int main(int argc, char* argv[]) {
string program = "pls2fasta";
string versionString = VERSION;
AppendPerforceChangelist(PERFORCE_VERSION_STRING, versionString);
string plsFileName, fastaOutName;
vector<string> plsFileNames;
bool trimByRegion, maskByRegion;
trimByRegion = false;
maskByRegion = false;
int argi = 3;
RegionTable regionTable;
string regionsFOFNName = "";
vector<string> regionFileNames;
bool splitSubreads = true;
int minSubreadLength = 0;
bool addSimulatedData = false;
bool printSimulatedCoordinate = false;
bool printSimulatedSequenceIndex = false;
bool printFastq = false;
bool printCcs = false;
int lineLength = 50;
int minReadScore = 0;
vector<int> holeNumbers;
CommandLineParser clp;
bool printOnlyBest = false;
clp.SetProgramName(program);
clp.SetVersion(versionString);
clp.RegisterStringOption("in.pls.h5", &plsFileName, "Input pls.h5/bax.h5/fofn file.", true);
clp.RegisterStringOption("out.fasta", &fastaOutName, "Output fasta/fastq file.", true);
clp.RegisterPreviousFlagsAsHidden();
clp.RegisterFlagOption("trimByRegion", &trimByRegion, "Trim away low quality regions.");
clp.RegisterFlagOption("maskByRegion", &maskByRegion, "Mask low quality regions with 'N'.");
clp.RegisterStringOption("regionTable", ®ionsFOFNName, "Optional HDF file with a /PulseData/Regions dataset.");
clp.RegisterIntOption("minSubreadLength", &minSubreadLength, "Do not write subreads less than the specified length.", CommandLineParser::PositiveInteger);
clp.RegisterFlagOption("noSplitSubreads", &splitSubreads, "Do not split reads on adapter sequences.");
clp.RegisterIntListOption("holeNumber", &holeNumbers, "Only print this hole number (or list of numbers).");
clp.RegisterFlagOption("fastq", &printFastq, "Print in FASTQ format with quality.");
clp.RegisterFlagOption("ccs", &printCcs, "Print de novo CCS sequences");
clp.RegisterIntOption("lineLength", &lineLength, "Specify fasta/fastq line length", CommandLineParser::PositiveInteger);
clp.RegisterIntOption("minReadScore", &minReadScore, "Minimum read score to print a read. The score is "
"a number between 0 and 1000 and represents the expected accuracy percentage * 10. "
"A typical value would be between 750 and 800. This does not apply to ccs reads.", CommandLineParser::NonNegativeInteger);
clp.RegisterFlagOption("best", &printOnlyBest, "If a CCS sequence exists, print this. Otherwise, print the longest"
"subread. This does not support fastq.");
string description = ("Converts pls.h5/bax.h5/fofn files to fasta or fastq files. Although fasta files are provided"
" with every run, they are not trimmed nor split into subreads. This program takes "
"additional annotation information, such as the subread coordinates and high quality regions "
"and uses them to create fasta sequences that are substrings of all bases called. Most of the time "
"you will want to trim low quality reads, so you should specify -trimByRegion.");
clp.SetProgramSummary(description);
clp.ParseCommandLine(argc, argv);
cerr << "[INFO] " << GetTimestamp() << " [" << program << "] started." << endl;
if (trimByRegion and maskByRegion) {
cout << "ERROR! You cannot both trim and mask regions. Use one or the other." << endl;
exit(1);
}
if (printFastq) {
// Setting lineLength to 0 flags to print on one line.
lineLength = 0;
}
if (FileOfFileNames::IsFOFN(plsFileName)) {
FileOfFileNames::FOFNToList(plsFileName, plsFileNames);
}
else {
plsFileNames.push_back(plsFileName);
}
if (regionsFOFNName == "") {
regionFileNames = plsFileNames;
}
else {
if (FileOfFileNames::IsFOFN(regionsFOFNName)) {
FileOfFileNames::FOFNToList(regionsFOFNName, regionFileNames);
}
else {
regionFileNames.push_back(regionsFOFNName);
}
}
ofstream fastaOut;
CrucialOpen(fastaOutName, fastaOut);
int plsFileIndex;
HDFRegionTableReader hdfRegionReader;
sort(holeNumbers.begin(), holeNumbers.end());
for (plsFileIndex = 0; plsFileIndex < plsFileNames.size(); plsFileIndex++) {
if (trimByRegion or maskByRegion or splitSubreads) {
hdfRegionReader.Initialize(regionFileNames[plsFileIndex]);
hdfRegionReader.ReadTable(regionTable);
regionTable.SortTableByHoleNumber();
}
ReaderAgglomerate reader;
HDFBasReader ccsReader;
if (printOnlyBest) {
ccsReader.SetReadBasesFromCCS();
ccsReader.Initialize(plsFileNames[plsFileIndex]);
}
if (printCcs == false) {
reader.IgnoreCCS();
}
else {
reader.hdfBasReader.SetReadBasesFromCCS();
}
if (addSimulatedData) {
reader.hdfBasReader.IncludeField("SimulatedCoordinate");
reader.hdfBasReader.IncludeField("SimulatedSequenceIndex");
}
if (reader.SetReadFileName(plsFileNames[plsFileIndex]) == 0) {
cout << "ERROR, could not determine file type."
<< plsFileNames[plsFileIndex] << endl;
exit(1);
}
if (reader.Initialize() == 0) {
cout << "ERROR, could not initialize file "
<< plsFileNames[plsFileIndex] << endl;
exit(1);
}
DNALength simulatedCoordinate;
DNALength simulatedSequenceIndex;
reader.SkipReadQuality();
SMRTSequence seq;
vector<ReadInterval> subreadIntervals;;
SMRTSequence ccsSeq;
while (reader.GetNext(seq)) {
if (printOnlyBest) {
ccsReader.GetNext(ccsSeq);
}
if (holeNumbers.size() != 0 and
binary_search(holeNumbers.begin(), holeNumbers.end(), seq.zmwData.holeNumber) == false) {
continue;
}
if (seq.length == 0) {
continue;
}
if (addSimulatedData) {
reader.hdfBasReader.simulatedCoordinateArray.Read(reader.hdfBasReader.curRead-1, reader.hdfBasReader.curRead, &simulatedCoordinate);
reader.hdfBasReader.simulatedSequenceIndexArray.Read(reader.hdfBasReader.curRead-1, reader.hdfBasReader.curRead, &simulatedSequenceIndex);
}
if (printCcs == true) {
if (printFastq == false) {
seq.PrintSeq(fastaOut);
}
else {
seq.PrintFastq(fastaOut, lineLength);
}
continue;
}
//
// Determine the high quality boundaries of the read. This is
// the full read is no hq regions exist, or it is stated to
// ignore regions.
//
DNALength hqReadStart, hqReadEnd;
int hqRegionScore;
if (GetReadTrimCoordinates(seq, seq.zmwData, regionTable, hqReadStart, hqReadEnd, hqRegionScore) == false or
(trimByRegion == false and maskByRegion == false)) {
hqReadStart = 0;
hqReadEnd = seq.length;
}
//
// Mask off the low quality portions of the reads.
//
if (maskByRegion) {
if (hqReadStart > 0) {
fill(&seq.seq[0], &seq.seq[hqReadStart], 'N');
}
if (hqReadEnd != seq.length) {
fill(&seq.seq[hqReadEnd], &seq.seq[seq.length], 'N');
}
}
//
// Now possibly print the full read with masking. This could be handled by making a
//
if (splitSubreads == false) {
ReadInterval wholeRead(0, seq.length);
// The set of subread intervals is just the entire read.
subreadIntervals.clear();
subreadIntervals.push_back(wholeRead);
}
else {
//
// Print subread coordinates no matter whether or not reads have subreads.
//
subreadIntervals.clear(); // clear old, new intervals are appended.
CollectSubreadIntervals(seq, ®ionTable, subreadIntervals);
}
//
// Output all subreads as separate sequences.
//
int intvIndex;
SMRTSequence bestSubreadSequence;
int bestSubreadScore = -1;
int bestSubreadIndex = 0;
int bestSubreadStart = 0, bestSubreadEnd = 0;
SMRTSequence bestSubread;
for (intvIndex = 0; intvIndex < subreadIntervals.size(); intvIndex++) {
SMRTSequence subreadSequence, subreadSequenceRC;
subreadSequence.subreadStart = subreadIntervals[intvIndex].start;
subreadSequence.subreadEnd = subreadIntervals[intvIndex].end;
//
// When trimming by region, only output the parts of the
// subread that overlap the hq region.
//
if (trimByRegion == true) {
subreadSequence.subreadStart = max((DNALength) subreadIntervals[intvIndex].start, hqReadStart);
subreadSequence.subreadEnd = min((DNALength) subreadIntervals[intvIndex].end, hqReadEnd);
}
if (subreadSequence.subreadStart >= subreadSequence.subreadEnd or
subreadSequence.subreadEnd - subreadSequence.subreadStart <= minSubreadLength) {
//
// There is no high qualty portion of this subread. Skip it.
//
continue;
}
if (hqRegionScore < minReadScore) {
continue;
}
//
// Print the subread, adding the coordinates as part of the title.
//
subreadSequence.ReferenceSubstring(seq, subreadSequence.subreadStart,
subreadSequence.subreadEnd - subreadSequence.subreadStart);
stringstream titleStream;
titleStream << seq.title;
if (splitSubreads) {
//
// Add the subread coordinates if splitting on subread.
//
titleStream << "/"
<< subreadSequence.subreadStart
<< "_" << subreadSequence.subreadEnd;
}
//
// If running on simulated data, add where the values were simulated from.
//
if (addSimulatedData) {
titleStream << ((FASTASequence*)&seq)->title << "/chrIndex_"
<< simulatedSequenceIndex << "/position_"<< simulatedCoordinate;
((FASTASequence*)&seq)->CopyTitle(titleStream.str());
}
subreadSequence.CopyTitle(titleStream.str());
//
// Eventually replace with WriterAgglomerate.
//
if (printOnlyBest == false) {
if (subreadSequence.length > 0) {
if (printFastq == false) {
((FASTASequence*)&subreadSequence)->PrintSeq(fastaOut);
}
else {
subreadSequence.PrintFastq(fastaOut, lineLength);
}
}
delete[] subreadSequence.title;
}
else {
int subreadWeightedScore = subreadSequence.length * hqRegionScore;
if (subreadWeightedScore > bestSubreadScore) {
bestSubreadIndex = intvIndex;
bestSubread = subreadSequence;
bestSubreadScore = subreadWeightedScore;
}
}
}
if (printOnlyBest) {
if (ccsSeq.length > 0) {
if (printFastq == false) {
ccsSeq.PrintSeq(fastaOut);
}
else {
ccsSeq.PrintFastq(fastaOut, ccsSeq.length);
}
}
else {
if (bestSubreadScore >= 0) {
if (printFastq == false) {
bestSubread.PrintSeq(fastaOut);
}
else {
bestSubread.PrintFastq(fastaOut, bestSubread.length);
}
bestSubread.Free();
}
}
ccsSeq.Free();
}
seq.Free();
}
reader.Close();
hdfRegionReader.Close();
}
cerr << "[INFO] " << GetTimestamp() << " [" << program << "] ended." << endl;
}
int main(int argc, char* argv[]) {
string inputFileName, outputFileName;
if (argc < 2) {
PrintUsage();
exit(0);
}
vector<string> inputFileNames;
inputFileName = argv[1];
outputFileName = argv[2];
int argi = 3;
RegionTable regionTable;
string regionsFOFNName = "";
vector<string> regionFileNames;
bool splitSubreads = true;
bool useCCS = false;
int minSubreadLength = 1;
while (argi < argc) {
if (strcmp(argv[argi], "-regionTable") == 0) {
regionsFOFNName = argv[++argi];
}
else if (strcmp(argv[argi], "-noSplitSubreads") == 0) {
splitSubreads = false;
}
else if (strcmp(argv[argi], "-minSubreadLength") == 0) {
minSubreadLength = atoi(argv[++argi]);
}
else if (strcmp(argv[argi], "-useccsdenovo") == 0) {
useCCS = true;
}
else {
PrintUsage();
cout << "ERROR! Option " << argv[argi] << " is not supported." << endl;
}
argi++;
}
if (FileOfFileNames::IsFOFN(inputFileName)) {
FileOfFileNames::FOFNToList(inputFileName, inputFileNames);
}
else {
inputFileNames.push_back(inputFileName);
}
if (regionsFOFNName == "") {
regionFileNames = inputFileNames;
}
else {
if (FileOfFileNames::IsFOFN(regionsFOFNName)) {
FileOfFileNames::FOFNToList(regionsFOFNName, regionFileNames);
}
else {
regionFileNames.push_back(regionsFOFNName);
}
}
ofstream fastaOut;
CrucialOpen(outputFileName, fastaOut);
int plsFileIndex;
HDFRegionTableReader hdfRegionReader;
AfgBasWriter afgWriter;
afgWriter.Initialize(outputFileName);
for (plsFileIndex = 0; plsFileIndex < inputFileNames.size(); plsFileIndex++) {
if (splitSubreads) {
hdfRegionReader.Initialize(regionFileNames[plsFileIndex]);
hdfRegionReader.ReadTable(regionTable);
regionTable.SortTableByHoleNumber();
}
ReaderAgglomerate reader;
// reader.SkipReadQuality(); // should have been taken care of by *Filter modules
if (useCCS){
reader.UseCCS();
} else {
reader.IgnoreCCS();
}
reader.Initialize(inputFileNames[plsFileIndex]);
CCSSequence seq;
int seqIndex = 0;
int numRecords = 0;
vector<ReadInterval> subreadIntervals;
while (reader.GetNext(seq)){
++seqIndex;
if (splitSubreads == false) {
if (seq.length >= minSubreadLength) {
afgWriter.Write(seq);
}
seq.Free();
continue;
}
DNALength hqReadStart, hqReadEnd;
int score;
GetReadTrimCoordinates(seq, seq.zmwData, regionTable, hqReadStart, hqReadEnd, score);
subreadIntervals.clear(); // clear old, new intervals are appended.
CollectSubreadIntervals(seq,®ionTable, subreadIntervals);
if (seq.length == 0 and subreadIntervals.size() > 0) {
cout << "WARNING! A high quality interval region exists for a read of length 0." <<endl;
cout << " The offending ZMW number is " << seq.zmwData.holeNumber << endl;
seq.Free();
continue;
}
for (int intvIndex = 0; intvIndex < subreadIntervals.size(); intvIndex++) {
SMRTSequence subreadSequence;
int subreadStart = subreadIntervals[intvIndex].start > hqReadStart ?
subreadIntervals[intvIndex].start : hqReadStart;
int subreadEnd = subreadIntervals[intvIndex].end < hqReadEnd ?
subreadIntervals[intvIndex].end : hqReadEnd;
int subreadLength = subreadEnd - subreadStart;
if (subreadLength < minSubreadLength) continue;
subreadSequence.subreadStart = subreadStart;
subreadSequence.subreadEnd = subreadEnd;
subreadSequence.ReferenceSubstring(seq, subreadStart, subreadLength);
stringstream titleStream;
titleStream << seq.title << "/" << subreadIntervals[intvIndex].start
<< "_" << subreadIntervals[intvIndex].end;
subreadSequence.CopyTitle(titleStream.str());
afgWriter.Write(subreadSequence);
delete[] subreadSequence.title;
}
seq.Free();
}
reader.Close();
hdfRegionReader.Close();
}
}