0; i < BAM_CORE_SIZE; i+=sizeof(uint32_t) ) 
            SwapEndian_32p(&x[i]); 
    }
    
    // set BamAlignment 'core' and 'support' data
    bAlignment.RefID    = BgzfData::UnpackSignedInt(&x[0]);  
    bAlignment.Position = BgzfData::UnpackSignedInt(&x[4]);
    
    unsigned int tempValue = BgzfData::UnpackUnsignedInt(&x[8]);
    bAlignment.Bin        = tempValue >> 16;
    bAlignment.MapQuality = tempValue >> 8 & 0xff;
    bAlignment.SupportData.QueryNameLength = tempValue & 0xff;

    tempValue = BgzfData::UnpackUnsignedInt(&x[12]);
    bAlignment.AlignmentFlag = tempValue >> 16;
    bAlignment.SupportData.NumCigarOperations = tempValue & 0xffff;

    bAlignment.SupportData.QuerySequenceLength = BgzfData::UnpackUnsignedInt(&x[16]);
    bAlignment.MateRefID    = BgzfData::UnpackSignedInt(&x[20]);
    bAlignment.MatePosition = BgzfData::UnpackSignedInt(&x[24]);
    bAlignment.InsertSize   = BgzfData::UnpackSignedInt(&x[28]);
    
    // set BamAlignment length
    bAlignment.Length = bAlignment.SupportData.QuerySequenceLength;
    
    // read in character data - make sure proper data size was read
    bool readCharDataOK = false;
    const unsigned int dataLength = bAlignment.SupportData.BlockLength - BAM_CORE_SIZE;
    char* allCharData = (char*)calloc(sizeof(char), dataLength);
    
    if ( mBGZF.Read(allCharData, dataLength) == (signed int)dataLength) { 
      
        // store 'allCharData' in supportData structure
        bAlignment.SupportData.AllCharData.assign((const char*)allCharData, dataLength);
        
        // set success flag
        readCharDataOK = true;
        
        // save CIGAR ops 
        // need to calculate this here so that  BamAlignment::GetEndPosition() performs correctly, 
        // even when BamReader::GetNextAlignmentCore() is called 
        const unsigned int cigarDataOffset = bAlignment.SupportData.QueryNameLength;
        uint32_t* cigarData = (uint32_t*)(allCharData + cigarDataOffset);
        CigarOp op;
        bAlignment.CigarData.clear();
        bAlignment.CigarData.reserve(bAlignment.SupportData.NumCigarOperations);
        for (unsigned int i = 0; i < bAlignment.SupportData.NumCigarOperations; ++i) {

            // swap if necessary
            if ( IsBigEndian ) SwapEndian_32(cigarData[i]);
          
            // build CigarOp structure
            op.Length = (cigarData[i] >> BAM_CIGAR_SHIFT);
            op.Type   = CIGAR_LOOKUP[ (cigarData[i] & BAM_CIGAR_MASK) ];

            // save CigarOp
            bAlignment.CigarData.push_back(op);
        }
    }

    free(allCharData);
    return readCharDataOK;
}

// loads reference data from BAM file
void BamReader::BamReaderPrivate::LoadReferenceData(void) {

    // get number of reference sequences
    char buffer[4];
    mBGZF.Read(buffer, 4);
    unsigned int numberRefSeqs = BgzfData::UnpackUnsignedInt(buffer);
    if ( IsBigEndian ) SwapEndian_32(numberRefSeqs);
    if ( numberRefSeqs == 0 ) return;
    References.reserve((int)numberRefSeqs);

    // iterate over all references in header
    for (unsigned int i = 0; i != numberRefSeqs; ++i) {

        // get length of reference name
        mBGZF.Read(buffer, 4);
        unsigned int refNameLength = BgzfData::UnpackUnsignedInt(buffer);
        if ( IsBigEndian ) SwapEndian_32(refNameLength);
        char* refName = (char*)calloc(refNameLength, 1);

        // get reference name and reference sequence length
        mBGZF.Read(refName, refNameLength);
        mBGZF.Read(buffer, 4);
        int refLength = BgzfData::UnpackSignedInt(buffer);
        if ( IsBigEndian ) SwapEndian_32(refLength); 

        // store data for reference
        RefData aReference;
        aReference.RefName   = (string)((const char*)refName);
        aReference.RefLength = refLength;
        References.push_back(aReference);

        // clean up calloc-ed temp variable
        free(refName);
    }
}

// mark references with no alignment data
void BamReader::BamReaderPrivate::MarkReferences(void) {
    
    // ensure index is available
    if ( !HasIndex ) return;
    
    // mark empty references
    for ( int i = 0; i < (int)References.size(); ++i ) 
	References.at(i).RefHasAlignments = Index->HasAlignments(i);
}

// opens BAM file (and index)
bool BamReader::BamReaderPrivate::Open(const string& filename, const string& indexFilename, const bool lookForIndex, const bool preferStandardIndex) {

    // store filenames
    Filename = filename;
    IndexFilename = indexFilename;

    // open the BGZF file for reading, return false on failure
    if ( !mBGZF.Open(filename, "rb") ) return false; 
    
    // retrieve header text & reference data
    LoadHeaderData();
    LoadReferenceData();

    // store file offset of first alignment
    AlignmentsBeginOffset = mBGZF.Tell();

    // if no index filename provided 
    if ( IndexFilename.empty() ) {
     
        // client did not specify that index SHOULD be found
        // useful for cases where sequential access is all that is required
        if ( !lookForIndex ) return true; 
          
        // otherwise, look for index file, return success/fail
        return LoadIndex(lookForIndex, preferStandardIndex) ;
    }
    
    // client supplied an index filename
    // attempt to load index data, return success/fail
    return LoadIndex(lookForIndex, preferStandardIndex);    
}

// returns BAM file pointer to beginning of alignment data
bool BamReader::BamReaderPrivate::Rewind(void) {
   
    // rewind to first alignment, return false if unable to seek
    if ( !mBGZF.Seek(AlignmentsBeginOffset) ) return false;
  
    // retrieve first alignment data, return false if unable to read
    BamAlignment al;
    if ( !LoadNextAlignment(al) ) return false;
      
    // reset default region info using first alignment in file
    Region.clear();
    HasAlignmentsInRegion = true;

    // rewind back to beginning of first alignment
    // return success/fail of seek
    return mBGZF.Seek(AlignmentsBeginOffset);
}

// change the index caching behavior
void BamReader::BamReaderPrivate::SetIndexCacheMode(const BamIndex::BamIndexCacheMode mode) {
    IndexCacheMode = mode;
    if ( Index == 0 ) return;
    Index->SetCacheMode(mode);
}

// asks Index to attempt a Jump() to specified region
// returns success/failure
bool BamReader::BamReaderPrivate::SetRegion(const BamRegion& region) {
      
    // clear out any prior BamReader region data
    //
    // N.B. - this is cleared so that BamIndex now has free reign to call
    // GetNextAlignmentCore() and do overlap checking without worrying about BamReader 
    // performing any overlap checking of its own and moving on to the next read... Calls 
    // to GetNextAlignmentCore() with no Region set, simply return the next alignment.
    // This ensures that the Index is able to do just that. (All without exposing 
    // LoadNextAlignment() to the public API, and potentially confusing clients with the nomenclature)
    Region.clear();
  
    // check for existing index 
    if ( !HasIndex ) return false; 
    
    // adjust region if necessary to reflect where data actually begins
    BamRegion adjustedRegion(region);
    AdjustRegion(adjustedRegion);
    
    // if no data present, return true
    // not an error, but BamReader knows that no data is there for future alignment access
    // (this is useful in a MultiBamReader setting where some BAM files may lack data in regions
    // that other BAMs have data)
    if ( !HasAlignmentsInRegion ) { 
	Region = adjustedRegion;
	return true;
    }
    
    // attempt jump to user-specified region return false if jump could not be performed at all
    // (invalid index, unknown reference, etc)
    //
    // Index::Jump() is allowed to modify the HasAlignmentsInRegion flag
    //  * This covers case where a region is requested that lies beyond the last alignment on a reference
    //    If this occurs, any subsequent calls to GetNexAlignment[Core] simply return false
    //    BamMultiReader is then able to successfully pull alignments from a region from multiple files
    //    even if one or more have no data.
    if ( !Index->Jump(adjustedRegion, &HasAlignmentsInRegion) ) return false;
    
    // save region and return success
    Region = adjustedRegion;
    return true;
}