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// ==========================================================================
// SeqAn - The Library for Sequence Analysis
// ==========================================================================
// Copyright (c) 2006-2013, Knut Reinert, FU Berlin
// All rights reserved.
//
// Redistribution and use in source and binary forms, with or without
// modification, are permitted provided that the following conditions are met:
//
// * Redistributions of source code must retain the above copyright
// notice, this list of conditions and the following disclaimer.
// * Redistributions in binary form must reproduce the above copyright
// notice, this list of conditions and the following disclaimer in the
// documentation and/or other materials provided with the distribution.
// * Neither the name of Knut Reinert or the FU Berlin nor the names of
// its contributors may be used to endorse or promote products derived
// from this software without specific prior written permission.
//
// THIS SOFTWARE IS PROVIDED BY THE COPYRIGHT HOLDERS AND CONTRIBUTORS "AS IS"
// AND ANY EXPRESS OR IMPLIED WARRANTIES, INCLUDING, BUT NOT LIMITED TO, THE
// IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE
// ARE DISCLAIMED. IN NO EVENT SHALL KNUT REINERT OR THE FU BERLIN BE LIABLE
// FOR ANY DIRECT, INDIRECT, INCIDENTAL, SPECIAL, EXEMPLARY, OR CONSEQUENTIAL
// DAMAGES (INCLUDING, BUT NOT LIMITED TO, PROCUREMENT OF SUBSTITUTE GOODS OR
// SERVICES; LOSS OF USE, DATA, OR PROFITS; OR BUSINESS INTERRUPTION) HOWEVER
// CAUSED AND ON ANY THEORY OF LIABILITY, WHETHER IN CONTRACT, STRICT
// LIABILITY, OR TORT (INCLUDING NEGLIGENCE OR OTHERWISE) ARISING IN ANY WAY
// OUT OF THE USE OF THIS SOFTWARE, EVEN IF ADVISED OF THE POSSIBILITY OF SUCH
// DAMAGE.
//
// ==========================================================================
// Author: Tobias Rausch <rausch@embl.de>
// Author: Manuel Holtgrewe <manuel.holtgrewe@fu-berlin.de>
// ==========================================================================
// Shows how to use the bam_io module to estimate the insert size from a SAM
// or BAM file.
// ==========================================================================
#include <iostream>
#include <vector>
#include <seqan/basic.h>
#include <seqan/sequence.h>
#include <seqan/stream.h>
#include <seqan/bam_io.h>
#if SEQAN_HAS_ZLIB
using namespace seqan;
// Stores information about a library.
struct LibraryInfo
{
// This is the notation introduced by Illumina.
//
// F+: >R1 R2>
// F-: >R2 R1>
// R+: >R1 R2<
// R-: <R2 R1>
enum Orientation
{
F_PLUS = 0,
F_MINUS = 1,
R_PLUS = 2,
R_MINUS = 3
};
unsigned median;
double stdDev;
unsigned maxNormalISize;
Orientation defaultOrient;
LibraryInfo() : median(0), stdDev(0), maxNormalISize(0), defaultOrient(F_PLUS)
{}
};
inline int
getStrandIndependentOrientation(BamAlignmentRecord const& rec)
{
if (!hasFlagRC(rec)) {
if (!(hasFlagNextRC(rec))) {
return (rec.beginPos < rec.pNext) ? LibraryInfo::F_PLUS : LibraryInfo::F_MINUS;
} else {
return (rec.beginPos < rec.pNext) ? LibraryInfo::R_PLUS : LibraryInfo::R_MINUS;
}
} else {
if (!hasFlagNextRC(rec)) {
return (rec.beginPos > rec.pNext) ? LibraryInfo::R_PLUS : LibraryInfo::R_MINUS;
} else {
return (rec.beginPos > rec.pNext) ? LibraryInfo::F_PLUS : LibraryInfo::F_MINUS;
}
}
}
bool endsWith(CharString const & str, CharString const & x)
{
typedef Size<CharString>::Type TSize;
TSize len = std::min(length(str), length(x));
TSize pos = length(str) - len;
return suffix(str, pos) == x;
}
template <typename TStreamOrReader, typename TFormatTag>
bool performEstimation(LibraryInfo & libInfo, TStreamOrReader & streamOrReader, TFormatTag const & formatTag)
{
typedef StringSet<String<char> > TNameStore;
typedef NameStoreCache<TNameStore> TNameStoreCache;
// Vector of all insert sizes.
typedef std::vector<unsigned int> TVecISize;
TVecISize vecISize;
// BAM Meta Info.
TNameStore nameStore;
TNameStoreCache nameStoreCache(nameStore);
BamIOContext<TNameStore> context(nameStore, nameStoreCache);
// Read Header.
BamHeader header;
readRecord(header, context, streamOrReader, formatTag);
// Orientations.
unsigned orientationCounters[4] = {0, 0, 0, 0};
// Running mean.
__uint64 runningMean = 0;
unsigned pairCount = 0;
// Count.
BamAlignmentRecord record;
while (!atEnd(streamOrReader))
{
if (readRecord(record, context, streamOrReader, formatTag) != 0)
{
std::cerr << "Error reading SAM/BAM file.\n";
return false;
}
// Skip all records except for first mate of properly mapped pairs.
if (!hasFlagMultiple(record) || hasFlagUnmapped(record) || hasFlagNextUnmapped(record) ||
hasFlagSecondary(record) || hasFlagFirst(record))
continue;
vecISize.push_back(abs(record.tLen));
runningMean += abs(record.tLen);
pairCount += 1;
orientationCounters[getStrandIndependentOrientation(record)] += 1;
}
// Get default orientation.
unsigned orientMax = 0;
for (unsigned i = 0; i < 4; ++i)
if (orientationCounters[i] > orientationCounters[orientMax])
orientMax = i;
LibraryInfo::Orientation orientations[4] = { LibraryInfo::F_PLUS, LibraryInfo::F_MINUS, LibraryInfo::R_PLUS, LibraryInfo::R_MINUS };
libInfo.defaultOrient = orientations[orientMax];
// Trim off the chimera peak in mate-pair libraries.
if (libInfo.defaultOrient == LibraryInfo::R_MINUS && (runningMean / pairCount) >= 1000u)
{
typedef typename TVecISize::const_iterator TVecISizeIter;
TVecISize vecISizeTmp;
for(TVecISizeIter it = vecISize.begin(); it < vecISize.end(); ++it)
if (*it > 1000)
vecISizeTmp.push_back(*it);
std::swap(vecISize, vecISizeTmp);
}
// Check that this is a proper paired-end library
if (vecISize.empty())
return true;
// Get library stats.
//
// Start with median.
typedef typename TVecISize::iterator TVecISizeIter;
TVecISizeIter begin = vecISize.begin();
TVecISizeIter end = vecISize.end();
std::nth_element(begin, begin + (end - begin) / 2, end);
begin = vecISize.begin();
end = vecISize.end();
libInfo.median = *(begin + (end - begin) / 2);
// Standard deviation is next.
//
// SD calculation cutoffs are 7 SDs to the left and right assuming 10% library deviation.
libInfo.stdDev = 0;
double cutoffMax = libInfo.median + 7 * 0.1 * libInfo.median;
double cutoffMin = libInfo.median - 7 * 0.1 * libInfo.median;
if ((cutoffMin < 0) || (cutoffMax < cutoffMin))
cutoffMin = 0;
unsigned int count = 0;
for(;begin < end; ++begin)
{
if ((*begin >= cutoffMin) && (*begin <= cutoffMax))
{
libInfo.stdDev += (*begin - libInfo.median) * (*begin - libInfo.median);
++count;
}
}
libInfo.stdDev = sqrt(libInfo.stdDev / count);
libInfo.maxNormalISize = static_cast<unsigned>(libInfo.median + 3 * libInfo.stdDev);
return true;
}
int main(int argc, char const ** argv)
{
if (argc != 2)
{
std::cerr << "Invalid arguments!\n"
<< "USAGE: bam_library_size {IN.sam,IN.bam}\n";
return 1;
}
LibraryInfo libInfo;
// Guess file type from extension.
if (endsWith(argv[1], ".sam"))
{
std::ifstream inSamStream(toCString(argv[1]), std::ios::binary | std::ios::in);
if (!inSamStream.good())
{
std::cerr << "Could not open input SAM file " << argv[1] << "\n";
return 1;
}
RecordReader<std::ifstream, SinglePass<> > reader(inSamStream);
if (!performEstimation(libInfo, reader, Sam()))
return 1;
}
else
{
Stream<Bgzf> inBamStream;
if (!open(inBamStream, toCString(argv[1]), "r"))
{
std::cerr << "Could not open input BAM file " << argv[1] << "\n";
return 1;
}
if (!performEstimation(libInfo, inBamStream, Bam()))
return 1;
}
// Print result.
std::cout << "Library Information\n\n"
<< "path: " << argv[1] << "\n"
<< "median: " << libInfo.median << "\n"
<< "standard deviation: " << libInfo.stdDev << "\n"
<< "maximum normal insert size: " << libInfo.maxNormalISize << "\n";
std::cout << "orientation: ";
switch (libInfo.defaultOrient)
{
case LibraryInfo::F_PLUS:
std::cout << "F+ R1 ---> ---> R2\n";
break;
case LibraryInfo::F_MINUS:
std::cout << "F- R1 ---> ---> R2\n";
break;
case LibraryInfo::R_PLUS:
std::cout << "R+ R1 ---> <--- R2\n";
break;
case LibraryInfo::R_MINUS:
std::cout << "R- R1 <--- ---> R2\n";
break;
}
return 0;
}
#else
int main(int, char const **)
{
std::cerr << "bam_library_size can only be compiled correctly with zlib." << std::endl;
return 0;
}
#endif // #if SEQAN_HAS_ZLIB
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