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#include <iostream>
#include <seqan/store.h>
#include <seqan/arg_parse.h>
#include <seqan/misc/interval_tree.h>
#include <seqan/parallel.h>
using namespace seqan2;
// define used types
typedef FragmentStore<> TStore;
typedef Value<TStore::TAnnotationStore>::Type TAnnotation;
typedef TAnnotation::TId TId;
typedef TAnnotation::TPos TPos;
typedef IntervalAndCargo<TPos, TId> TInterval;
typedef IntervalTree<TPos, TId> TIntervalTree;
typedef Value<TStore::TAlignedReadStore>::Type TAlignedRead;
//
// 1. Load annotations and alignments from files
//
bool loadFiles(TStore & store, std::string const & annotationFileName, std::string const & alignmentFileName)
{
BamFileIn alignmentFile;
if (!open(alignmentFile, alignmentFileName.c_str()))
{
std::cerr << "Couldn't open alignment file " << alignmentFileName << std::endl;
return false;
}
std::cout << "Loading read alignments ..... " << std::flush;
readRecords(store, alignmentFile);
std::cout << "[" << length(store.alignedReadStore) << "]" << std::endl;
// load annotations
GffFileIn annotationFile;
if (!open(annotationFile, toCString(annotationFileName)))
{
std::cerr << "Couldn't open annotation file" << annotationFileName << std::endl;
return false;
}
std::cout << "Loading genome annotation ... " << std::flush;
readRecords(store, annotationFile);
std::cout << "[" << length(store.annotationStore) << "]" << std::endl;
return true;
}
//
// 2. Extract intervals from gene annotations (grouped by contigId)
//
void extractGeneIntervals(String<String<TInterval> > & intervals, TStore const & store)
{
// extract intervals from gene annotations (grouped by contigId)
resize(intervals, length(store.contigStore));
Iterator<TStore const, AnnotationTree<> >::Type it = begin(store, AnnotationTree<>());
if (!goDown(it))
return;
do
{
SEQAN_ASSERT_EQ(getType(it), "gene");
TPos beginPos = getAnnotation(it).beginPos;
TPos endPos = getAnnotation(it).endPos;
TId contigId = getAnnotation(it).contigId;
if (beginPos > endPos)
std::swap(beginPos, endPos);
// insert forward-strand interval of the gene and its annotation id
appendValue(intervals[contigId], TInterval(beginPos, endPos, value(it)));
}
while (goRight(it));
}
//
// 3. Construct interval trees
//
void constructIntervalTrees(String<TIntervalTree> & intervalTrees,
String<String<TInterval> > & intervals)
{
int numContigs = length(intervals);
resize(intervalTrees, numContigs);
SEQAN_OMP_PRAGMA(parallel for)
for (int i = 0; i < numContigs; ++i)
createIntervalTree(intervalTrees[i], intervals[i]);
}
//
// 4. Count reads per gene
//
void countReadsPerGene(String<unsigned> & readsPerGene, String<TIntervalTree> const & intervalTrees, TStore const & store)
{
resize(readsPerGene, length(store.annotationStore), 0);
String<TId> result;
int numAlignments = length(store.alignedReadStore);
// iterate aligned reads and get search their begin and end positions
SEQAN_OMP_PRAGMA(parallel for private (result))
for (int i = 0; i < numAlignments; ++i)
{
TAlignedRead const & ar = store.alignedReadStore[i];
TPos queryBegin = _min(ar.beginPos, ar.endPos);
TPos queryEnd = _max(ar.beginPos, ar.endPos);
// search read-overlapping genes
findIntervals(result, intervalTrees[ar.contigId], queryBegin, queryEnd);
// increase read counter for each overlapping annotation given the id in the interval tree
for (unsigned j = 0; j < length(result); ++j)
{
SEQAN_OMP_PRAGMA(atomic)
readsPerGene[result[j]] += 1;
}
}
}
//![solution]
//
// 5. Output RPKM values
//
void outputGeneCoverage(String<unsigned> const & readsPerGene, TStore const & store)
{
// output abundances for covered genes
Iterator<TStore const, AnnotationTree<> >::Type transIt = begin(store, AnnotationTree<>());
Iterator<TStore const, AnnotationTree<> >::Type exonIt;
double millionMappedReads = length(store.alignedReadStore) / 1000000.0;
std::cout << "#gene name\tRPKM value" << std::endl;
for (unsigned j = 0; j < length(readsPerGene); ++j)
{
if (readsPerGene[j] == 0)
continue;
unsigned mRNALengthMax = 0;
goTo(transIt, j);
// determine maximal mRNA length (which we use as gene length)
SEQAN_ASSERT_NOT(isLeaf(transIt));
goDown(transIt);
do
{
exonIt = nodeDown(transIt);
unsigned mRNALength = 0;
// determine mRNA length, sum up the lengths of its exons
do
{
if (getAnnotation(exonIt).typeId == store.ANNO_EXON)
mRNALength += abs((int)getAnnotation(exonIt).beginPos - (int)getAnnotation(exonIt).endPos);
}
while (goRight(exonIt));
if (mRNALengthMax < mRNALength)
mRNALengthMax = mRNALength;
}
while (goRight(transIt));
// RPKM is number of reads mapped to a gene divided by its gene length in kbps
// and divided by millions of total mapped reads
std::cout << store.annotationNameStore[j] << '\t';
std::cout << readsPerGene[j] / (mRNALengthMax / 1000.0) / millionMappedReads << std::endl;
}
}
//![solution]
//![main]
int main(int, char const **)
{
TStore store;
String<String<TInterval> > intervals;
String<TIntervalTree> intervalTrees;
String<unsigned> readsPerGene;
std::string annotationFileName = getAbsolutePath("demos/tutorial/simple_rna_seq/example.gtf");
std::string alignmentFileName = getAbsolutePath("demos/tutorial/simple_rna_seq/example.sam");
if (!loadFiles(store, annotationFileName, alignmentFileName))
return 1;
extractGeneIntervals(intervals, store);
constructIntervalTrees(intervalTrees, intervals);
countReadsPerGene(readsPerGene, intervalTrees, store);
outputGeneCoverage(readsPerGene, store);
return 0;
}
//![main]
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