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#include "SoloReadBarcode.h"
#include "serviceFuns.cpp"
#include "SequenceFuns.h"
#include "ErrorWarning.h"
#include "GlobalVariables.h"
void SoloReadBarcode::matchCBtoWL(string &cbSeq1, string &cbQual1, vector<uint64> &cbWL, int32 &cbMatch1, vector<uint64> &cbMatchInd1, string &cbMatchString1)
{
cbMatch1=-1;
cbMatchString1="";
cbMatchInd1.clear();
//convert CB and check for Ns
uint64 cbB1;
int64 posN=convertNuclStrToInt64(cbSeq1,cbB1);
if (!pSolo.cbWLyes) {//no whitelist - no search
if (posN!=-1) {//Ns are present, discard this read
cbMatch1=-2;
//stats.V[stats.nNinBarcode]++;
} else {//no Ns
//cbI=(int64) cbB1;
cbMatchInd1.push_back(cbB1);//all possible barcodes are accepted. This will overflow if CB is longer than 31b
cbMatchString1 = to_string(cbB1);
cbMatch1=0;
};
return;
};
if (posN==-2) {//>2 Ns, might already be filtered by Illumina
cbMatch1=-2;
//stats.V[stats.nNinBarcode]++;
return;
} else if (posN==-1) {//no Ns, count only for featureType==gene
int64 cbI=binarySearchExact<uint64>(cbB1,cbWL.data(),cbWL.size());
if (cbI>=0) {//exact match
cbMatchInd1.push_back((uint64) cbI);
cbMatchString1 = to_string(cbMatchInd1[0]);
cbMatch1=0;
return;
};
};
if (!pSolo.CBmatchWL.mm1) //only exact matches allowed
return;
cbMatch1=0;
if (posN>=0) {//one N
uint32 posNshift=2*(cbSeq1.size()-1-posN);//shift bits for posN
bool matched = false;
for (uint32 jj=0; jj<4; jj++) {
uint64 cbB11=cbB1^(jj<<posNshift);
int64 cbI1=binarySearchExact<uint64>(cbB11,cbWL.data(),cbWL.size());
if (cbI1>=0) {//found match
if (!pSolo.CBmatchWL.mm1_multi_Nbase && matched) {
cbMatchInd1.clear();
cbMatch1=-3;
break; //this is 2nd match, not allowed for N-bases
};
matched = true;
//output all
cbMatchInd1.push_back(cbI1);
++cbMatch1;
cbMatchString1 += ' ' +to_string(cbI1) + ' ' + cbQual1[posN];
};
};
} else {//look for 1MM; posN==-1, no Ns
for (uint32 ii=0; ii<cbSeq1.size(); ii++) {
for (uint32 jj=1; jj<4; jj++) {
int64 cbI1=binarySearchExact<uint64>(cbB1^(jj<<(ii*2)),cbWL.data(),cbWL.size());
if (cbI1>=0) {//found match
//output all
cbMatchInd1.push_back(cbI1);
++cbMatch1;
cbMatchString1 += ' ' +to_string(cbI1) + ' ' + cbQual1.at(cbSeq1.size()-1-ii);
};
};
};
};
if (cbMatch1==0) {//no matches
//stats.V[stats.nNoMatch]++;
cbMatch1=-1;
} else if (cbMatch1==1) {//1 match, no need to record the quality
cbMatchString1 = to_string(cbMatchInd1[0]);
} else if (!pSolo.CBmatchWL.mm1_multi) {//>1 matches, but this is not allowed
//stats.V[stats.nTooMany]++;
cbMatch1=-3;
cbMatchInd1.clear();
cbMatchString1="";
};// else cbMatch contains number of matches, and cbMatchString has CBs and qualities
};
void SoloReadBarcode::addStats(const int32 cbMatch1)
{
if (!pSolo.cbWLyes) //no stats if no WL
return;
if (cbMatch1>1) {
stats.V[stats.nMismatchToMultWL]++;
return;
};
switch (cbMatch1) {
case 0:
cbReadCountExact[cbMatchInd[0]]++;//note that this simply counts reads per exact CB, no checks of genes or UMIs
stats.V[stats.nExactMatch]++;
break;
case 1:
stats.V[stats.nMismatchOneWL]++;
break;
case -1 :
stats.V[stats.nNoMatch]++;
break;
case -2 :
stats.V[stats.nNinCB]++;
break;
case -3 :
stats.V[stats.nTooMany]++;
break;
case -11 :
stats.V[stats.nNoCB]++;
break;
case -12 :
stats.V[stats.nMismatchesInMultCB]++;
break;
};
};
///////////////////////////////////////////////////////////////////////////////////////
bool SoloReadBarcode::convertCheckUMI()
{//check UMIs, return if bad UMIs
if (convertNuclStrToInt64(umiSeq,umiB)!=-1) {//convert and check for Ns
stats.V[stats.nNinUMI]++;//UMIs are not allowed to have Ns
umiCheck=-23;
return false;
};
if (umiB==homoPolymer[0] || umiB==homoPolymer[1] || umiB==homoPolymer[2] || umiB==homoPolymer[3]) {
stats.V[stats.nUMIhomopolymer]++;
umiCheck=-24;
return false;
};
return true;
};
//////////////////////////////////////////////////////////////////////////////////////
void SoloReadBarcode::getCBandUMI(char **readSeq, char **readQual, uint64 *readLen, const string &readNameExtraIn, const uint32 &readFilesIndex, const char *readName)
{
if (pSolo.type==0)
return;
///////////////////////////SmartSeq
if (pSolo.type==pSolo.SoloTypes::SmartSeq) {
cbSeq=cbQual=cbSeqCorrected=""; //TODO make cbSeq=file label
cbMatch=0;
cbMatchInd={readFilesIndex};
cbMatchString=to_string(cbMatchInd[0]);
addStats(cbMatch);
return;
};
cbMatch=-1;
cbMatchString="";
cbMatchInd.clear();
///////////////////////////////////////////////////////////////////////////////////////////////////////
////////// bSeq and bQual
if (P.readFilesTypeN != 10) {//not SAM: barcode seq/qual are at the beginning of readNameExtra
bSeq=std::string(readSeq[pSolo.barcodeRead], readLen[pSolo.barcodeRead]);
bQual=std::string(readQual[pSolo.barcodeRead], readLen[pSolo.barcodeRead]);
} else {//SAM: barcode seq/qual is collected from SAM tags
uint32 pos1 = readNameExtraIn.find_first_not_of(" \t"); //to remove whitespace
string readNameExtraT;
if (pos1==0) {
readNameExtraT = '\t' + readNameExtraIn;//\t needs to be in front of readNameExtra for efficient search
} else {
readNameExtraT = readNameExtraIn;
readNameExtraT[pos1] = '\t';
};
bSeq = {};
for (auto &tag: pSolo.samAtrrBarcodeSeq) {
size_t pos1 = readNameExtraT.find(tag); //find tag
if ( pos1 == std::string::npos ) {
ostringstream errOut;
errOut << "EXITING because of FATAL ERROR in input read file: could not find barcode sequence SAM attribute " << tag << " in read " <<readName <<"\n" ;
errOut << "with SAM attributes: "<< readNameExtraT <<"\n";
errOut << "SOLUTION: make sure that all reads in the input SAM/BAM have all attributes from --soloInputSAMattrBarcodeSeq\n";
exitWithError(errOut.str(),std::cerr, P.inOut->logMain, EXIT_CODE_INPUT_FILES, P);
};
pos1 +=6; //skip 6 chars, e.g. \tCB:Z:
size_t pos2 = readNameExtraT.find('\t', pos1); //find next \t
bSeq += readNameExtraT.substr(pos1,pos2-pos1);
};
bQual = {};
if (pSolo.samAtrrBarcodeQual.size()==0) {//if quality tags are not supplied
bQual.resize(bSeq.size(), 'H');
} else {
for (auto &tag: pSolo.samAtrrBarcodeQual) {
size_t pos1 = readNameExtraT.find(tag); //find tag, and skip 6 chars, e.g. \tCB:Z:
if ( pos1 == std::string::npos ) {
ostringstream errOut;
errOut << "EXITING because of FATAL ERROR in input read file: could not find barcode qualities SAM attribute " << tag << " in read " <<readName <<"\n" ;
errOut << "with SAM attributes: "<< readNameExtraT <<"\n";
errOut << "SOLUTION: make sure that all reads in the input SAM/BAM have all attributes from --soloInputSAMattrBarcodeQual\n";
exitWithError(errOut.str(),std::cerr, P.inOut->logMain, EXIT_CODE_INPUT_FILES, P);
};
pos1 += 6; //skip 6 chars, e.g. \tCB:Z:
size_t pos2 = readNameExtraT.find('\t', pos1); //find next \t
bQual += readNameExtraT.substr(pos1,pos2-pos1);
};
};
if (bQual.size() != bSeq.size()) {
ostringstream errOut;
errOut << "EXITING because of FATAL ERROR in input read file: the total length of barcode qualities is " << bQual.size() << " not equal to the sequence length " << bSeq.size() <<"\n" ;
errOut << "Read ID="<< readName <<" ; Qualities="<< bQual <<" ; Sequence="<< bSeq << " ; Read SAM attributes: "<< readNameExtraT <<"\n";
errOut << "SOLUTION: make sure correct attributes are listed in --soloInputSAMattrBarcodeQual\n";
exitWithError(errOut.str(),std::cerr, P.inOut->logMain, EXIT_CODE_INPUT_FILES, P);
};
};
if ( bSeq.size() != P.pSolo.bL ) {//check barcodeRead length. bSeq.size == bQual.size here, this should have been checked before
if (P.pSolo.bL > 0) {
ostringstream errOut;
errOut << "EXITING because of FATAL ERROR in input read file: the total length of barcode sequence is " << bSeq.size() << " not equal to expected " << P.pSolo.bL <<"\n" <<
"Read ID=" << readName <<" ; Sequence=" << bSeq <<'\n' <<
"SOLUTION: check the formatting of input read files.\n" <<
"If UMI+CB length is not equal to the barcode read length, specify barcode read length with --soloBarcodeReadLength\n" <<
"To avoid checking of barcode read length, specify --soloBarcodeReadLength 0" ;
exitWithError(errOut.str(),std::cerr, P.inOut->logMain, EXIT_CODE_INPUT_FILES, P);
} else if (bSeq.size()<P.pSolo.cbumiL) {//barcode sequence too short - append Ns, will work only if P.pSolo.cbumiL>0
bSeq.append(P.pSolo.cbumiL-bSeq.size(), 'N');
bQual.append(P.pSolo.cbumiL-bQual.size(), 'H');
};
};
if ( pSolo.type != pSolo.SoloTypes::CB_UMI_Simple ) {
for (uint64 ix=0; ix<( P.pSolo.bL>0 ? P.pSolo.bL : bQual.size() ); ix++) {//bL==0 use the whole barcode read for quality scores histogram
qualHist[(uint8)bQual[ix]]++;
};
};
/////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////////
////////////////// cbSeq and umiSeq, and match CB to WL, different CB_UMI types
///////////////////////////CB_UMI_Simple a.k.a Droplet
if ( pSolo.type==pSolo.SoloTypes::CB_UMI_Simple ) {
cbSeq=bSeq.substr(pSolo.cbS-1,pSolo.cbL);
umiSeq=bSeq.substr(pSolo.umiS-1,pSolo.umiL);
cbQual=bQual.substr(pSolo.cbS-1,pSolo.cbL);
umiQual=bQual.substr(pSolo.umiS-1,pSolo.umiL);
for (uint64 ix=0; ix<cbQual.size(); ix++) {
qualHist[(uint8)cbQual[ix]]++;
};
for (uint64 ix=0; ix<umiQual.size(); ix++) {
qualHist[(uint8)umiQual[ix]]++;
};
matchCBtoWL(cbSeq, cbQual, pSolo.cbWL, cbMatch, cbMatchInd, cbMatchString);
if (!convertCheckUMI()) {//UMI conversion
#ifdef MATCH_CellRanger
// this is what CR does - will not use it to avoid differences with / 2.7.6a
// this affects <10 reads raw, only 1 UMI count filtered
if (cbMatch==0)
cbReadCountExact[cbMatchInd[0]]++; //still need to count it as exact before return, even if UMI is not good
#endif
cbMatch=umiCheck;
cbMatchString="";
cbMatchInd.clear();
return;
};
///////////////////////////CB_samTagOut
} else if ( pSolo.type==pSolo.SoloTypes::CB_samTagOut ) {//similar to CB_UMI_Simple, but no UMI, and define cbSeqCorrected
cbSeq=bSeq.substr(pSolo.cbS-1,pSolo.cbL);
umiSeq=bSeq.substr(pSolo.umiS-1,pSolo.umiL);
cbQual=bQual.substr(pSolo.cbS-1,pSolo.cbL);
umiQual=bQual.substr(pSolo.umiS-1,pSolo.umiL);
matchCBtoWL(cbSeq, cbQual, pSolo.cbWL, cbMatch, cbMatchInd, cbMatchString);
if ( cbMatch==0 || cbMatch==1 ) {
cbSeqCorrected=pSolo.cbWLstr[cbMatchInd[0]];
} else {
cbSeqCorrected="";
};
///////////////////////////CB_UMI_Complex
} else if ( pSolo.type==pSolo.SoloTypes::CB_UMI_Complex ) {
cbSeq="";
cbQual="";
umiSeq="";
umiQual="";
uint32 adapterStart=0;
if (pSolo.adapterYes) {
if (localAlignHammingDist(bSeq, pSolo.adapterSeq, adapterStart) > pSolo.adapterMismatchesNmax) {
stats.V[stats.nNoAdapter]++;
cbMatch=-21;
return; //no adapter found
};
};
if (!pSolo.umiV.extractBarcode(bSeq, bQual, adapterStart, umiSeq, umiQual)) {
stats.V[stats.nNoUMI]++;
cbMatch=-22;
return;
};
bool cbMatchGood=true;
if (!convertCheckUMI()) {
cbMatchGood = false;//CB matching will not be done, just extract the sequences
cbMatch = umiCheck;
};
cbMatchInd={0};
for (auto &cb : pSolo.cbV) {//cycle over multiple barcodes
string cbSeq1, cbQual1;
if ( !cb.extractBarcode(bSeq, bQual, adapterStart, cbSeq1, cbQual1)
|| cbSeq1.size() < cb.minLen || cbSeq1.size() >= cb.wl.size() || cb.wl[cbSeq1.size()].size()==0 ) {
//barcode cannot be extracted
if (cbMatchGood) {
cbMatch=-11;
cbMatchGood=false;
};
};
cbSeq += cbSeq1 + "_";
cbQual += cbQual1 + "_";
if (!cbMatchGood)
continue; //continue - to be able to record full cbSeq, cbQual, but no need to match to the WL
int32 cbMatch1;
vector<uint64> cbMatchInd1;
matchCBtoWL(cbSeq1, cbQual1, cb.wl[cbSeq1.size()], cbMatch1, cbMatchInd1, cbMatchString); //cbMatchString is not used for now, multiple matches are not allowed
if (cbMatch1<0) {//no match
cbMatchGood=false;
cbMatch = cbMatch1;
} else if (cbMatch1>0 && cbMatch>0) {//this barcode has >1 1MM match, or previous barcode had a mismatch
cbMatchGood=false;
cbMatch = -12; //marks mismatches in multiple barcodes
} else {//good match
cbMatchInd[0] += cb.wlFactor*(cbMatchInd1[0]+cb.wlAdd[cbSeq1.size()]);
cbMatch=max(cbMatch,cbMatch1);//1 wins over 0
};
};
cbSeq.pop_back();//remove last "_" from file
cbQual.pop_back();
if (cbMatchGood) {
cbMatchString=to_string(cbMatchInd[0]);
};
};
addStats(cbMatch);
};
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