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#include <cctype>
#include <iostream>
#include <map>
#include <random>
#include <seqan/basic.h>
#include <seqan/align.h>
#include <seqan/graph_align.h>
#include <seqan/sequence.h>
#include <seqan/stream.h>
#include <seqan/seq_io.h>
#include <seqan/bam_io.h>
#include <seqan/arg_parse.h>
#include <seqan/parallel.h>
// Data structure for options.
struct Options
{
// Verbosity: 0 -- quiet, 1 -- normal, 2 -- verbose, 3 -- very verbose.
int verbosity;
// Number of threads to use.
int numThreads;
// Number of elements in one chunk
unsigned chunkSize;
// Path to genome.
seqan2::CharString pathGenome;
// Path to pre-correction SAM file.
seqan2::CharString pathSamPreCorrection;
// Path to post-correction SAM file.
seqan2::CharString pathSamPostCorrection;
// Path to post-correction FASTA/FASTQ file.
seqan2::CharString pathFastaFastqPostCorrection;
// Path to list with fixed and introduced errors.
seqan2::CharString pathCorrectionLog;
// Whether to log all alignments.
bool logAll;
// Minimal number of unclipped bases that a read must have such that it is not ignored. 0 to disable clipping.
int minUnclippedBases;
// Maximal error rate in initial alignment such that the read is not ignored. 0 to disable ignoring.
int maxErrorRate;
// Maximal number of errors in initial alignment. Set to -1 to disable. See also maxErrorRate.
int maxErrorCount;
// Whether or not to allow indels.
bool indels;
// Additional characters to take out of genome left and right of alignment, in percent of the original read length.
int padding;
// Bandwidth to use for alignment.
int bandwidth;
// Check sorting.
bool checkSorting;
// Read only the first maxChunks chunks (default, maxValue<uint64_t>())
uint64_t maxChunks;
Options() :
verbosity(1), numThreads(1), chunkSize(0), logAll(false), minUnclippedBases(0), maxErrorRate(0),
maxErrorCount(0), indels(true), padding(5), bandwidth(2 * padding), checkSorting(true),
maxChunks(0)
{}
};
// Less-than string comparison from samtools.
static inline int strnum_cmp(const char *a, const char *b)
{
char *pa, *pb;
pa = (char*)a; pb = (char*)b;
while (*pa && *pb) {
if (isdigit(*pa) && isdigit(*pb)) {
long ai, bi;
ai = strtol(pa, &pa, 10);
bi = strtol(pb, &pb, 10);
if (ai != bi) return ai<bi? -1 : ai>bi? 1 : 0;
} else {
if (*pa != *pb) break;
++pa; ++pb;
}
}
if (*pa == *pb)
return (pa-a) < (pb-b)? -1 : (pa-a) > (pb-b)? 1 : 0;
return *pa<*pb? -1 : *pa>*pb? 1 : 0;
}
// Data structure to collect statistics in.
struct Stats
{
// Number of unmapped records before, after, and both before and after correction.
uint64_t numUnmappedPre;
uint64_t numUnmappedPost;
uint64_t numIgnoredPre;
// Number of alignment pairs that were evaluated.
uint64_t numTotal;
// Number of bases aligned pre-correction/post-correction.
uint64_t numBasesPre;
uint64_t numBasesPost;
// Number of errors pre-correction/post-correction.
uint64_t numErrorsPre;
uint64_t numErrorsPost;
// Number of erroneous reads pre-correction/post-correction.
uint64_t numErrorReadsPre;
uint64_t numErrorReadsPost;
uint64_t numReads;
// Usual TP/FP/FN values.
uint64_t tp, fp, tn, fn;
// Histogram of differences (before/after correction).
std::map<int, unsigned> histo;
// Histogram of read error rate distribution in percent.
std::map<int, unsigned> preErrorHisto;
// Histogram of errors at a certain position.
typedef seqan2::Tuple<unsigned, 4> TPositionalCounts;
TPositionalCounts zeroCounts;
seqan2::String<TPositionalCounts> preErrorsAtPos; // 0..mismatch, 1..insert, 2..deletion in the read compared to reference
seqan2::String<TPositionalCounts> postErrorsAtPos; // 3..total number of reads having a base at that position (not cropped)
// We can later compute the gain from actualErrorSum / diffErrorSum.
//
// Summed up edit distance errors in the pre-correction alignment.
int64_t actualErrorSum;
// Summed up difference between pre-correction and post-correction errors.
int64_t diffErrorSum;
// Number of introduced and removed errors.
uint64_t numErrorsIntroduced;
uint64_t numErrorsRemoved;
// Reusable data structures
seqan2::BamAlignmentRecord _recordPre;
seqan2::BamAlignmentRecord _recordPost;
seqan2::String<seqan2::BamAlignmentRecord> _chunkPre;
seqan2::String<seqan2::Dna5String> _chunkPost;
seqan2::Dna5String _genomeInfixPre;
seqan2::Dna5String _genomeInfixPost;
seqan2::Dna5String _preRead;
seqan2::Dna5String _postRead;
typedef seqan2::Align<seqan2::Dna5String> TAlign;
TAlign _preAlign;
TAlign _postAlign;
Stats() : numUnmappedPre(0), numUnmappedPost(0), numIgnoredPre(0), numTotal(0),
numBasesPre(0), numBasesPost(0), numErrorsPre(0), numErrorsPost(0), numErrorReadsPre(0),
numErrorReadsPost(0), numReads(0), tp(0), fp(0), tn(0), fn(0), actualErrorSum(0), diffErrorSum(0),
numErrorsIntroduced(0), numErrorsRemoved(0)
{
zeroCounts[0] = 0;
zeroCounts[1] = 0;
zeroCounts[2] = 0;
zeroCounts[3] = 0;
}
};
typedef seqan2::StringSet<seqan2::CharString> TNameStore;
typedef seqan2::NameStoreCache<TNameStore> TNameStoreCache;
// Error in alignment.
struct AlignmentError
{
int pos; // position in genome
int offset; // offset in case of long gaps in genome
char genomeBase; // base in genome
char readBase; // base in the read
AlignmentError() : pos(-1), offset(-1), genomeBase('X'), readBase('X') {}
AlignmentError(int pos, int offset, char genomeBase, char readBase) :
pos(pos), offset(offset), genomeBase(genomeBase), readBase(readBase)
{}
bool operator<(AlignmentError const & other) const
{
return ((pos < other.pos) ||
(pos == other.pos && offset < other.offset) ||
(pos == other.pos && offset == other.offset && genomeBase < other.genomeBase) ||
(pos == other.pos && offset == other.offset && genomeBase == other.genomeBase &&
readBase < other.readBase));
}
};
std::ostream & operator<<(std::ostream & out, AlignmentError const & ae)
{
return out << "AlignmentError(pos=" << ae.pos << ", offset=" << ae.offset << ", genome=" << ae.genomeBase
<< ", read=" << ae.readBase << ")";
}
void computeErrors(std::vector<AlignmentError> & result,
seqan2::Align<seqan2::Dna5String> const & align,
int genomeOffset)
{
using namespace seqan2;
result.clear();
int leadingGaps = countGaps(begin(row(align, 1), Standard()));
int trailingGaps = 0;
while (trailingGaps < (int)length(row(align, 1)) && isGap(row(align, 1), length(row(align, 1)) - trailingGaps - 1))
++trailingGaps;
typedef Iterator<Row<Align<Dna5String> const>::Type, Standard>::Type TIterator;
TIterator itC = iter(row(align, 0), leadingGaps, Standard()); // contig
TIterator itCEnd = iter(row(align, 0), length(row(align, 0)) - trailingGaps, Standard()); // contig
TIterator itR = iter(row(align, 1), leadingGaps, Standard()); // read
for (int pos = genomeOffset, offset = 0; itC != itCEnd; ++itC, ++itR)
if (isGap(itC) && !isGap(itR))
{
result.push_back(AlignmentError(pos, offset, '-', convert<char>(*itR)));
++offset;
}
else if (!isGap(itC) && isGap(itR))
{
result.push_back(AlignmentError(pos, offset, convert<char>(*itC), '-'));
++pos;
offset = 0;
}
else if (!isGap(itC) && !isGap(itR))
{
if (convert<Dna5>(*itC) != convert<Dna5>(*itR))
result.push_back(AlignmentError(pos, offset, convert<char>(*itC), convert<char>(*itR)));
++pos;
offset = 0;
}
}
int countTrueNegatives(std::vector<AlignmentError> const & errorsPre,
std::vector<AlignmentError> const & errorsPost,
seqan2::Align<seqan2::Dna5String> const & alignPre,
int genomeOffsetPre)
{
using namespace seqan2;
int leadingGaps = countGaps(begin(row(alignPre, 1), Standard()));
int trailingGaps = 0;
while (trailingGaps < (int)length(row(alignPre, 1)) && isGap(row(alignPre, 1), length(row(alignPre, 1)) - trailingGaps - 1))
++trailingGaps;
int offset = genomeOffsetPre + leadingGaps;
// Compute at which positions there was no error before and after.
std::vector<bool> noError(length(row(alignPre, 0)) - leadingGaps - trailingGaps, false);
for (std::vector<AlignmentError>::const_iterator it = errorsPre.begin(); it != errorsPre.end(); ++it)
if (it->pos >= offset && it->pos - offset < (int)noError.size())
noError.at(it->pos - offset) = false;
for (std::vector<AlignmentError>::const_iterator it = errorsPost.begin(); it != errorsPost.end(); ++it)
if (it->pos >= offset && it->pos - offset < (int)noError.size())
noError.at(it->pos - offset) = false;
int result = 0;
for (std::vector<bool>::const_iterator it = noError.begin(); it != noError.end(); ++it)
result += !*it;
return result;
}
// Allows the trimming of strings after the first whitespace.
void trimSeqHeaderToId(seqan2::CharString & header)
{
unsigned i = 0;
for (; i < length(header); ++i)
if (isspace(header[i]))
break;
resize(header, i);
}
// Returns "yes"/"no" for a bool.
char const * getYesNo(bool b)
{
return b ? "YES" : "NO";
}
// Computes gain between two SAM records and updates Stats object.
void updateStats(Stats & stats,
std::ofstream & correctionLog,
seqan2::BamAlignmentRecord const & preRecord,
seqan2::Dna5String & postRead, // rc-ed to same as preRecord.
seqan2::String<unsigned> const & idMap,
seqan2::StringSet<seqan2::Dna5String> const & seqs,
Options const & options)
{
using namespace seqan2;
stats.numUnmappedPre += hasFlagUnmapped(preRecord);
if (hasFlagUnmapped(preRecord))
return; // Ignore.
stats.numTotal += 1;
stats.numReads += 1;
// In the following, we compute scores with globalAlignment() using edit distance and convert them into distances by
// computing -score.
unsigned seqIdx = idMap[preRecord.rID];
// Get additional shifts in genomic position if there is soft clipping.
int beginShift = 0, endShift = 0;
if (!empty(preRecord.cigar) && front(preRecord.cigar).operation == 'S')
beginShift = front(preRecord.cigar).count;
if (length(preRecord.cigar) > 1u && back(preRecord.cigar).operation == 'S')
endShift = back(preRecord.cigar).count;
if (options.minUnclippedBases != 0 && ((int)length(preRecord.seq) - (beginShift + endShift) < options.minUnclippedBases))
{
stats.numIgnoredPre += 1;
return; // Ignore, too few unclipped bases.
}
// Compute begin and positions of genome infix, with padding, but not going over the end of the genome.
unsigned beginPos = preRecord.beginPos;
unsigned endPos = beginPos + length(preRecord.seq);
unsigned beginPosPre = beginPos, beginPosPost = beginPos;
unsigned endPosPre = endPos, endPosPost = endPos;
Dna5String & preRead = stats._preRead;
preRead = preRecord.seq;
// speed-up heuristic
bool skipAlignPre = (infix(seqs[seqIdx], beginPos, endPos) == preRead);
bool skipAlignPost = (infix(seqs[seqIdx], beginPos, endPos) == postRead);
int padding = (int)ceil(0.01 * options.padding * length(preRecord.seq));
if (options.indels)
{
if ((int)beginPos > padding + beginShift)
beginPos -= (padding + beginShift);
else
beginPos = 0;
endPos = preRecord.beginPos + getAlignmentLengthInRef(preRecord);
endPos += (padding + endShift);
if (endPos > length(seqs[seqIdx]))
endPos = length(seqs[seqIdx]);
if (!skipAlignPre)
{
beginPosPre = beginPos;
endPosPre = endPos;
}
if (!skipAlignPost)
{
beginPosPost = beginPos;
endPosPost = endPos;
}
}
// Get genome infix, and convert read seqs (CharString) into Dna5Strings.
Dna5String & genomeInfixPre = stats._genomeInfixPre;
genomeInfixPre = infix(seqs[seqIdx], beginPosPre, endPosPre);
Dna5String & genomeInfixPost = stats._genomeInfixPost;
genomeInfixPost = infix(seqs[seqIdx], beginPosPost, endPosPost);
if (hasFlagRC(preRecord))
{
// bring sequences to original read direction
reverseComplement(genomeInfixPre);
reverseComplement(genomeInfixPost);
reverseComplement(preRead);
reverseComplement(postRead);
}
// else
// {
// std::cout << preRead << std::endl;
// std::cout << postRead << std::endl;
// }
// Create alignment objects and AlignConfig object.
typedef Align<Dna5String> TAlign;
TAlign & preAlign = stats._preAlign;
TAlign & postAlign = stats._postAlign;
resize(rows(preAlign), 2);
setSource(row(preAlign, 0), genomeInfixPre);
setSource(row(preAlign, 1), preRead);
resize(rows(postAlign), 2);
setSource(row(postAlign, 0), genomeInfixPost);
setSource(row(postAlign, 1), postRead);
// Compute distance before and after correction.
int diffPre = 0;
int diffPost = 0;
// Sorted vectors (i.e. sets) of alignment errors computed for FP/TP rate.
std::vector<AlignmentError> errorsPre, errorsPost, errorsTmp;
if (options.indels)
{
// In the case of indels, we have to perform an edit distance alignment.
AlignConfig<true, false, false, true> alignConfig;
Score<int, Simple> scoringScheme(0, -1000, -1001);
/////////
// PRE //
/////////
// Align pre record to genome.
if (!skipAlignPre)
diffPre = -globalAlignment(preAlign, scoringScheme, alignConfig, NeedlemanWunsch()) / 1000;
computeErrors(errorsPre, preAlign, beginPosPre);
int diffPreRate = (int)ceil(100.0 * diffPre / length(preRead));
if ((options.maxErrorRate != 0 && diffPreRate > options.maxErrorRate) ||
(options.maxErrorCount != -1 && diffPre > options.maxErrorCount))
{
stats.numIgnoredPre += 1;
return; // Ignore alignment with too many errors.
}
stats.preErrorHisto[diffPreRate] += 1;
// update positional error counts PRE
resize(stats.preErrorsAtPos, std::max(length(stats.preErrorsAtPos), length(preRead)), stats.zeroCounts);
if (diffPre != 0)
{
typedef Row<TAlign>::Type TRow;
typedef Iterator<TRow, Standard>::Type TGapsIter;
int viewBegin = toViewPosition(row(preAlign, 1), 0);
int viewEnd = toViewPosition(row(preAlign, 1), length(preRead) - 1) + 1;
TGapsIter genomeIter = begin(row(preAlign, 0), Standard()) + viewBegin;
TGapsIter readIter = begin(row(preAlign, 1), Standard()) + viewBegin;
unsigned readPos = 0;
for (int i = viewBegin; i < viewEnd; ++i, goNext(genomeIter), goNext(readIter))
{
// errro types: 0..mismatch, 1..insert, 2..deletion in the read compared to reference
if (isGap(genomeIter))
{
++stats.preErrorsAtPos[readPos][1];
}
else if (isGap(readIter))
{
++stats.preErrorsAtPos[readPos][2];
continue; // don't increment readPos here
}
else if (*genomeIter != *readIter)
{
++stats.preErrorsAtPos[readPos][0];
}
// increment number of total bases
++stats.preErrorsAtPos[readPos][3];
++readPos;
}
SEQAN_CHECK(atEnd(genomeIter) == atEnd(readIter), "Invalid pairwise alignment!");
}
else
{
// increment at least number of total bases (in case of no error)
for (unsigned i = 0; i < length(preRead); ++i)
++stats.preErrorsAtPos[i][3];
}
//////////
// POST //
//////////
// Align post record to genome.
if (!skipAlignPost)
diffPost = -globalAlignment(postAlign, scoringScheme, alignConfig, NeedlemanWunsch()) / 1000;
computeErrors(errorsPost, postAlign, beginPosPost);
uint64_t tp = 0, fp = 0, tn = 0, fn = 0;
errorsTmp.clear();
std::set_difference(errorsPre.begin(), errorsPre.end(), errorsPost.begin(), errorsPost.end(),
std::back_inserter(errorsTmp));
tp += errorsTmp.size();
errorsTmp.clear();
std::set_difference(errorsPost.begin(), errorsPost.end(), errorsPre.begin(), errorsPre.end(),
std::back_inserter(errorsTmp));
fp += errorsTmp.size();
tn += countTrueNegatives(errorsPre, errorsPost, preAlign, beginPosPre);
errorsTmp.clear();
std::set_intersection(errorsPost.begin(), errorsPost.end(), errorsPre.begin(), errorsPre.end(),
std::back_inserter(errorsTmp));
fn += errorsTmp.size();
stats.tp += tp;
stats.fp += fp;
stats.tn += tn;
stats.fn += fn;
if (options.verbosity >= 3 || (options.verbosity >= 2 && (abs(diffPre) > 10 || abs(diffPost) > 10)))
{
std::cerr << "RECORD: " << preRecord.qName << "\n"
<< "BEFORE, score == " << diffPre << "\n" << preAlign << '\n';
std::cerr << "Alignment Errors PRE\n";
std::copy(errorsPre.begin(), errorsPre.end(), std::ostream_iterator<AlignmentError>(std::cerr, "\n"));
std::cerr << "---\n";
std::cerr << "AFTER, score == " << diffPost << "\n" << postAlign << '\n';
std::cerr << "Alignment Errors POST\n";
std::copy(errorsPost.begin(), errorsPost.end(), std::ostream_iterator<AlignmentError>(std::cerr, "\n"));
std::cerr << "---\n";
std::cerr << " --> BEFORE - AFTER == " << diffPre - diffPost << '\n'
<< "TP = " << tp << ", FP = " << fp << ", TN = " << tn << ", FN = " << fn << '\n';
}
if (correctionLog.is_open() && (diffPre != diffPost || options.logAll))
{
correctionLog << "RECORD: " << preRecord.qName << "\n"
<< "\n"
<< "BEFORE score = " << diffPre << "\n"
<< preAlign << "\n"
<< "\n"
<< "AFTER score = " << diffPost << "\n"
<< postAlign << "\n"
<< "------------------------------------------------------------------------------\n";
}
// update positional error counts POST
resize(stats.postErrorsAtPos, std::max(length(stats.postErrorsAtPos), length(postRead)), stats.zeroCounts);
if (diffPost != 0)
{
typedef Row<TAlign>::Type TRow;
typedef Iterator<TRow, Standard>::Type TGapsIter;
int viewBegin = toViewPosition(row(postAlign, 1), 0);
int viewEnd = toViewPosition(row(postAlign, 1), length(postRead) - 1) + 1;
TGapsIter genomeIter = begin(row(postAlign, 0), Standard()) + viewBegin;
TGapsIter readIter = begin(row(postAlign, 1), Standard()) + viewBegin;
unsigned readPos = 0;
for (int i = viewBegin; i < viewEnd; ++i, goNext(genomeIter), goNext(readIter))
{
// errro types: 0..mismatch, 1..insert, 2..deletion in the read compared to reference
if (isGap(genomeIter))
{
++stats.postErrorsAtPos[readPos][1];
}
else if (isGap(readIter))
{
++stats.postErrorsAtPos[readPos][2];
continue; // don't increment readPos here
}
else if (*genomeIter != *readIter)
{
++stats.postErrorsAtPos[readPos][0];
}
// increment number of total bases
++stats.postErrorsAtPos[readPos][3];
++readPos;
}
SEQAN_CHECK(atEnd(genomeIter) == atEnd(readIter), "Invalid pairwise alignment!");
}
else
{
// increment at least number of total bases (in case of no error)
for (unsigned i = 0; i < length(postRead); ++i)
++stats.postErrorsAtPos[i][3];
}
}
else
{
// In the case of Hamming distance, we can simply count matching and mismatching bases.
if (options.verbosity >= 3)
std::cerr << "infix == " << genomeInfixPre << "\tpre == " << preRead << "\tpost == " << postRead << '\n';
//SEQAN_CHECK(length(genomeInfixPre) >= length(preRead), "Must have geq length with Hamming distance.");
//SEQAN_CHECK(length(genomeInfixPre) >= length(postRead), "Must have geq length with Hamming distance.");
//SEQAN_CHECK(length(preRead) >= length(postRead), "Must have the same length with Hamming distance.");
// For Hamming distance, we compare at most m characters where m is the smaller one of the
// lengths of postRead and preRead.
unsigned minLen = std::min(length(postRead), length(preRead));
std::string flags;
resize(stats.preErrorsAtPos, std::max(length(stats.preErrorsAtPos), length(preRead)), stats.zeroCounts);
resize(stats.postErrorsAtPos, std::max(length(stats.postErrorsAtPos), length(postRead)), stats.zeroCounts);
unsigned numIntroduced = 0;
unsigned numRemoved = 0;
for (unsigned i = 0; i < minLen; ++i)
{
bool badPre = (genomeInfixPre[i] != preRead[i]);
bool badPost = (genomeInfixPre[i] != postRead[i]);
if (badPre && !badPost)
{
flags.push_back('-');
numRemoved += 1;
}
else if (!badPre && badPost)
{
flags.push_back('+');
numIntroduced += 1;
}
else
{
flags.push_back(' ');
}
stats.preErrorsAtPos[i][0] += badPre;
stats.postErrorsAtPos[i][0] += badPost;
++stats.preErrorsAtPos[i][3];
++stats.postErrorsAtPos[i][3];
diffPre += badPre;
diffPost += badPost;
}
// If preRead is longer than postRead then matches between pre read and genome are counted as introduced
// errors. Mismatches are counted as removed errors.
for (unsigned i = minLen; i < length(preRead); ++i)
{
if (genomeInfixPre[i] == preRead[i])
{
diffPost += 1;
numIntroduced += 1;
}
else
{
diffPre += 1;
numRemoved += 1;
++stats.preErrorsAtPos[i][0];
}
++stats.preErrorsAtPos[i][3];
}
// If postRead is longer than preRead then matches between the post read are counted as removed errors
// and mismatches are counted as removed errors. If the read reaches over the genome then this is counted
// as mismatches.
for (unsigned i = minLen; i < length(postRead); ++i)
{
if (beginPos + i > length(seqs[seqIdx]))
{
diffPost += length(postRead) - i;
numIntroduced += length(postRead) - i;
break; // End of genome.
}
if (genomeInfixPre[i] == postRead[i])
{
diffPre += 1;
numRemoved += 1;
}
else
{
diffPost += 1;
numIntroduced += 1;
++stats.postErrorsAtPos[i][0];
}
++stats.postErrorsAtPos[i][3];
}
// Update histogram of error
int diffPreRate = (int)ceil(100.0 * diffPre / length(preRead));
stats.preErrorHisto[diffPreRate] += 1;
if (options.verbosity >= 3 || (options.verbosity >= 2 && (abs(diffPre) > 10 || abs(diffPost) > 10)))
std::cerr << "RECORD: " << preRecord.qName << "\n"
<< "GENOME (PRE) " << genomeInfixPre << "\n"
<< "GENOME (POST) " << genomeInfixPost << "\n"
<< "BEFORE " << preRead << "\tscore == " << diffPre << '\n'
<< "AFTER " << postRead << "\tscore == " << diffPost << '\n'
<< "REMOVED " << numRemoved << "\n"
<< "INTRODUCED " << numIntroduced << "\n"
<< " --> BEFORE - AFTER == " << diffPre - diffPost << '\n';
if (correctionLog.good() && diffPre != diffPost)
{
correctionLog << "RECORD: " << preRecord.qName << "\n"
<< "INTRODUCED " << numIntroduced << "\tREMOVED\t" << numRemoved << "\n"
<< "\n"
<< "BEFORE\n"
<< genomeInfixPre << "\n";
for (unsigned i = 0; i < minLen; ++i)
correctionLog << ((genomeInfixPre[i] == preRead[i]) ? '|' : ' ');
correctionLog << "\n"
<< preRead << "\n\n"
<< "AFTER\n"
<< genomeInfixPost << "\n";
for (unsigned i = 0; i < minLen; ++i)
correctionLog << ((genomeInfixPost[i] == postRead[i]) ? '|' : ' ');
correctionLog << "\n"
<< postRead << "\n"
<< flags << "\n"
<< "==============================================================================\n";
}
stats.numErrorsRemoved += numRemoved;
stats.numErrorsIntroduced += numIntroduced;
}
stats.numBasesPre += length(preRead);
stats.numBasesPost += length(postRead);
stats.numErrorsPre += diffPre;
stats.numErrorsPost += diffPost;
stats.numErrorReadsPre += (diffPre != 0);
stats.numErrorReadsPost += (diffPost != 0);
SEQAN_CHECK(diffPre >= 0, "Edit distance must be >= 0!");
SEQAN_CHECK(diffPost >= 0, "Edit distance must be >= 0!");
stats.actualErrorSum += diffPre;
stats.diffErrorSum += diffPre - diffPost;
stats.histo[diffPre - diffPost] += 1;
}
seqan2::ArgumentParser::ParseResult
parseCommandLine(Options & options, int argc, char const ** argv)
{
// Setup ArgumentParser.
seqan2::ArgumentParser parser("compute_gain");
setShortDescription(parser, "Compute read correction metric GAIN.");
setCategory(parser, "Error Correction");
setVersion(parser, "0.2");
setDate(parser, "August 2012");
addUsageLine(parser,
"[\\fIOPTIONS\\fP] \\fB-g\\fP GENOME.fa \\fB--pre\\fP \\fIPRE.{sam,bam}\\fP \\fB--post\\fP "
"\\fIPOST.sam\\fP");
addDescription(parser,
"This program computes the read correction tool metric GAIN. It takes a genome FASTA file and "
"two SAM file with read alignments before and after correction. It then computes various "
"statistics and computes the GAIN, based on the edit or Hamming distance for reach read before "
"and after correction.");
addOption(parser, seqan2::ArgParseOption("q", "quiet", "Disable most output."));
addOption(parser, seqan2::ArgParseOption("v", "verbose", "Enable more verbose output."));
addOption(parser, seqan2::ArgParseOption("vv", "very-verbose", "Enable even more verbose output."));
#ifdef _OPENMP
addOption(parser, seqan2::ArgParseOption("nt", "num-threads", "Number of threads to use.", seqan2::ArgParseOption::INTEGER, "THREADS"));
setDefaultValue(parser, "num-threads", options.numThreads);
setMinValue(parser, "num-threads", "1");
#endif
addOption(parser, seqan2::ArgParseOption("", "chunk-size", "Chunk size.", seqan2::ArgParseOption::INTEGER, "THREADS"));
setDefaultValue(parser, "chunk-size", "10000");
setMinValue(parser, "chunk-size", "100");
addOption(parser, seqan2::ArgParseOption("", "max-chunks", "Maximal number of chunks to read (0=disabled).", seqan2::ArgParseOption::INTEGER));
setMinValue(parser, "max-chunks", "0");
setDefaultValue(parser, "max-chunks", "0");
// Argument Section -- Comparison
//
addSection(parser, "Comparison");
addOption(parser, seqan2::ArgParseOption("", "padding", "Additional genome characters to use for alignment in percent of the origina read length.", seqan2::ArgParseOption::INTEGER, "PADDING"));
setDefaultValue(parser, "padding", 5);
setMinValue(parser, "padding", "0");
addOption(parser, seqan2::ArgParseOption("", "bandwidth", "Bandwidth to use for alignment.", seqan2::ArgParseOption::INTEGER, "BAND"));
setDefaultValue(parser, "bandwidth", 10);
setMinValue(parser, "bandwidth", "1");
hideOption(parser, "bandwidth");
addOption(parser, seqan2::ArgParseOption("", "metric", "The metric type to use.", seqan2::ArgParseOption::STRING,
"METRIC"));
setDefaultValue(parser, "metric", "edit");
setValidValues(parser, "metric", "hamming edit");
// Argument Section -- Filtration
addSection(parser, "Input / Output");
addOption(parser, seqan2::ArgParseOption("", "min-unclipped-bases", "Reads with fewer unclipped bases are ignored. "
"Set to 0 to disable ignoring because of this.",
seqan2::ArgParseOption::INTEGER, "NUM"));
setDefaultValue(parser, "min-unclipped-bases", "0");
setMinValue(parser, "min-unclipped-bases", "0");
addOption(parser, seqan2::ArgParseOption("", "max-error-rate", "Reads with a higher error rate in the initial mapping "
"are ignored. Given in percent. Set to 0 to disable ignoring because of "
"this.", seqan2::ArgParseOption::INTEGER, "NUM"));
setDefaultValue(parser, "max-error-rate", "0");
setMinValue(parser, "max-error-rate", "0");
addOption(parser, seqan2::ArgParseOption("", "max-error-count", "Reads with a higher error count in the initial mapping "
"are ignored. Given in percent. Set to -1 to disable ignoring because of "
"this.", seqan2::ArgParseOption::INTEGER, "NUM"));
setDefaultValue(parser, "max-error-count", "-1");
setMinValue(parser, "max-error-count", "-1");
// Argument Section -- Input / Output
addSection(parser, "Input / Output");
addOption(parser, seqan2::ArgParseOption("g", "genome", "Genome file.", seqan2::ArgParseOption::INPUT_FILE,
"GENOME.fa"));
setRequired(parser, "genome");
setValidValues(parser, "genome", seqan2::SeqFileIn::getFileExtensions());
addOption(parser, seqan2::ArgParseOption("", "pre", "Pre-correction SAM file.", seqan2::ArgParseOption::INPUT_FILE,
"PRE.{sam,bam}"));
setRequired(parser, "pre");
setValidValues(parser, "pre", "sam bam");
addOption(parser, seqan2::ArgParseOption("", "post-sam", "Post-correction SAM file.", seqan2::ArgParseOption::INPUT_FILE,
"POST.sam"));
setValidValues(parser, "post-sam", "sam");
addOption(parser, seqan2::ArgParseOption("", "post", "Post-correction FASTQ or FASTA file.", seqan2::ArgParseOption::INPUT_FILE,
"POST.fq"));
setValidValues(parser, "post", "fastq fq fastq.gz fq.gz fasta fa fasta.gz fa.gz");
addOption(parser, seqan2::ArgParseOption("", "correction-log", "Write log about introduced/removed errors to this file.",
seqan2::ArgParseOption::OUTPUT_FILE, "OUT.txt"));
addOption(parser, seqan2::ArgParseOption("", "log-all", "Log all not only introduced/removed errors."));
addOption(parser, seqan2::ArgParseOption("", "no-check-sorting", "No checking for reads being sorted."));
// Parse command line.
seqan2::ArgumentParser::ParseResult res = seqan2::parse(parser, argc, argv);
// Only extract options if the program will continue after parseCommandLine()
if (res != seqan2::ArgumentParser::PARSE_OK)
return res;
// Extract option values.
if (isSet(parser, "quiet"))
options.verbosity = 0;
else if (isSet(parser, "verbose"))
options.verbosity = 2;
else if (isSet(parser, "very-verbose"))
options.verbosity = 3;
if (!isSet(parser, "post-sam") && !isSet(parser, "post"))
{
std::cerr << "ERROR: Neither --post-sam nor --post was set!\n";
return seqan2::ArgumentParser::PARSE_ERROR;
}
#ifdef _OPENMP
getOptionValue(options.numThreads, parser, "num-threads");
#endif
getOptionValue(options.chunkSize, parser, "chunk-size");
getOptionValue(options.pathGenome, parser, "genome");
getOptionValue(options.pathSamPreCorrection, parser, "pre");
getOptionValue(options.pathSamPostCorrection, parser, "post-sam");
getOptionValue(options.pathFastaFastqPostCorrection, parser, "post");
getOptionValue(options.pathCorrectionLog, parser, "correction-log");
getOptionValue(options.minUnclippedBases, parser, "min-unclipped-bases");
getOptionValue(options.maxErrorRate, parser, "max-error-rate");
getOptionValue(options.maxErrorCount, parser, "max-error-count");
getOptionValue(options.maxChunks, parser, "max-chunks");
options.logAll = isSet(parser, "log-all");
seqan2::CharString metricValue;
getOptionValue(metricValue, parser, "metric");
options.indels = (metricValue == "edit");
options.checkSorting = !isSet(parser, "no-check-sorting");
return seqan2::ArgumentParser::PARSE_OK;
}
int main(int argc, char const ** argv)
{
using namespace seqan2;
// -----------------------------------------------------------------------
// Program Initialization
// -----------------------------------------------------------------------
// Checking command line parameters.
Options options;
seqan2::ArgumentParser::ParseResult argParseRes = parseCommandLine(options, argc, argv);
// If parsing was not successful then exit with code 1 if there were errors.
// Otherwise, exit with code 0 (e.g. help was printed).
if (argParseRes != seqan2::ArgumentParser::PARSE_OK)
return argParseRes == seqan2::ArgumentParser::PARSE_ERROR;
// Opening file and record reader.
seqan2::BamFileIn inPre;
if (!open(inPre, toCString(options.pathSamPreCorrection)))
{
std::cerr << "ERROR: Could not open pre-correction file.\n";
return 1;
}
BamHeader header;
try
{
readHeader(header, inPre);
}
catch (seqan2::ParseError const & e)
{
std::cerr << "ERROR: Problem parsing pre SAM/BAM header.\n";
return 1;
}
seqan2::BamFileIn inPostBam(inPre);
seqan2::SeqFileIn inPostFastq;
bool success;
bool postBam = !empty(options.pathSamPostCorrection); // Whether or not to read post SAM.
if (postBam)
success = open(inPostBam, toCString(options.pathSamPostCorrection));
else
success = open(inPostFastq, toCString(options.pathFastaFastqPostCorrection));
if (!success)
{
std::cerr << "ERROR: Could not open post-correction file.\n";
return 1;
}
// Read pre-correction SAM header.
std::cerr << "SAM headers...\n";
// Read post-correction SAM header and forget it immediately. We COULD check for consistency with pre-correction
// header but this tool is for internal use only...
if (postBam)
{
try
{
BamHeader header;
readHeader(header, inPostBam);
}
catch (seqan2::ParseError const & e)
{
std::cerr << "ERROR: Problem parsing post SAM/BAM header\n";
return 1;
}
}
// Read genome and compute mapping from SAM record reference ids to seqs index.
std::cerr << "Read Genome...\n";
seqan2::SeqFileIn inGenome;
if (!open(inGenome, toCString(options.pathGenome)))
{
std::cerr << "ERROR: Could not open genome file\n";
return 1;
}
StringSet<CharString> ids;
StringSet<Dna5String> seqs;
try
{
readRecords(ids, seqs, inGenome);
}
catch (seqan2::ParseError const & e)
{
std::cerr << "ERROR: Problem reading genome file.\n";
return 1;
}
String<unsigned> idMap;
resize(idMap, length(contigNames(context(inPre))), std::numeric_limits<unsigned>::max());
for (unsigned i = 0; i < length(ids); ++i)
{
trimSeqHeaderToId(ids[i]);
unsigned idx = 0;
if (!getIdByName(idx, contigNamesCache(context(inPre)), ids[i]))
{
std::cerr << "Reference " << ids[i] << " not in SAM references!\n";
return 1;
}
idMap[idx] = i;
}
// -----------------------------------------------------------------------
// Open file for correction log.
// -----------------------------------------------------------------------
std::ofstream correctionLog;
if (!empty(options.pathCorrectionLog))
correctionLog.open(toCString(options.pathCorrectionLog));
// -----------------------------------------------------------------------
// Statistics Computation
// -----------------------------------------------------------------------
std::cerr << "Compute Statistics...\n";
seqan2::String<Stats> stats;
resize(stats, options.numThreads);
// Whether or not to break out of the loop below with or without an error, required in this way because we return
// from OpenMP block.
bool stop = false;
bool error = false;
uint64_t chunksLeftToRead = options.maxChunks;
--chunksLeftToRead;
// to reduce the number of threads waiting in front of the critical section
std::mt19937 rng(42);
std::uniform_real_distribution<double> distChunkSizeNoise(options.chunkSize, 2 * options.chunkSize);
SEQAN_OMP_PRAGMA(parallel num_threads(options.numThreads))
while (!stop && !error)
{
int const tid = omp_get_thread_num();
BamAlignmentRecord &recordPre = stats[tid]._recordPre;
BamAlignmentRecord &recordPost = stats[tid]._recordPost;
seqan2::String<BamAlignmentRecord> &chunkPre = stats[tid]._chunkPre;
seqan2::String<seqan2::Dna5String> &chunkPost = stats[tid]._chunkPost;
clear(chunkPre);
clear(chunkPost);
// Read next chunk atomically.
//
// The stop/error variables are only updated in the critical section which implies a flush on all variables
// anyway. Thus there is no need to flush manually again.
SEQAN_OMP_PRAGMA(critical (read_chunk))
{
int const tid = omp_get_thread_num();
unsigned myChunkSize = (unsigned)distChunkSizeNoise(rng);
seqan2::CharString prevName;
seqan2::CharString postId;
clear(recordPre.qName);
clear(recordPost.qName);
while (!stop && !error && !atEnd(inPre))
{
if (postBam)
stop = atEnd(inPostBam);
else
stop = atEnd(inPostFastq);
if (stop)
break;
// Read next record into chunk.
try
{
readRecord(recordPre, inPre);
}
catch (seqan2::IOError const & e)
{
error = true;
continue;
}
if (hasFlagSecondary(recordPre))
continue; // Skip, this happens for bwasw input.
// check sorting
if (options.checkSorting && !empty(prevName) && strnum_cmp(toCString(prevName), toCString(recordPre.qName)) >= 0)
{
std::cerr << "ERROR: Expected sorted by quername but was.\n"
<< "But was: " << recordPre.qName << " >= " << prevName << "\n";
stop = error = false;
}
prevName = recordPre.qName;
// read post-records as long as they are less than the last pre-record
while (!stop && (empty(recordPost.qName) || strnum_cmp(toCString(recordPre.qName), toCString(recordPost.qName)) > 0))
{
try
{
if (postBam)
{
readRecord(recordPost, inPostBam);
stats[tid].numUnmappedPost += hasFlagUnmapped(recordPost);
stop = atEnd(inPostBam);
}
else
{
readRecord(recordPost.qName, recordPost.seq, inPostFastq);
trimSeqHeaderToId(recordPost.qName);
stop = atEnd(inPostFastq);
}
}
catch (seqan2::IOError const & e)
{
error = true;
continue;
}
stats[tid].numUnmappedPre++;
}
// found matching pair of qnames?
if (recordPre.qName == recordPost.qName)
{
appendValue(chunkPre, recordPre);
appendValue(chunkPost, recordPost.seq);
if (postBam)
{
if (hasFlagRC(recordPost) != hasFlagRC(recordPre))
reverseComplement(back(chunkPost)); // transform post-read to the same orientation as pre-read
}
else
{
if (hasFlagRC(recordPre))
reverseComplement(back(chunkPost)); // transform post-read to the same orientation as pre-read
}
stats[tid].numUnmappedPre--; // we overcounted unmapped pre-records by the one that matches the post-record
}
// Break if chunk full.
if (length(chunkPre) > myChunkSize)
break;
}
if (atEnd(inPre) || chunksLeftToRead == 0ul)
stop = true; // Do not read any more, this thread finishes its computation.
else
--chunksLeftToRead;
}
// TODO(holtgrew): Defer RC for post chunk until here with array of flags.
// Process chunk.
for (unsigned i = 0; i < length(chunkPre); ++i)
updateStats(stats[tid], correctionLog, chunkPre[i], chunkPost[i], idMap, seqs, options);
}
if (!atEnd(inPre) || (postBam && !atEnd(inPostBam)) || (!postBam && !atEnd(inPostFastq)))
std::cerr << "WARNING: Files not read completely!\n";
if (error)
{
std::cerr << "An error occurred. Bailing out.\n";
return 1;
}
// Count remaining records, are unmapped/nonpresent in other file.
if (chunksLeftToRead != 0ul)
{
SEQAN_CHECK(atEnd(inPre), "Pre-correction reader must be at end!");
if (postBam)
SEQAN_CHECK(atEnd(inPostBam), "Post-correction reader must be at end!");
else
SEQAN_CHECK(atEnd(inPostFastq), "Post-correction reader must be at end!");
}
// -----------------------------------------------------------------------
// Write Output
// -----------------------------------------------------------------------
// Compute global stats.
Stats globalStats;
for (unsigned i = 0; i < length(stats); ++i)
{
globalStats.numUnmappedPre += stats[i].numUnmappedPre;
globalStats.numIgnoredPre += stats[i].numIgnoredPre;
globalStats.numTotal += stats[i].numTotal;
globalStats.numBasesPre += stats[i].numBasesPre;
globalStats.numBasesPost += stats[i].numBasesPost;
globalStats.numErrorsPre += stats[i].numErrorsPre;
globalStats.numErrorsPost += stats[i].numErrorsPost;
globalStats.numErrorReadsPre += stats[i].numErrorReadsPre;
globalStats.numErrorReadsPost += stats[i].numErrorReadsPost;
globalStats.numReads += stats[i].numReads;
globalStats.actualErrorSum += stats[i].actualErrorSum;
globalStats.diffErrorSum += stats[i].diffErrorSum;
globalStats.numErrorsIntroduced += stats[i].numErrorsIntroduced;
globalStats.numErrorsRemoved += stats[i].numErrorsRemoved;
globalStats.tp += stats[i].tp;
globalStats.fp += stats[i].fp;
globalStats.tn += stats[i].tn;
globalStats.fn += stats[i].fn;
for (std::map<int, unsigned>::const_iterator it = stats[i].histo.begin(); it != stats[i].histo.end(); ++it)
globalStats.histo[it->first] += it->second;
for (std::map<int, unsigned>::const_iterator it = stats[i].preErrorHisto.begin(); it != stats[i].preErrorHisto.end(); ++it)
globalStats.preErrorHisto[it->first] += it->second;
resize(globalStats.preErrorsAtPos, length(stats[i].preErrorsAtPos), globalStats.zeroCounts);
for (unsigned pos = 0; pos < length(stats[i].preErrorsAtPos); ++pos)
for (int err = 0; err < 4; ++err)
globalStats.preErrorsAtPos[pos][err] += stats[i].preErrorsAtPos[pos][err];
resize(globalStats.postErrorsAtPos, length(stats[i].postErrorsAtPos), globalStats.zeroCounts);
for (unsigned pos = 0; pos < length(stats[i].postErrorsAtPos); ++pos)
for (int err = 0; err < 4; ++err)
globalStats.postErrorsAtPos[pos][err] += stats[i].postErrorsAtPos[pos][err];
}
// The quick stats are what we need for the table in the paper.
std::cout << "QUICK STATS\n"
<< "gain\tbase error rate pre\tread error rate pre\tbase error rate post\tread error rate post"
<< "\tsensitivity\tspecificity\tTP\tFP\tTN\tFN";
if (!options.indels)
std::cout << "errors removed\terrors introduced";
std::cout << "\n";
double sens = (globalStats.tp + globalStats.fn) ? (100.0 * globalStats.tp / (globalStats.tp + globalStats.fn)) : 100.0;
double spec = (globalStats.fp + globalStats.tn) ? (100.0 * globalStats.tn / (globalStats.fp + globalStats.tn)) : 100.0;
fprintf(stdout, "%2.5f\t%2.5f\t%2.5f\t%2.5f\t%2.5f\t%.2f\t%.2f",
100.0 * globalStats.diffErrorSum / globalStats.actualErrorSum,
100.0 * globalStats.numErrorsPre / globalStats.numBasesPre,
100.0 * globalStats.numErrorReadsPre / globalStats.numReads,
100.0 * globalStats.numErrorsPost / globalStats.numBasesPost,
100.0 * globalStats.numErrorReadsPost / globalStats.numReads,
sens,
spec);
std::cout << "\t" << globalStats.tp << "\t" << globalStats.fp << "\t" << globalStats.tn << "\t" << globalStats.fn;
if (!options.indels)
std::cout << "\t" << globalStats.numErrorsRemoved << "\t" << globalStats.numErrorsIntroduced;
std::cout << "\n\n";
// Print detailed statistics and histogram.
std::cout << "STATISTICS\n"
<< "total read count " << globalStats.numTotal << "\t\t(excludes unmapped reads)\n"
<< "unmapped pre count " << globalStats.numUnmappedPre << "\n"
<< "ignored pre count " << globalStats.numIgnoredPre << "\n"
<< "\n"
<< "gain " << 100.0 * globalStats.diffErrorSum / globalStats.actualErrorSum << "\n"
<< "\n"
<< "base error rate pre " << 100.0 * globalStats.numErrorsPre / globalStats.numBasesPre << "\n"
<< "read error rate pre " << 100.0 * globalStats.numErrorReadsPre / globalStats.numReads << "\n"
<< "base error rate post " << 100.0 * globalStats.numErrorsPost / globalStats.numBasesPost << "\n"
<< "read error rate post " << 100.0 * globalStats.numErrorReadsPost / globalStats.numReads << "\n"
<< "\n"
<< "sensitivity " << sens << "\n"
<< "specificity " << spec << "\n"
<< "\n"
<< "true positives " << globalStats.tp << "\n"
<< "false positives " << globalStats.fp << "\n"
<< "true negatives " << globalStats.tn << "\n"
<< "false negatives " << globalStats.fn << "\n"
<< "\n"
//<< "unmapped post count " << globalStats.numUnmappedPost << "\n"
//<< "unmapped both count " << globalStats.numUnmappedBoth << "\n"
<< "\n"
<< "CORRECTION HISTOGRAM\n"
<< "\n"
<< "E.g. a value of 2 means 2 errors corrected, a value of -2 means two errors introduced.\n"
<< "\n"
<< "diff\tcount\t\tpercentage of reads\n";
for (std::map<int, unsigned>::const_iterator it = globalStats.histo.begin(); it != globalStats.histo.end(); ++it)
fprintf(stdout, "%3d\t%12u\t%5.2f\n", it->first, it->second, 100.0 * it->second / globalStats.numTotal);
std::cout << "\n\nPRE-CORRECTION DISTRIBUTION\n\n"
<< "Distribution of errors before correction\n\n"
<< "error rate\tcount\t\tpercentage of reads\n";
for (std::map<int, unsigned>::const_iterator it = globalStats.preErrorHisto.begin(); it != globalStats.preErrorHisto.end(); ++it)
fprintf(stdout, "%3d\t%12u\t%5.2f\n", it->first, it->second, 100.0 * it->second / globalStats.numTotal);
// Print positional error distributions PRE/POST
std::cout << "\n\nPRE/POST-CORRECTION POSITIONAL ERROR DISTRIBUTION\n\n"
<< "position\tpre-mis\tpre-del\tpre-ins\tpre-bases\tpost-mis\tpost-del\tpost-ins\tpost-bases\n";
unsigned maxLen = std::max(length(globalStats.preErrorsAtPos), length(globalStats.postErrorsAtPos));
resize(globalStats.preErrorsAtPos, maxLen, globalStats.zeroCounts);
resize(globalStats.postErrorsAtPos, maxLen, globalStats.zeroCounts);
for (unsigned pos = 0; pos < maxLen; ++pos)
{
fprintf(stdout, "%3d", pos);
for (unsigned err = 0; err < 4; ++err)
fprintf(stdout, "\t%12u", globalStats.preErrorsAtPos[pos][err]);
for (unsigned err = 0; err < 4; ++err)
fprintf(stdout, "\t%12u", globalStats.postErrorsAtPos[pos][err]);
std::cout << '\n';
}
// Print configuration.
std::cout << "\n\nCONFIGURATION\n\n"
<< " GENOME \t" << options.pathGenome << "\n"
<< " SAM PRE \t" << options.pathSamPreCorrection << "\n"
<< " SAM POST \t" << options.pathSamPostCorrection << "\n"
<< " FA/FQ POST \t" << options.pathFastaFastqPostCorrection << "\n"
<< " INDELS \t" << getYesNo(options.indels) << "\n"
<< " PADDING \t" << options.padding << "\n"
<< " BANDWIDTH \t" << options.padding << "\n"
<< " MIN UNCLIPPED BASES\t" << options.minUnclippedBases << "\n"
<< " MAX ERROR RATE \t" << options.maxErrorRate << "\n"
<< " MAX ERROR COUNT \t" << options.maxErrorCount << "\n"
<< "\n"
<< " NUM THREADS \t" << options.numThreads << "\n"
<< " CORRECTION LOG \t" << options.pathCorrectionLog << "\n"
<< " LOG ALL \t" << getYesNo(options.logAll) << "\n"
<< " BANDWIDTH \t" << options.bandwidth << "\n"
<< "\n";
return 0;
}
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