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SalmonAlevin.cpp
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/**
>HEADER
Copyright (c) 2013, 2014, 2015, 2016 Rob Patro rob.patro@cs.stonybrook.edu
This file is part of Salmon.
Salmon is free software: you can redistribute it and/or modify
it under the terms of the GNU General Public License as published by
the Free Software Foundation, either version 3 of the License, or
(at your option) any later version.
Salmon is distributed in the hope that it will be useful,
but WITHOUT ANY WARRANTY; without even the implied warranty of
MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the
GNU General Public License for more details.
You should have received a copy of the GNU General Public License
along with Salmon. If not, see <http://www.gnu.org/licenses/>.
<HEADER
**/
#include <algorithm>
#include <atomic>
#include <cassert>
#include <cmath>
#include <cstdio>
#include <exception>
#include <functional>
#include <iterator>
#include <map>
#include <memory>
#include <mutex>
#include <queue>
#include <random>
#include <sstream>
#include <thread>
#include <unordered_map>
#include <unordered_set>
#include <vector>
// C++ string formatting library
#include "spdlog/fmt/fmt.h"
// C Includes for BWA
#include <cctype>
#include <cstdio>
#include <cstdlib>
#include <cstring>
#include <unistd.h>
// Jellyfish 2 include
// #include "jellyfish/mer_dna.hpp"
// Boost Includes
#include <boost/container/flat_map.hpp>
#include <boost/dynamic_bitset/dynamic_bitset.hpp>
#include <boost/filesystem.hpp>
#include <boost/lockfree/queue.hpp>
#include <boost/program_options.hpp>
#include <boost/range/irange.hpp>
#include <boost/thread/thread.hpp>
// TBB Includes
#include "tbb/blocked_range.h"
#include "tbb/concurrent_queue.h"
#include "tbb/concurrent_unordered_map.h"
#include "tbb/concurrent_unordered_set.h"
#include "tbb/concurrent_vector.h"
#include "tbb/parallel_for.h"
#include "tbb/parallel_for_each.h"
#include "tbb/parallel_reduce.h"
#include "tbb/partitioner.h"
// logger includes
#include "spdlog/spdlog.h"
// Cereal includes
#include "cereal/archives/binary.hpp"
#include "cereal/types/vector.hpp"
#include "concurrentqueue.h"
#include <cuckoohash_map.hh>
// core includes
#include "core/range.hpp"
// radicl includes
#include "radicl/BasicBinWriter.hpp"
#include "radicl/RADHeader.hpp"
//alevin include
#include "AlevinOpts.hpp"
#include "AlevinUtils.hpp"
#include "SingleCellProtocols.hpp"
#include "CollapsedCellOptimizer.hpp"
#include "BarcodeGroup.hpp"
// salmon includes
#include "ClusterForest.hpp"
#include "FastxParser.hpp"
#include "IOUtils.hpp"
#include "LibraryFormat.hpp"
#include "ReadLibrary.hpp"
#include "SalmonConfig.hpp"
#include "SalmonIndex.hpp"
#include "SalmonMath.hpp"
#include "SalmonUtils.hpp"
#include "Transcript.hpp"
#include "AlignmentGroup.hpp"
#include "BiasParams.hpp"
#include "CollapsedEMOptimizer.hpp"
#include "CollapsedGibbsSampler.hpp"
#include "EquivalenceClassBuilder.hpp"
#include "ForgettingMassCalculator.hpp"
#include "FragmentLengthDistribution.hpp"
#include "GZipWriter.hpp"
#include "SalmonMappingUtils.hpp"
#include "ReadExperiment.hpp"
#include "SalmonOpts.hpp"
#include "PairedAlignmentFormatter.hpp"
#include "pufferfish/Util.hpp"
#include "pufferfish/MemCollector.hpp"
#include "pufferfish/MemChainer.hpp"
#include "pufferfish/SAMWriter.hpp"
#include "pufferfish/PuffAligner.hpp"
#include "pufferfish/ksw2pp/KSW2Aligner.hpp"
#include "pufferfish/metro/metrohash64.h"
#include "parallel_hashmap/phmap.h"
#include "pufferfish/chobo/static_vector.hpp"
#include "pufferfish/SelectiveAlignmentUtils.hpp"
namespace alevin{
/****** QUASI MAPPING DECLARATIONS *********/
using MateStatus = pufferfish::util::MateStatus;
using QuasiAlignment = pufferfish::util::QuasiAlignment;
/****** QUASI MAPPING DECLARATIONS *******/
using paired_parser = fastx_parser::FastxParser<fastx_parser::ReadPair>;
using single_parser = fastx_parser::FastxParser<fastx_parser::ReadSeq>;
using TranscriptID = uint32_t;
using TranscriptIDVector = std::vector<TranscriptID>;
using KmerIDMap = std::vector<TranscriptIDVector>;
constexpr uint32_t miniBatchSize{5000};
// NOTE: Consider the implications of using uint32_t below.
// Currently, this should not limit the barcode length to <= 16 in
// "fry" mode (either sla or sketch) since we never actually put the
// barcode in the corresponding variable of the AlignmentGroup class.
// In traditional alevin, this should be referring to a barcode *index*
// rather than the sequence itself, so it should be OK so long as we have
// < std::numeric_limits<uint32_t>::max() distinct barcodes / reads.
// However, that assumption should be tested more thoroughly.
using CellBarcodeT = uint32_t;
using UMIBarcodeT = uint64_t;
template <typename AlnT> using AlevinAlnGroup = AlignmentGroup<AlnT, CellBarcodeT, UMIBarcodeT>;
template <typename AlnT> using AlnGroupVec = std::vector<AlevinAlnGroup<AlnT>>;
template <typename AlnT>
using AlnGroupVecRange = core::range<typename AlnGroupVec<AlnT>::iterator>;
#define __MOODYCAMEL__
#if defined(__MOODYCAMEL__)
template <typename AlnT>
using AlnGroupQueue = moodycamel::ConcurrentQueue<AlevinAlnGroup<AlnT>*>;
#else
template <typename AlnT>
using AlnGroupQueue = tbb::concurrent_queue<AlevinAlnGroup<AlnT>*>;
#endif
//#include "LightweightAlignmentDefs.hpp"
}
//have to create new namespace because of multiple definition
using namespace alevin;
/* ALEVIN DECLERATIONS*/
using bcEnd = BarcodeEnd;
namespace aut = alevin::utils;
using BlockedIndexRange = tbb::blocked_range<size_t>;
using ReadExperimentT = ReadExperiment<EquivalenceClassBuilder<SCTGValue>>;
/////// REDUNDANT CODE END//
template <typename AlnT>
void processMiniBatchSimple(ReadExperimentT& readExp, ForgettingMassCalculator& fmCalc,
ReadLibrary& readLib,
const SalmonOpts& salmonOpts,
AlnGroupVecRange<AlnT> batchHits,
std::vector<Transcript>& transcripts,
ClusterForest& clusterForest,
FragmentLengthDistribution& fragLengthDist,
std::atomic<uint64_t>& numAssignedFragments,
bool initialRound,
std::atomic<bool>& burnedIn, double& maxZeroFrac){
using salmon::math::LOG_0;
using salmon::math::LOG_1;
using salmon::math::LOG_EPSILON;
using salmon::math::LOG_ONEHALF;
using salmon::math::logAdd;
using salmon::math::logSub;
const uint64_t numBurninFrags = salmonOpts.numBurninFrags;
auto& log = salmonOpts.jointLog;
size_t numTranscripts{transcripts.size()};
size_t localNumAssignedFragments{0};
size_t priorNumAssignedFragments{numAssignedFragments};
std::uniform_real_distribution<> uni(
0.0, 1.0 + std::numeric_limits<double>::min());
std::vector<uint64_t> libTypeCounts(LibraryFormat::maxLibTypeID() + 1);
bool hasCompatibleMapping{false};
uint64_t numCompatibleFragments{0};
std::vector<FragmentStartPositionDistribution>& fragStartDists =
readExp.fragmentStartPositionDistributions();
bool updateCounts = initialRound;
double incompatPrior = salmonOpts.incompatPrior;
bool useReadCompat = incompatPrior != salmon::math::LOG_1;
// If we're auto detecting the library type
auto* detector = readLib.getDetector();
bool autoDetect = (detector != nullptr) ? detector->isActive() : false;
// If we haven't detected yet, nothing is incompatible
if (autoDetect) { incompatPrior = salmon::math::LOG_1; }
double logForgettingMass{0.0};
uint64_t currentMinibatchTimestep{0};
// logForgettingMass and currentMinibatchTimestep are OUT parameters!
fmCalc.getLogMassAndTimestep(logForgettingMass, currentMinibatchTimestep);
auto expectedLibraryFormat = readLib.format();
uint64_t zeroProbFrags{0};
// EQClass
auto& eqBuilder = readExp.equivalenceClassBuilder();
// Build reverse map from transcriptID => hit id
using HitID = uint32_t;
int i{0};
{
// Iterate over each group of alignments (a group consists of all alignments
// reported
// for a single read). Distribute the read's mass to the transcripts
// where it potentially aligns.
for (auto& alnGroup : batchHits) {
// If we had no alignments for this read, then skip it
if (alnGroup.size() == 0) {
continue;
}
//extract barcode of the read
uint32_t barcode = alnGroup.barcode();
uint64_t umi = alnGroup.umi();
// We start out with probability 0
double sumOfAlignProbs{LOG_0};
// Record whether or not this read is unique to a single transcript.
bool transcriptUnique{true};
auto firstTranscriptID = alnGroup.alignments().front().transcriptID();
std::vector<uint32_t> txpIDs;
uint32_t numInGroup{0};
uint32_t prevTxpID{0};
hasCompatibleMapping = false;
// For each alignment of this read
for (auto& aln : alnGroup.alignments()) {
auto transcriptID = aln.transcriptID();
auto& transcript = transcripts[transcriptID];
transcriptUnique =
transcriptUnique and (transcriptID == firstTranscriptID);
if (autoDetect) {
detector->addSample(aln.libFormat());
if (detector->canGuess()) {
detector->mostLikelyType(readLib.getFormat());
expectedLibraryFormat = readLib.getFormat();
incompatPrior = salmonOpts.incompatPrior;
autoDetect = false;
} else if (!detector->isActive()) {
expectedLibraryFormat = readLib.getFormat();
incompatPrior = salmonOpts.incompatPrior;
autoDetect = false;
}
}
// The probability that the fragments align to the given strands in
// the given orientations.
bool isCompat =
salmon::utils::isCompatible(
aln.libFormat(),
expectedLibraryFormat,
static_cast<int32_t>(aln.pos),
aln.fwd,
aln.mateStatus);
double logAlignCompatProb = isCompat ? LOG_1 : incompatPrior;
aln.logProb = logAlignCompatProb;
if (!isCompat and salmonOpts.ignoreIncompat) {
aln.logProb = salmon::math::LOG_0;
continue;
}
// Increment the count of this type of read that we've seen
++libTypeCounts[aln.libFormat().formatID()];
if (!hasCompatibleMapping and logAlignCompatProb == LOG_1) { hasCompatibleMapping = true; }
// If this alignment had a zero probability, then skip it
if (std::abs(aln.logProb) == LOG_0) {
continue;
}
sumOfAlignProbs = logAdd(sumOfAlignProbs, aln.logProb);
if (transcriptID < prevTxpID) {
std::cerr << "[ERROR] Transcript IDs are not in sorted order; "
"please report this bug on GitHub!\n";
}
prevTxpID = transcriptID;
txpIDs.push_back(transcriptID);
}
// If this fragment has a zero probability,
// go to the next one
if (sumOfAlignProbs == LOG_0) {
++zeroProbFrags;
continue;
} else { // otherwise, count it as assigned
++localNumAssignedFragments;
if (hasCompatibleMapping) { ++numCompatibleFragments; }
}
auto eqSize = txpIDs.size();
if (eqSize > 0) {
TranscriptGroup tg(txpIDs);
eqBuilder.addBarcodeGroup(std::move(tg), barcode, umi);
}
// update the single target transcript
if (transcriptUnique) {
if (updateCounts) {
transcripts[firstTranscriptID].addUniqueCount(1);
}
} else { // or the appropriate clusters
clusterForest.mergeClusters<AlnT>(alnGroup.alignments().begin(),
alnGroup.alignments().end());
clusterForest.updateCluster(
alnGroup.alignments().front().transcriptID(), 1.0,
logForgettingMass, updateCounts);
}
} // end read group
} // end timer
if (zeroProbFrags > 0) {
auto batchReads = batchHits.size();
maxZeroFrac = std::max(maxZeroFrac, static_cast<double>(100.0 * zeroProbFrags) / batchReads);
}
numAssignedFragments += localNumAssignedFragments;
if (numAssignedFragments >= numBurninFrags and !burnedIn) {
// NOTE: only one thread should succeed here, and that
// thread will set burnedIn to true.
readExp.updateTranscriptLengthsAtomic(burnedIn);
fragLengthDist.cacheCMF();
}
if (initialRound) {
readLib.updateLibTypeCounts(libTypeCounts);
readLib.updateCompatCounts(numCompatibleFragments);
}
}
/**
* Perform sketch mapping of sc reads and produce output file.
**/
template <typename IndexT, typename ProtocolT>
void process_reads_sc_sketch(paired_parser* parser, ReadExperimentT& readExp, ReadLibrary& rl,
AlnGroupVec<QuasiAlignment>& structureVec,
std::atomic<uint64_t>& numObservedFragments,
std::atomic<uint64_t>& numAssignedFragments,
std::atomic<uint64_t>& numUniqueMappings,
std::atomic<uint64_t>& validHits,
std::atomic<uint32_t>& smallSeqs,
std::atomic<uint32_t>& nSeqs,
IndexT* qidx, std::vector<Transcript>& transcripts,
FragmentLengthDistribution& fragLengthDist,
SalmonOpts& salmonOpts,
std::atomic<uint64_t>& num_chunks,
std::ofstream& rad_file,
std::ofstream& ubc_file, // unmapped barcode count
std::mutex& fileMutex,
std::mutex& ubc_file_mutex, // mutex for barcode count
std::mutex& iomutex,
AlevinOpts<ProtocolT>& alevinOpts,
MappingStatistics& mstats) {
(void) numAssignedFragments;
(void) fragLengthDist;
uint64_t count_fwd = 0, count_bwd = 0;
uint64_t prevObservedFrags{1};
uint64_t leftHitCount{0};
uint64_t hitListCount{0};
salmon::utils::ShortFragStats shortFragStats;
double maxZeroFrac{0.0};
// Write unmapped reads
fmt::MemoryWriter unmappedNames;
bool writeUnmapped = salmonOpts.writeUnmappedNames;
spdlog::logger* unmappedLogger = (writeUnmapped) ? salmonOpts.unmappedLog.get() : nullptr;
// Write unmapped reads
fmt::MemoryWriter orphanLinks;
bool writeOrphanLinks = salmonOpts.writeOrphanLinks;
spdlog::logger* orphanLinkLogger = (writeOrphanLinks) ? salmonOpts.orphanLinkLog.get() : nullptr;
BasicBinWriter bw;
uint32_t num_reads_in_chunk{0};
bw << num_reads_in_chunk;
bw << num_reads_in_chunk;
size_t minK = qidx->k();
size_t locRead{0};
size_t rangeSize{0};
uint64_t localNumAssignedFragments{0};
uint64_t localNumUniqueMappings{0};
bool consistentHits = salmonOpts.consistentHits;
bool quiet = salmonOpts.quiet;
size_t maxNumHits{salmonOpts.maxReadOccs};
size_t readLenLeft{0};
size_t readLenRight{0};
constexpr const int32_t invalidScore = std::numeric_limits<int32_t>::min();
MemCollector<IndexT> memCollector(qidx);
ksw2pp::KSW2Aligner aligner;
pufferfish::util::AlignmentConfig aconf;
pufferfish::util::MappingConstraintPolicy mpol;
bool initOK = salmon::mapping_utils::initMapperSettings(salmonOpts, memCollector, aligner, aconf, mpol);
PuffAligner puffaligner(qidx->refseq_, qidx->refAccumLengths_, qidx->k(), aconf, aligner);
pufferfish::util::CachedVectorMap<size_t, std::vector<pufferfish::util::MemCluster>, std::hash<size_t>> hits;
std::vector<pufferfish::util::MemCluster> recoveredHits;
std::vector<pufferfish::util::JointMems> jointHits;
PairedAlignmentFormatter<IndexT*> formatter(qidx);
pufferfish::util::QueryCache qc;
bool mimicStrictBT2 = salmonOpts.mimicStrictBT2;
bool mimicBT2 = salmonOpts.mimicBT2;
bool noDovetail = !salmonOpts.allowDovetail;
bool useChainingHeuristic = !salmonOpts.disableChainingHeuristic;
pufferfish::util::HitCounters hctr;
salmon::utils::MappingType mapType{salmon::utils::MappingType::UNMAPPED};
bool hardFilter = salmonOpts.hardFilter;
fmt::MemoryWriter sstream;
auto* qmLog = salmonOpts.qmLog.get();
bool writeQuasimappings = (qmLog != nullptr);
struct SimpleHit {
bool is_fw{false};
int32_t pos{-1};
float score{0.0};
uint32_t num_hits{0};
uint32_t tid{std::numeric_limits<uint32_t>::max()};
bool valid_pos(int32_t read_len, uint32_t txp_len, int32_t max_over) {
int32_t signed_txp_len = static_cast<int32_t>(txp_len);
return (pos > -max_over) and ((pos + read_len) < (signed_txp_len + max_over));
}
};
enum class HitDirection : uint8_t {FW, RC, BOTH};
struct SketchHitInfo {
// add a hit to the current target that occurs in the forward
// orientation with respect to the target.
bool add_fw(int32_t ref_pos, int32_t read_pos, int32_t max_stretch, float score_inc) {
bool added{false};
// since hits are collected by moving _forward_ in the
// read, if this is a fw hit, it should be moving
// forward in the reference. Only add it if this is
// the case. This ensure that we don't
// double-count a k-mer that might occur twice on
// this target.
if (ref_pos > last_ref_pos_fw and read_pos > last_read_pos_fw) {
if (last_read_pos_fw == -1) {
approx_pos_fw = ref_pos - read_pos;
} else {
if ((ref_pos - approx_pos_fw) > max_stretch) { return false; }
}
//if (last_ref_pos_fw > -1 and (ref_pos > last_ref_pos_fw + 15)) { return false; }
last_ref_pos_fw = ref_pos;
last_read_pos_fw = read_pos;
fw_score += score_inc;
++fw_hits;
added = true;
}
return added;
}
// add a hit to the current target that occurs in the forward
// orientation with respect to the target.
bool add_rc(int32_t ref_pos, int32_t read_pos, int32_t max_stretch, float score_inc) {
bool added{false};
// since hits are collected by moving _forward_ in the
// read, if this is an rc hit, it should be moving
// backwards in the reference. Only add it if this is
// the case.
// This ensures that we don't double-count a k-mer that
// might occur twice on this target.
if (ref_pos < last_ref_pos_rc and read_pos > last_read_pos_rc) {
approx_pos_rc = ref_pos;
if (last_read_pos_rc == -1) {
approx_end_pos_rc = ref_pos + read_pos;
} else {
if (approx_end_pos_rc - approx_pos_rc > max_stretch) { return false;}
}
//if (last_ref_pos_rc > -1 and ref_pos < last_ref_pos_rc - 15) { return false; }
last_ref_pos_rc = ref_pos;
last_read_pos_rc = read_pos;
rc_score += score_inc;
++rc_hits;
added = true;
}
return added;
}
// true if forward, false if rc
// second element is score
inline HitDirection best_hit_direction() {
int32_t fw_minus_rc = static_cast<int32_t>(fw_hits) - static_cast<int32_t>(rc_hits);
return (fw_minus_rc > 0) ? HitDirection::FW :
((fw_minus_rc < 0) ? HitDirection::RC : HitDirection::BOTH);
}
inline SimpleHit get_fw_hit() {
return SimpleHit{true, approx_pos_fw, fw_score, fw_hits, std::numeric_limits<uint32_t>::max()};
}
inline SimpleHit get_rc_hit() {
return SimpleHit{false, approx_pos_rc, rc_score, rc_hits, std::numeric_limits<uint32_t>::max()};
}
inline SimpleHit get_best_hit() {
auto best_direction = best_hit_direction();
return (best_direction != HitDirection::RC) ?
SimpleHit{true, approx_pos_fw, fw_score, fw_hits, std::numeric_limits<uint32_t>::max()} :
SimpleHit{false, approx_pos_rc, rc_score, rc_hits, std::numeric_limits<uint32_t>::max()};
}
inline std::string to_string() {
std::stringstream ss;
ss << "fw_hits: " << fw_hits << ", fw_score : " << fw_score << ", fw_pos : " << approx_pos_fw
<< " || rc_hits: " << rc_hits << ", rc_score: " << rc_score << ", rc_pos: " << approx_pos_rc;
return ss.str();
}
int32_t last_read_pos_fw{-1};
int32_t last_read_pos_rc{-1};
int32_t last_ref_pos_fw{-1};
int32_t last_ref_pos_rc{std::numeric_limits<int32_t>::max()};
int32_t approx_pos_fw{-1};
int32_t approx_pos_rc{-1};
int32_t approx_end_pos_rc{-1};
uint32_t fw_hits{0};
uint32_t rc_hits{0};
float fw_score{0.0};
float rc_score{0.0};
};
// map to recall the number of unmapped reads we see
// for each barcode
phmap::flat_hash_map<uint64_t, uint32_t> unmapped_bc_map;
// map from transcript id to hit info
phmap::flat_hash_map<uint32_t, SketchHitInfo> hit_map;
std::vector<SimpleHit> accepted_hits;
//////////////////////
// NOTE: validation mapping based new parameters
std::string rc1; rc1.reserve(300);
size_t numMappingsDropped{0};
size_t numDecoyFrags{0};
const double decoyThreshold = salmonOpts.decoyThreshold;
salmon::mapping_utils::MappingScoreInfo msi(decoyThreshold);
// we only collect detailed decoy information if we will be
// writing output to SAM.
msi.collect_decoys(writeQuasimappings);
std::string readBuffer;
std::string umi(alevinOpts.protocol.umiLength, 'N');
std::string barcode(alevinOpts.protocol.barcodeLength, 'N');
//////////////////////
bool tryAlign{salmonOpts.validateMappings};
auto rg = parser->getReadGroup();
while (parser->refill(rg)) {
rangeSize = rg.size();
if (rangeSize > structureVec.size()) {
salmonOpts.jointLog->error("rangeSize = {}, but structureVec.size() = {} "
"--- this shouldn't happen.\n"
"Please report this bug on GitHub",
rangeSize, structureVec.size());
std::exit(1);
}
LibraryFormat expectedLibraryFormat = rl.format();
std::string extraBAMtags;
if(writeQuasimappings) {
size_t reserveSize { alevinOpts.protocol.barcodeLength + alevinOpts.protocol.umiLength + 12};
extraBAMtags.reserve(reserveSize);
}
auto localProtocol = alevinOpts.protocol;
for (size_t i = 0; i < rangeSize; ++i) { // For all the read in this batch
auto& rp = rg[i];
readLenLeft = rp.first.seq.length();
readLenRight= rp.second.seq.length();
bool tooShortRight = (readLenRight < (minK+alevinOpts.trimRight));
//localUpperBoundHits = 0;
auto& jointHitGroup = structureVec[i];
jointHitGroup.clearAlignments();
auto& jointAlignments= jointHitGroup.alignments();
hits.clear();
jointHits.clear();
memCollector.clear();
hit_map.clear();
accepted_hits.clear();
//jointAlignments.clear();
//readSubSeq.clear();
mapType = salmon::utils::MappingType::UNMAPPED;
//////////////////////////////////////////////////////////////
// extracting barcodes
size_t barcodeLength = alevinOpts.protocol.barcodeLength;
size_t umiLength = alevinOpts.protocol.umiLength;
//umi.clear();
//barcode.clear();
nonstd::optional<uint32_t> barcodeIdx;
extraBAMtags.clear();
bool seqOk = false;
// keep track of the *least* freqeuntly
// occurring hit in this fragment to consider
// alternative processing of fragments where
// all observed hits have high frequency.
uint64_t alt_max_occ = 0;
if (alevinOpts.protocol.end == bcEnd::FIVE ||
alevinOpts.protocol.end == bcEnd::THREE){
// If the barcode sequence could be extracted, then this is set to true,
// but the barcode sequence itself may still be invalid (e.g. contain `N` characters).
// However, if extracted_barcode is false here, there is no hope to even recover the
// barcode and we shouldn't attempt it.
bool extracted_barcode = aut::extractBarcode(rp.first.seq, rp.second.seq, localProtocol, barcode);
// If we could pull out something where the barcode sequence should have been
// then continue to process it.
if (extracted_barcode) {
// if the barcode consisted of valid nucleotides, then seqOk is true
// otherwise false
seqOk = aut::sequenceCheck(barcode, Sequence::BARCODE);
if (not seqOk){
// If the barcode contained invalid nucleotides
// this attempts to replace the first one with an `A`.
// If this returns true, there was only one `N` and we
// replaced it; otherwise there was more than one `N`
// and the barcode sequence should be treated as invalid.
seqOk = aut::recoverBarcode(barcode);
}
}
// If we have a valid barcode
if (seqOk) {
bool umi_ok = aut::extractUMI(rp.first.seq, rp.second.seq, localProtocol, umi);
if ( !umi_ok ) {
smallSeqs += 1;
} else {
alevin::types::AlevinUMIKmer umiIdx;
bool isUmiIdxOk = umiIdx.fromChars(umi);
if (isUmiIdxOk) {
jointHitGroup.setUMI(umiIdx.word(0));
bool rh = false;
std::string* readSubSeq = aut::getReadSequence(alevinOpts.protocol, rp.first.seq, rp.second.seq, readBuffer);
rh = tooShortRight
? false
: memCollector.get_raw_hits_sketch(*readSubSeq,
qc,
true, // isLeft
false // verbose
);
// if there were hits
if (rh) {
uint32_t num_valid_hits{0};
uint64_t total_occs{0};
uint64_t largest_occ{0};
float perfect_score{0.0};
auto& raw_hits = memCollector.get_left_hits();
// SANITY
decltype(raw_hits[0].first) prev_read_pos = -1;
// the maximum span the supporting k-mers of a
// mapping position are allowed to have.
// NOTE this is still > read_length b/c the stretch is measured wrt the
// START of the terminal k-mer.
int32_t max_stretch = static_cast<int32_t>(readSubSeq->length() * 1.0);
// a raw hit is a pair of read_pos and a projected hit
// the least frequent hit for this fragment.
uint64_t min_occ = std::numeric_limits<uint64_t>::max();
// this is false by default and will be set to true
// if *every* collected hit for this fragment occurs
// salmonOpts.maxReadOccs times or more.
bool had_alt_max_occ = false;
for (auto& raw_hit : raw_hits) {
auto& read_pos = raw_hit.first;
auto& proj_hits = raw_hit.second;
auto& refs = proj_hits.refRange;
uint64_t num_occ = static_cast<uint64_t>(refs.size());
min_occ = std::min(min_occ, num_occ);
// SANITY
if (read_pos <= prev_read_pos) {
salmonOpts.jointLog->warn("read_pos : {}, prev_read_pos : {}", read_pos, prev_read_pos);
}
prev_read_pos = read_pos;
if (num_occ < salmonOpts.maxReadOccs) {
++num_valid_hits;
total_occs += num_occ;
largest_occ = (num_occ > largest_occ) ? num_occ : largest_occ;
float score_inc = 1.0 / num_occ;
perfect_score += score_inc;
for (auto &pos_it : refs) {
const auto& ref_pos_ori = proj_hits.decodeHit(pos_it);
uint32_t tid = static_cast<uint32_t>(qidx->getRefId(pos_it.transcript_id()));
int32_t pos = static_cast<int32_t>(ref_pos_ori.pos);
bool ori = ref_pos_ori.isFW;
if (ori) {
hit_map[tid].add_fw(pos, static_cast<int32_t>(read_pos), max_stretch, score_inc);
} else {
hit_map[tid].add_rc(pos, static_cast<int32_t>(read_pos), max_stretch, score_inc);
}
} // DONE: for (auto &pos_it : refs)
} // DONE : if (static_cast<uint64_t>(refs.size()) < salmonOpts.maxReadOccs)
} // DONE : for (auto& raw_hit : raw_hits)
// If our default threshold was too stringent, then set a more liberal
// threshold and look up the k-mers that occur the least frequently.
// Specifically, if the min occuring hits have frequency < min_thresh_prime (2500 by default)
// times, then collect the min occuring hits to get the mapping.
// TODO: deal with code duplication below.
size_t max_allowed_occ = 2500;
if ((min_occ >= salmonOpts.maxReadOccs) and (min_occ < max_allowed_occ)) {
prev_read_pos = -1;
max_allowed_occ = min_occ;
for (auto& raw_hit : raw_hits) {
auto& read_pos = raw_hit.first;
auto& proj_hits = raw_hit.second;
auto& refs = proj_hits.refRange;
uint64_t num_occ = static_cast<uint64_t>(refs.size());
min_occ = std::min(min_occ, num_occ);
had_alt_max_occ = true;
// SANITY
if (read_pos <= prev_read_pos) {
salmonOpts.jointLog->warn(
"read_pos : {}, prev_read_pos : {}", read_pos,
prev_read_pos);
}
prev_read_pos = read_pos;
if (num_occ <= max_allowed_occ) {
++num_valid_hits;
total_occs += num_occ;
largest_occ =
(num_occ > largest_occ) ? num_occ : largest_occ;
float score_inc = 1.0 / num_occ;
perfect_score += score_inc;
for (auto& pos_it : refs) {
const auto& ref_pos_ori = proj_hits.decodeHit(pos_it);
uint32_t tid = static_cast<uint32_t>(
qidx->getRefId(pos_it.transcript_id()));
int32_t pos = static_cast<int32_t>(ref_pos_ori.pos);
bool ori = ref_pos_ori.isFW;
if (ori) {
hit_map[tid].add_fw(pos,
static_cast<int32_t>(read_pos),
max_stretch, score_inc);
} else {
hit_map[tid].add_rc(pos,
static_cast<int32_t>(read_pos),
max_stretch, score_inc);
}
} // DONE: for (auto &pos_it : refs)
} // DONE : if (static_cast<uint64_t>(refs.size()) <
// salmonOpts.maxReadOccs)
} // DONE : for (auto& raw_hit : raw_hits)
}
//float perfect_score = static_cast<float>(num_valid_hits) / total_occs;
float acceptable_score = (num_valid_hits == 1) ? perfect_score :
perfect_score - (1.0f / largest_occ);
uint32_t best_alt_hits = 0;
int32_t signed_read_len = static_cast<int32_t>(readSubSeq->length());
bool saw_acceptable_score = false;
for (auto& kv : hit_map) {
auto best_hit_dir = kv.second.best_hit_direction();
// if the best direction is FW or BOTH, add the fw hit
// otherwise add the RC.
auto simple_hit = (best_hit_dir != HitDirection::RC) ?
kv.second.get_fw_hit() : kv.second.get_rc_hit();
if (simple_hit.num_hits >= num_valid_hits) {
simple_hit.tid = kv.first;
accepted_hits.emplace_back(simple_hit);
// if we had equally good hits in both directions
// add the rc hit here (since we added the fw)
// above if the best hit was either FW or BOTH
if (best_hit_dir == HitDirection::BOTH) {
auto second_hit = kv.second.get_rc_hit();
second_hit.tid = kv.first;
accepted_hits.emplace_back(second_hit);
}
} else {
//best_alt_score = simple_hit.score > best_alt_score ? simple_hit.score : best_alt_score;
best_alt_hits = simple_hit.num_hits > best_alt_hits ? simple_hit.num_hits : best_alt_hits;
}
}
alt_max_occ = had_alt_max_occ ? accepted_hits.size() : salmonOpts.maxReadOccs;
/*
if (accepted_hits.empty() and (num_valid_hits > 1) and (best_alt_hits >= num_valid_hits - 1)) {
for (auto& kv : hit_map) {
auto simple_hit = kv.second.get_best_hit();
if (simple_hit.num_hits >= best_alt_hits) {
//if (simple_hit.valid_pos(signed_read_len, transcripts[kv.first].RefLength, 10)) {
simple_hit.tid = kv.first;
accepted_hits.emplace_back(simple_hit);
//}
}
}
}
*/
} // DONE : if (rh)
} else {
nSeqs += 1;
}
}
}
} else {
salmonOpts.jointLog->error( "wrong barcode-end parameters.\n"
"Please report this bug on Github");
salmonOpts.jointLog->flush();
spdlog::drop_all();
std::exit(1);
}
//////////////////////////////////////////////////////////////
// Consider a read as too short if the ``non-barcode'' end is too short
if (tooShortRight) {
++shortFragStats.numTooShort;
shortFragStats.shortest = std::min(shortFragStats.shortest,
std::max(readLenLeft, readLenRight));
}
// If the read mapped to > maxReadOccs places, discard it
if (accepted_hits.size() > alt_max_occ ) {
accepted_hits.clear();
} else if (!accepted_hits.empty()) {
mapType = salmon::utils::MappingType::SINGLE_MAPPED;
}
alevin::types::AlevinCellBarcodeKmer bck;
bool barcode_ok = bck.fromChars(barcode);
// NOTE: Think if we should put decoy mappings in the RAD file
if (mapType == salmon::utils::MappingType::SINGLE_MAPPED) {
// num aln
bw << static_cast<uint32_t>(accepted_hits.size());
// bc
// if we can fit the barcode into an integer
if ( barcodeLength <= 32 ) {
if (barcode_ok) {
if ( barcodeLength <= 16 ) { // can use 32-bit int
uint32_t shortbck = static_cast<uint32_t>(0x00000000FFFFFFFF & bck.word(0));
bw << shortbck;
} else { // must use 64-bit int
bw << bck.word(0);
}
}
} else { // must use a string for the barcode
bw << barcode;
}
// umi
if ( umiLength <= 16 ) { // if we can use 32-bit int
uint64_t umiint = jointHitGroup.umi();
uint32_t shortumi = static_cast<uint32_t>(0x00000000FFFFFFFF & umiint);
bw << shortumi;
} else if ( umiLength <= 32 ) { // if we can use 64-bit int
uint64_t umiint = jointHitGroup.umi();
bw << umiint;
} else { // must use string
bw << umi;
}
for (auto& aln : accepted_hits) {
uint32_t fw_mask = aln.is_fw ? 0x80000000 : 0x00000000;
bw << (aln.tid | fw_mask);
}
++num_reads_in_chunk;
} else { // if read was not mapped
if (barcode_ok) {
unmapped_bc_map[bck.word(0)] += 1;
}
}
if (num_reads_in_chunk > 5000) {
++num_chunks;
uint32_t num_bytes = bw.num_bytes();
bw.write_integer_at_offset(0, num_bytes);
bw.write_integer_at_offset(sizeof(num_bytes), num_reads_in_chunk);
fileMutex.lock();
rad_file << bw;
fileMutex.unlock();
bw.clear();
num_reads_in_chunk = 0;
// reserve space for headers of next chunk
bw << num_reads_in_chunk;
bw << num_reads_in_chunk;
}
/*
if (writeQuasimappings) {
writeAlignmentsToStream(rp, formatter, jointAlignments, sstream, true, true, extraBAMtags);
}
*/
validHits += accepted_hits.size();
localNumAssignedFragments += (accepted_hits.size() > 0);
localNumUniqueMappings += (accepted_hits.size() == 1) ? 1 : 0;
locRead++;
++numObservedFragments;
jointHitGroup.clearAlignments();
if (!quiet and numObservedFragments % 500000 == 0) {
iomutex.lock();
const char RESET_COLOR[] = "\x1b[0m";
char green[] = "\x1b[30m";
green[3] = '0' + static_cast<char>(fmt::GREEN);
char red[] = "\x1b[30m";
red[3] = '0' + static_cast<char>(fmt::RED);
fmt::print(
stderr, "\033[A\r\r{}processed{} {} Million {}fragments{}\n",
green, red, numObservedFragments / 1000000, green, RESET_COLOR);
fmt::print(stderr, "hits: {}, hits per frag: {}", validHits,
validHits / static_cast<float>(prevObservedFrags));
iomutex.unlock();
}
} // end for i < j->nb_filled