int main(int argc, const char *argv[]) {
OptArgs opts;
opts.ParseCmdLine(argc, argv);
string queryFile, goldFile;
double epsilon;
bool help = false;
bool version = false;
int allowedWrong = 0;
double maxAbsVal = 0;
double minCorrelation = 1;
opts.GetOption(queryFile, "", 'q', "query-wells");
opts.GetOption(goldFile, "", 'g', "gold-wells");
opts.GetOption(epsilon, "0.0", 'e', "epsilon");
opts.GetOption(allowedWrong, "0", 'm', "max-mismatch");
opts.GetOption(minCorrelation, "1", 'c', "min-cor");
opts.GetOption(maxAbsVal, "1e3", '-', "max-val");
opts.GetOption(help, "false", 'h', "help");
opts.GetOption(version, "false", 'v', "version");
opts.CheckNoLeftovers();
if (version) {
fprintf (stdout, "%s", IonVersion::GetFullVersion("RawWellsEquivalent").c_str());
exit(0);
}
if (queryFile.empty() || goldFile.empty() || help) {
cout << "RawWellsEquivalent - Check to see how similar two wells files are to each other" << endl
<< "options: " << endl
<< " -g,--gold-wells trusted wells to compare against." << endl
<< " -q,--query-wells new wells to check." << endl
<< " -e,--epsilon maximum allowed difference to be considered equivalent." << endl
<< " -m,--max-mixmatch maximum number of non-equivalent entries to allow." << endl
<< " -c,--min-cor minimum correlation allowed to be considered equivalent." << endl
<< " --max-val maximum absolute value considered (avoid extreme values)." << endl
<< " -h,--help this message." << endl
<< "" << endl
<< "usage: " << endl
<< " RawWellsEquivalent -e 10 --query-wells query.wells --gold-wells gold.wells " << endl;
exit(1);
}
NumericalComparison<double> compare = CompareWells(queryFile, goldFile, epsilon, maxAbsVal);
cout << compare.GetCount() << " total values. " << endl
<< compare.GetNumSame() << " (" << (100.0 * compare.GetNumSame())/compare.GetCount() << "%) are equivalent. " << endl
<< compare.GetNumDiff() << " (" << (100.0 * compare.GetNumDiff())/compare.GetCount() << "%) are not equivalent. " << endl
<< "Correlation of: " << compare.GetCorrelation() << endl;
if((compare.GetCount() - allowedWrong) >= compare.GetNumSame() ||
compare.GetCorrelation() < minCorrelation) {
cout << "Wells files not equivalent for allowed mismatch: " << allowedWrong
<< " minimum correlation: " << minCorrelation << endl;
return 1;
}
cout << "Wells files equivalent for allowed mismatch: " << allowedWrong
<< " minimum correlation: " << minCorrelation << endl;
return 0;
}
int main(int argc, const char *argv[]) {
OptArgs opts;
opts.ParseCmdLine(argc, argv);
string queryFile, goldFile;
double epsilon;
bool help = false;
bool version = false;
int allowedWrong = 0;
double maxAbsVal = 0;
double minCorrelation = 1;
bool dumpMisMatch = false;
opts.GetOption(queryFile, "", 'q', "query-wells");
opts.GetOption(goldFile, "", 'g', "gold-wells");
opts.GetOption(epsilon, "0.0", 'e', "epsilon");
opts.GetOption(allowedWrong, "0", 'm', "max-mismatch");
opts.GetOption(minCorrelation, "1", 'c', "min-cor");
opts.GetOption(maxAbsVal, "1e3", '-', "max-val");
opts.GetOption(help, "false", 'h', "help");
opts.GetOption(version, "false", 'v', "version");
opts.GetOption(dumpMisMatch, "false", 'o', "dump-mismatch");
opts.CheckNoLeftovers();
if (version) {
fprintf (stdout, "%s", IonVersion::GetFullVersion("RawWellsEquivalent").c_str());
exit(0);
}
if (queryFile.empty() || goldFile.empty() || help) {
printUsage();
exit(1);
}
DumpMismatches dump(dumpMisMatch);
NumericalComparison<double> compare = CompareWells(queryFile, goldFile, epsilon, maxAbsVal, dump);
cout << compare.GetCount() << " total values. " << endl
<< compare.GetNumSame() << " (" << (100.0 * compare.GetNumSame())/compare.GetCount() << "%) are equivalent. " << endl
<< compare.GetNumDiff() << " (" << (100.0 * compare.GetNumDiff())/compare.GetCount() << "%) are not equivalent. " << endl
<< "Correlation of: " << compare.GetCorrelation() << endl;
if((compare.GetCount() - allowedWrong) > compare.GetNumSame() ||
(compare.GetCorrelation() < minCorrelation && compare.GetCount() != compare.GetNumSame())) {
cout << "Wells files not equivalent for allowed mismatch: " << allowedWrong
<< " minimum correlation: " << minCorrelation << endl;
return 1;
}
cout << "Wells files equivalent for allowed mismatch: " << allowedWrong
<< " minimum correlation: " << minCorrelation << endl;
return 0;
}
int main (int argc, const char *argv[])
{
time_t program_start_time;
time(&program_start_time);
Json::Value calibration_json(Json::objectValue);
DumpStartingStateOfProgram (argc,argv,program_start_time, calibration_json["Calibration"]);
//
// Step 1. Process command line options
//
OptArgs opts;
opts.ParseCmdLine(argc, argv);
CalibrationContext calib_context;
if (not calib_context.InitializeFromOpts(opts)){
PrintHelp_CalModules();
}
HistogramCalibration master_histogram(opts, calib_context);
calib_context.hist_calibration_master = &master_histogram;
LinearCalibrationModel master_linear_model(opts, calib_context);
calib_context.linear_model_master = &master_linear_model;
opts.CheckNoLeftovers();
//
// Step 2. Execute threaded calibration
//
time_t calibration_start_time;
time(&calibration_start_time);
pthread_mutex_init(&calib_context.read_mutex, NULL);
pthread_mutex_init(&calib_context.write_mutex, NULL);
pthread_t worker_id[calib_context.num_threads];
for (int worker = 0; worker < calib_context.num_threads; worker++)
if (pthread_create(&worker_id[worker], NULL, CalibrationWorker, &calib_context)) {
cerr << "Calibration ERROR: Problem starting thread" << endl;
exit (EXIT_FAILURE);
}
for (int worker = 0; worker < calib_context.num_threads; worker++)
pthread_join(worker_id[worker], NULL);
pthread_mutex_destroy(&calib_context.read_mutex);
pthread_mutex_destroy(&calib_context.write_mutex);
time_t calibration_end_time;
time(&calibration_end_time);
//
// Step 3. Create models, write output, and close modules
//
// HP histogram calibration
if (master_histogram.CreateCalibrationModel())
master_histogram.ExportModelToJson(calibration_json["HPHistogram"]);
// Linear Model
if (master_linear_model.CreateCalibrationModel())
master_linear_model.ExportModelToJson(calibration_json["LinearModel"], "");
// Transfer stuff from calibration context and close bam reader
calib_context.Close(calibration_json["Calibration"]);
time_t program_end_time;
time(&program_end_time);
calibration_json["Calibration"]["end_time"] = get_time_iso_string(program_end_time);
calibration_json["Calibration"]["total_duration"] = (Json::Int)difftime(program_end_time,program_start_time);
calibration_json["Calibration"]["calibration_duration"] = (Json::Int)difftime(calibration_end_time,calibration_start_time);
SaveJson(calibration_json, calib_context.filename_json);
return EXIT_SUCCESS;
}
int main (int argc, const char *argv[])
{
printf ("------------- bamrealignment --------------\n");
OptArgs opts;
opts.ParseCmdLine(argc, argv);
vector<int> score_vals(4);
string input_bam = opts.GetFirstString ('i', "input", "");
string output_bam = opts.GetFirstString ('o', "output", "");
opts.GetOption(score_vals, "4,-6,-5,-2", 's', "scores");
int clipping = opts.GetFirstInt ('c', "clipping", 2);
bool anchors = opts.GetFirstBoolean ('a', "anchors", true);
int bandwidth = opts.GetFirstInt ('b', "bandwidth", 10);
bool verbose = opts.GetFirstBoolean ('v', "verbose", false);
bool debug = opts.GetFirstBoolean ('d', "debug", false);
int format = opts.GetFirstInt ('f', "format", 1);
int num_threads = opts.GetFirstInt ('t', "threads", 8);
string log_fname = opts.GetFirstString ('l', "log", "");
if (input_bam.empty() or output_bam.empty())
return PrintHelp();
opts.CheckNoLeftovers();
std::ofstream logf;
if (log_fname.size ())
{
logf.open (log_fname.c_str ());
if (!logf.is_open ())
{
fprintf (stderr, "bamrealignment: Failed to open log file %s\n", log_fname.c_str());
return 1;
}
}
BamReader reader;
if (!reader.Open(input_bam)) {
fprintf(stderr, "bamrealignment: Failed to open input file %s\n", input_bam.c_str());
return 1;
}
SamHeader header = reader.GetHeader();
RefVector refs = reader.GetReferenceData();
BamWriter writer;
writer.SetNumThreads(num_threads);
if (format == 1)
writer.SetCompressionMode(BamWriter::Uncompressed);
else
writer.SetCompressionMode(BamWriter::Compressed);
if (!writer.Open(output_bam, header, refs)) {
fprintf(stderr, "bamrealignment: Failed to open output file %s\n", output_bam.c_str());
return 1;
}
// The meat starts here ------------------------------------
if (verbose)
cout << "Verbose option is activated, each alignment will print to screen." << endl
<< " After a read hit RETURN to continue to the next one," << endl
<< " or press q RETURN to quit the program," << endl
<< " or press s Return to silence verbose," << endl
<< " or press c RETURN to continue printing without further prompt." << endl << endl;
unsigned int readcounter = 0;
unsigned int mapped_readcounter = 0;
unsigned int realigned_readcounter = 0;
unsigned int modified_alignment_readcounter = 0;
unsigned int pos_update_readcounter = 0;
unsigned int failed_clip_realigned_readcount = 0;
unsigned int already_perfect_readcount = 0;
unsigned int bad_md_tag_readcount = 0;
unsigned int error_recreate_ref_readcount = 0;
unsigned int error_clip_anchor_readcount = 0;
unsigned int error_sw_readcount = 0;
unsigned int error_unclip_readcount = 0;
unsigned int start_position_shift;
int orig_position;
int new_position;
string md_tag, new_md_tag, input = "x";
vector<CigarOp> new_cigar_data;
vector<MDelement> new_md_data;
bool position_shift = false;
time_t start_time = time(NULL);
Realigner aligner;
aligner.verbose_ = verbose;
aligner.debug_ = debug;
if (!aligner.SetScores(score_vals))
cout << "bamrealignment: Four scores need to be provided: match, mismatch, gap open, gap extend score!" << endl;
aligner.SetAlignmentBandwidth(bandwidth);
BamAlignment alignment;
while(reader.GetNextAlignment(alignment)){
readcounter ++;
position_shift = false;
if ( (readcounter % 100000) == 0 )
cout << "Processed " << readcounter << " reads. Elapsed time: " << (time(NULL) - start_time) << endl;
if (alignment.IsMapped()) {
orig_position = alignment.Position;
mapped_readcounter++;
aligner.SetClipping(clipping, !alignment.IsReverseStrand());
if (aligner.verbose_) {
cout << endl;
if (alignment.IsReverseStrand())
cout << "The read is from the reverse strand." << endl;
else
cout << "The read is from the forward strand." << endl;
}
if (!alignment.GetTag("MD", md_tag)) {
if (aligner.verbose_)
cout << "Warning: Skipping read " << alignment.Name << ". It is mapped but missing MD tag." << endl;
if (logf.is_open ())
logf << alignment.Name << '\t' << alignment.IsReverseStrand() << '\t' << alignment.RefID << '\t' << setfill ('0') << setw (8) << orig_position << '\t' << "MISSMD" << '\n';
bad_md_tag_readcount++;
} else if (aligner.CreateRefFromQueryBases(alignment.QueryBases, alignment.CigarData, md_tag, anchors)) {
bool clipfail = false;
if (Realigner::CR_ERR_CLIP_ANCHOR == aligner.GetCreateRefError ())
{
clipfail = true;
failed_clip_realigned_readcount ++;
}
if (!aligner.computeSWalignment(new_cigar_data, new_md_data, start_position_shift)) {
if (aligner.verbose_)
cout << "Error in the alignment! Not updating read information." << endl;
if (logf.is_open ())
logf << alignment.Name << '\t' << alignment.IsReverseStrand() << '\t' << alignment.RefID << '\t' << setfill ('0') << setw (8) << orig_position << '\t' << "SWERR" << '\n';
error_sw_readcount++;
writer.SaveAlignment(alignment); // Write alignment unchanged
continue;
}
if (!aligner.addClippedBasesToTags(new_cigar_data, new_md_data, alignment.QueryBases.size())) {
if (aligner.verbose_)
cout << "Error when adding clipped anchors back to tags! Not updating read information." << endl;
if (logf.is_open ())
logf << alignment.Name << '\t' << alignment.IsReverseStrand() << '\t' << alignment.RefID << '\t' << setfill ('0') << setw (8) << orig_position << '\t' << "UNCLIPERR" << '\n';
writer.SaveAlignment(alignment); // Write alignment unchanged
error_unclip_readcount ++;
continue;
}
new_md_tag = aligner.GetMDstring(new_md_data);
realigned_readcounter++;
// adjust start position of read
if (!aligner.LeftAnchorClipped() and start_position_shift != 0) {
new_position = aligner.updateReadPosition(alignment.CigarData, (int)start_position_shift, alignment.Position);
if (new_position != alignment.Position) {
pos_update_readcounter++;
position_shift = true;
alignment.Position = new_position;
}
}
if (position_shift || alignment.CigarData.size () != new_cigar_data.size () || md_tag != new_md_tag)
{
if (logf.is_open ())
{
logf << alignment.Name << '\t' << alignment.IsReverseStrand() << '\t' << alignment.RefID << '\t' << setfill ('0') << setw (8) << orig_position << '\t' << "MOD";
if (position_shift)
logf << "-SHIFT";
if (clipfail)
logf << " NOCLIP";
logf << '\n';
}
modified_alignment_readcounter++;
}
else
{
if (logf.is_open ())
{
logf << alignment.Name << '\t' << alignment.IsReverseStrand() << '\t' << alignment.RefID << '\t' << setfill ('0') << setw (8) << orig_position << '\t' << "UNMOD";
if (clipfail)
logf << " NOCLIP";
logf << '\n';
}
}
if (aligner.verbose_){
cout << alignment.Name << endl;
cout << "------------------------------------------" << endl;
// Wait for input to continue or quit program
if (input.size() == 0)
input = 'x';
else if (input[0] != 'c' and input[0] != 'C')
getline(cin, input);
if (input.size()>0){
if (input[0] == 'q' or input[0] == 'Q')
return 1;
else if (input[0] == 's' or input[0] == 'S')
aligner.verbose_ = false;
}
}
// Finally update alignment information
alignment.CigarData = new_cigar_data;
alignment.EditTag("MD", "Z" , new_md_tag);
} // end of CreateRef else if
else {
switch (aligner.GetCreateRefError ())
{
case Realigner::CR_ERR_RECREATE_REF:
if (logf.is_open ())
logf << alignment.Name << '\t' << alignment.IsReverseStrand() << '\t' << alignment.RefID << '\t' << setfill ('0') << setw (8) << orig_position << '\t' << "RECRERR" << '\n';
error_recreate_ref_readcount++;
break;
case Realigner::CR_ERR_CLIP_ANCHOR:
if (logf.is_open ())
logf << alignment.Name << '\t' << alignment.IsReverseStrand() << '\t' << alignment.RefID << '\t' << setfill ('0') << setw (8) << orig_position << '\t' << "CLIPERR" << '\n';
error_clip_anchor_readcount++;
break;
default:
// On a good run this writes way too many reads to the log file - don't want to create a too large txt file
// if (logf.is_open ())
//logf << alignment.Name << '\t' << alignment.IsReverseStrand() << '\t' << alignment.RefID << '\t' << setfill ('0') << setw (8) << orig_position << '\t' << "PERFECT" << '\n';
already_perfect_readcount++;
break;
}
if (aligner.verbose_) {
cout << alignment.Name << endl;
cout << "------------------------------------------" << endl;
// Wait for input to continue or quit program
if (input.size() == 0)
input = 'x';
else if (input[0] != 'c' and input[0] != 'C')
getline(cin, input);
if (input.size()>0){
if (input[0] == 'q' or input[0] == 'Q')
return 1;
else if (input[0] == 's' or input[0] == 'S')
aligner.verbose_ = false;
}
}
}
// --- Debug output for Rajesh ---
if (debug && aligner.invalid_cigar_in_input) {
aligner.verbose_ = true;
cout << "Invalid cigar string / md tag pair in read " << alignment.Name << endl;
// Rerun reference generation to display error
aligner.CreateRefFromQueryBases(alignment.QueryBases, alignment.CigarData, md_tag, anchors);
aligner.verbose_ = verbose;
aligner.invalid_cigar_in_input = false;
}
// --- --- ---
} // end of if isMapped
writer.SaveAlignment(alignment);
} // end while loop over reads
if (aligner.invalid_cigar_in_input)
cerr << "WARNING bamrealignment: There were invalid cigar string / md tag pairs in the input bam file." << endl;
// ----------------------------------------------------------------
// program end -- output summary information
cout << " File: " << input_bam << endl
<< " Total reads: " << readcounter << endl
<< " Mapped reads: " << mapped_readcounter << endl;
if (bad_md_tag_readcount)
cout << " Skipped: bad MD tags: " << bad_md_tag_readcount << endl;
if (error_recreate_ref_readcount)
cout << " Skipped: unable to recreate ref: " << error_recreate_ref_readcount << endl;
if (error_clip_anchor_readcount)
cout << " Skipped: error clipping anchor: " << error_clip_anchor_readcount << endl;
cout << " Skipped: already perfect: " << already_perfect_readcount << endl
<< " Total reads realigned: " << mapped_readcounter - already_perfect_readcount - bad_md_tag_readcount - error_recreate_ref_readcount - error_clip_anchor_readcount << endl;
if (failed_clip_realigned_readcount)
cout << " (including " << failed_clip_realigned_readcount << " that failed to clip)" << endl;
if (error_sw_readcount)
cout << " Failed to complete SW alignment: " << error_sw_readcount << endl;
if (error_unclip_readcount)
cout << " Failed to unclip anchor: " << error_unclip_readcount << endl;
cout << " Succesfully realigned: " << realigned_readcounter << endl
<< " Modified alignments: " << modified_alignment_readcounter << endl
<< " Shifted position: " << pos_update_readcounter << endl;
cout << "Processing time: " << (time(NULL)-start_time) << " seconds." << endl;
cout << "INFO: The output BAM file may be unsorted." << endl;
cout << "------------------------------------------" << endl;
return 0;
}
int PrepareHotspots(int argc, const char *argv[])
{
OptArgs opts;
opts.ParseCmdLine(argc, argv);
string input_bed_filename = opts.GetFirstString ('b', "input-bed", "");
string input_vcf_filename = opts.GetFirstString ('v', "input-vcf", "");
string input_real_vcf_filename = opts.GetFirstString ('p', "input-real-vcf", "");
string output_hot_vcf = opts.GetFirstString ('q', "output-fake-hot-vcf", "");
string output_bed_filename = opts.GetFirstString ('d', "output-bed", "");
string output_vcf_filename = opts.GetFirstString ('o', "output-vcf", "");
string reference_filename = opts.GetFirstString ('r', "reference", "");
string unmerged_bed = opts.GetFirstString ('u', "unmerged-bed", "");
bool left_alignment = opts.GetFirstBoolean('a', "left-alignment", false);
bool filter_bypass = opts.GetFirstBoolean('f', "filter-bypass", false);
bool allow_block_substitutions = opts.GetFirstBoolean('s', "allow-block-substitutions", true);
bool strict_check = opts.GetFirstBoolean('S', "strict-check", true);
opts.CheckNoLeftovers();
if((input_bed_filename.empty() == (input_vcf_filename.empty() and input_real_vcf_filename.empty())) or
(output_bed_filename.empty() and output_vcf_filename.empty()) or reference_filename.empty()) {
PrepareHotspotsHelp();
return 1;
}
if ((not input_real_vcf_filename.empty()) and (output_vcf_filename.empty() or not input_vcf_filename.empty())) {
PrepareHotspotsHelp();
return 1;
}
// Populate chromosome list from reference.fai
// Use mmap to fetch the entire reference
int ref_handle = open(reference_filename.c_str(),O_RDONLY);
struct stat ref_stat;
fstat(ref_handle, &ref_stat);
char *ref = (char *)mmap(0, ref_stat.st_size, PROT_READ, MAP_SHARED, ref_handle, 0);
FILE *fai = fopen((reference_filename+".fai").c_str(), "r");
if (!fai) {
fprintf(stderr, "ERROR: Cannot open %s.fai\n", reference_filename.c_str());
return 1;
}
vector<Reference> ref_index;
map<string,int> ref_map;
char line[1024], chrom_name[1024];
while (fgets(line, 1024, fai) != NULL) {
Reference ref_entry;
long chr_start;
if (5 != sscanf(line, "%1020s\t%ld\t%ld\t%d\t%d", chrom_name, &ref_entry.size, &chr_start,
&ref_entry.bases_per_line, &ref_entry.bytes_per_line))
continue;
ref_entry.chr = chrom_name;
ref_entry.start = ref + chr_start;
ref_index.push_back(ref_entry);
ref_map[ref_entry.chr] = (int) ref_index.size() - 1;
}
fclose(fai);
junction junc;
if (!unmerged_bed.empty()) {
FILE *fp = fopen(unmerged_bed.c_str(), "r");
if (!fp) {
fprintf(stderr, "ERROR: Cannot open %s\n", unmerged_bed.c_str());
return 1;
}
char line2[65536];
junc.init(ref_index.size());
bool line_overflow = false;
while (fgets(line2, 65536, fp) != NULL) {
if (line2[0] and line2[strlen(line2)-1] != '\n' and strlen(line2) == 65535) {
line_overflow = true;
continue;
}
if (line_overflow) {
line_overflow = false;
continue;
}
if (strstr(line2, "track")) continue;
char chr[100];
int b, e;
sscanf(line2, "%s %d %d", chr, &b, &e);
junc.add(ref_map[chr], b, e);
}
fclose(fp);
}
// Load input BED or load input VCF, group by chromosome
deque<LineStatus> line_status;
vector<deque<Allele> > alleles(ref_index.size());
if (!input_bed_filename.empty()) {
FILE *input = fopen(input_bed_filename.c_str(),"r");
if (!input) {
fprintf(stderr,"ERROR: Cannot open %s\n", input_bed_filename.c_str());
return 1;
}
char line2[65536];
int line_number = 0;
bool line_overflow = false;
while (fgets(line2, 65536, input) != NULL) {
if (line2[0] and line2[strlen(line2)-1] != '\n' and strlen(line2) == 65535) {
line_overflow = true;
continue;
}
line_number++;
if (line_overflow) {
line_overflow = false;
line_status.push_back(LineStatus(line_number));
line_status.back().filter_message_prefix = "Malformed hotspot BED line: line length exceeds 64K";
continue;
}
if (strncmp(line2, "browser", 7) == 0)
continue;
if (strncmp(line2, "track", 5) == 0) {
if (string::npos != string(line2).find("allowBlockSubstitutions=true"))
allow_block_substitutions = true;
continue;
}
// OID= table has special meaning
if (string::npos != string(line2).find("OID=")) {
line_status.push_back(LineStatus(line_number));
line_status.back().filter_message_prefix = "Bed line contains OID=";
continue;
}
char *current_chr = strtok(line2, "\t\r\n");
char *current_start = strtok(NULL, "\t\r\n");
char *current_end = strtok(NULL, "\t\r\n");
char *current_id = strtok(NULL, "\t\r\n");
char *penultimate = strtok(NULL, "\t\r\n");
char *ultimate = strtok(NULL, "\t\r\n");
for (char *next = strtok(NULL, "\t\r\n"); next; next = strtok(NULL, "\t\r\n")) {
penultimate = ultimate;
ultimate = next;
}
if (!current_chr or !current_start or !current_end or !current_id or !penultimate or !ultimate) {
line_status.push_back(LineStatus(line_number));
line_status.back().filter_message_prefix = "Malformed hotspot BED line: expected at least 6 fields";
continue;
}
Allele allele;
string string_chr(current_chr);
if (ref_map.find(string_chr) != ref_map.end())
allele.chr_idx = ref_map[string_chr];
else if (ref_map.find("chr"+string_chr) != ref_map.end())
allele.chr_idx = ref_map["chr"+string_chr];
else if (string_chr == "MT" and ref_map.find("chrM") != ref_map.end())
allele.chr_idx = ref_map["chrM"];
else {
line_status.push_back(LineStatus(line_number));
line_status.back().filter_message_prefix = "Unknown chromosome name: ";
line_status.back().filter_message = string_chr;
continue;
}
allele.pos = strtol(current_start,NULL,10);
allele.id = current_id;
char *current_ref = NULL;
char *current_alt = NULL;
for (char *next = strtok(penultimate, ";"); next; next = strtok(NULL, ";")) {
if (strncmp(next,"REF=",4) == 0)
current_ref = next;
else if (strncmp(next,"OBS=",4) == 0)
current_alt = next;
else if (strncmp(next,"ANCHOR=",7) == 0) {
// ignore ANCHOR
} else {
char *value = next;
while (*value and *value != '=')
++value;
if (*value == '=')
*value++ = 0;
allele.custom_tags[next] = value;
}
}
if (!current_ref or !current_alt) {
line_status.push_back(LineStatus(line_number));
line_status.back().filter_message_prefix = "Malformed hotspot BED line: REF and OBS fields required in penultimate column";
continue;
}
for (char *pos = current_ref+4; *pos; ++pos)
allele.ref += toupper(*pos);
for (char *pos = current_alt+4; *pos; ++pos)
allele.alt += toupper(*pos);
// here is the place to check the length of the hotspot cover the amplicon junction. ZZ
/*
if (junc.contain(allele.chr_idx, allele.pos, (unsigned int) allele.ref.size())) {
line_status.push_back(LineStatus(line_number));
line_status.back().filter_message_prefix = "hotspot BED line contain the complete overlapping region of two amplicon, the variant cannot be detected by tvc";
continue;
}
if (not junc.contained_in_ampl(allele.chr_idx, allele.pos, (unsigned int) allele.ref.size())) {
line_status.push_back(LineStatus(line_number));
line_status.back().filter_message_prefix = "hotspot BED line is not contained in any amplicon, the variant cannot be detected by tvc";
continue;
}
*/
allele.filtered = false;
line_status.push_back(LineStatus(line_number));
allele.line_status = &line_status.back();
allele.opos = allele.pos;
allele.oref = allele.ref;
allele.oalt = allele.alt;
alleles[allele.chr_idx].push_back(allele);
//line_status.back().allele = &alleles[allele.chr_idx].back();
line_status.back().chr_idx = allele.chr_idx;
line_status.back().opos = allele.opos;
line_status.back().id = allele.id;
}
fclose(input);
}
if (!input_vcf_filename.empty() or !input_real_vcf_filename.empty()) {
bool real_vcf = false;
FILE *input;
FILE *out_real = NULL;
FILE *out_hot = NULL;
int fake_ = 0;
int hn = 1;
if (!input_real_vcf_filename.empty()) {
real_vcf = true;
input = fopen(input_real_vcf_filename.c_str(),"r");
if (!input) {
fprintf(stderr,"ERROR: Cannot open %s\n", input_real_vcf_filename.c_str());
return 1;
}
out_real = fopen(output_vcf_filename.c_str(), "w");
if (!out_real) {
fprintf(stderr,"ERROR: Cannot open %s\n", output_vcf_filename.c_str());
return 1;
}
if (!output_hot_vcf.empty()) {
out_hot = fopen(output_hot_vcf.c_str(), "w");
if (!out_hot) {
fprintf(stderr,"ERROR: Cannot open %s\n", output_hot_vcf.c_str());
return 1;
}
} else out_hot = stdout;
fprintf(out_hot, "##fileformat=VCFv4.1\n##allowBlockSubstitutions=true\n#CHROM POS ID REF ALT QUAL FILTER INFO\n");
} else {
input = fopen(input_vcf_filename.c_str(),"r");
if (!input) {
fprintf(stderr,"ERROR: Cannot open %s\n", input_vcf_filename.c_str());
return 1;
}
}
char line2[65536];
char line3[65536];
int line_number = 0;
bool line_overflow = false;
list<one_vcfline> vcflist;
char last_chr[1024] = "";
while (fgets(line2, 65536, input) != NULL) {
if (line2[0] and line2[strlen(line2)-1] != '\n' and strlen(line2) == 65535) {
line_overflow = true;
continue;
}
line_number++;
if (line_overflow) {
line_overflow = false;
line_status.push_back(LineStatus(line_number));
line_status.back().filter_message_prefix = "Malformed hotspot VCF line: line length exceeds 64K";
continue;
}
if (strncmp(line2, "##allowBlockSubstitutions=true", 30) == 0) {
allow_block_substitutions = true;
continue;
}
if (line2[0] == '#') {
if (out_real) { fprintf(out_real, "%s", line2);}
continue;
}
if (real_vcf) strcpy(line3, line2);
char *current_chr = strtok(line2, "\t\r\n");
char *current_start = strtok(NULL, "\t\r\n");
char *current_id = strtok(NULL, "\t\r\n");
char *current_ref = strtok(NULL, "\t\r\n");
char *current_alt = strtok(NULL, "\t\r\n");
strtok(NULL, "\t\r\n"); // Ignore QUAL
strtok(NULL, "\t\r\n"); // Ignore FILTER
char *current_info = strtok(NULL, "\t\r\n");
strtok(NULL, "\t\r\n");
char *gt = strtok(NULL, "\t\r\n");
if (!current_chr or !current_start or !current_id or !current_ref or !current_alt) {
line_status.push_back(LineStatus(line_number));
if (real_vcf) line_status.back().filter_message_prefix = "Malformed real VCF line: expected at least 5 fields";
else line_status.back().filter_message_prefix = "Malformed hotspot VCF line: expected at least 5 fields";
continue;
}
string string_chr(current_chr);
int chr_idx = 0;
if (ref_map.find(string_chr) != ref_map.end())
chr_idx = ref_map[string_chr];
else if (ref_map.find("chr"+string_chr) != ref_map.end())
chr_idx = ref_map["chr"+string_chr];
else if (string_chr == "MT" and ref_map.find("chrM") != ref_map.end())
chr_idx = ref_map["chrM"];
else {
line_status.push_back(LineStatus(line_number));
line_status.back().filter_message_prefix = "Unknown chromosome name: ";
line_status.back().filter_message = string_chr;
continue;
}
for (char *pos = current_ref; *pos; ++pos)
*pos = toupper(*pos);
for (char *pos = current_alt; *pos; ++pos)
*pos = toupper(*pos);
// Process custom tags
vector<string> bstrand;
vector<string> hp_max_length;
string raw_oid;
string raw_omapalt;
string raw_oalt;
string raw_oref;
string raw_opos;
if (current_info) {
string raw_bstrand;
string raw_hp_max_length;
for (char *next = strtok(current_info, ";"); next; next = strtok(NULL, ";")) {
char *value = next;
while (*value and *value != '=')
++value;
if (*value == '=')
*value++ = 0;
if (strcmp(next, "TYPE") == 0)
continue;
if (strcmp(next, "HRUN") == 0)
continue;
if (strcmp(next, "HBASE") == 0)
continue;
if (strcmp(next, "FR") == 0)
continue;
if (strcmp(next, "OPOS") == 0) {
raw_opos = value;
continue;
}
if (strcmp(next, "OREF") == 0) {
raw_oref = value;
continue;
}
if (strcmp(next, "OALT") == 0) {
raw_oalt = value;
continue;
}
if (strcmp(next, "OID") == 0) {
raw_oid = value;
continue;
}
if (strcmp(next, "OMAPALT") == 0) {
raw_omapalt = value;
continue;
}
if (strcmp(next, "BSTRAND") == 0) {
raw_bstrand = value;
continue;
}
if (strcmp(next, "hp_max_length") == 0) {
raw_hp_max_length = value;
continue;
}
}
if (not raw_bstrand.empty())
split(raw_bstrand, ',', bstrand);
if (not raw_hp_max_length.empty())
split(raw_hp_max_length, ',', hp_max_length);
}
if (real_vcf) {
//fprintf(stderr, "%s\n", gt);
if (gt == NULL) continue;
// get gt
int g1 = atoi(gt), g2;
gt = strchr(gt, '/');
if (gt) g2 = atoi(gt+1);
else {fprintf(stderr, "GT not formatted right\n"); exit(1);}
//if (g1 == 0 and g2 == 0) continue;
unsigned int cur_pos = atoi(current_start);
one_vcfline newline(current_ref, current_alt, cur_pos, g1, g2, line3);
bool new_chr = false;
if (strcmp(current_chr, last_chr) != 0) {
new_chr = true;
}
while (not vcflist.empty()) {
if ((not new_chr) and vcflist.front().pos+strlen(vcflist.front().ref) > cur_pos) break;
if (vcflist.front().produce_hot_vcf(last_chr, out_real, hn, out_hot)) fake_++;
vcflist.pop_front();
}
if (new_chr) strcpy(last_chr, current_chr);
for (list<one_vcfline>::iterator it = vcflist.begin(); it != vcflist.end(); it++) {
it->check_subset(newline);
}
if (not newline.alts.empty()) vcflist.push_back(newline);
continue;
}
unsigned int allele_idx = 0;
for (char *sub_alt = strtok(current_alt,","); sub_alt; sub_alt = strtok(NULL,",")) {
Allele allele;
allele.chr_idx = chr_idx;
allele.ref = current_ref;
allele.alt = sub_alt;
allele.pos = strtol(current_start,NULL,10)-1;
allele.id = current_id;
if (allele.id == ".")
allele.id = "hotspot";
allele.filtered = false;
line_status.push_back(LineStatus(line_number));
allele.line_status = &line_status.back();
allele.opos = allele.pos;
allele.oref = allele.ref;
allele.oalt = allele.alt;
if (allele_idx < bstrand.size()) {
if (bstrand[allele_idx] != ".")
allele.custom_tags["BSTRAND"] = bstrand[allele_idx];
}
if (allele_idx < hp_max_length.size()) {
if (hp_max_length[allele_idx] != ".")
allele.custom_tags["hp_max_length"] = hp_max_length[allele_idx];
}
alleles[allele.chr_idx].push_back(allele);
//line_status.back().allele = &alleles[allele.chr_idx].back();
line_status.back().chr_idx = allele.chr_idx;
line_status.back().opos = allele.opos;
line_status.back().id = allele.id;
allele_idx++;
}
}
fclose(input);
if (real_vcf) {
while (not vcflist.empty()) {
if (vcflist.front().produce_hot_vcf(last_chr, out_real, hn, out_hot)) fake_++;
vcflist.pop_front();
}
fclose(out_real);
fclose(out_hot);
if (fake_ > 0)
return 0;
else return 1;
}
}
// Process by chromosome:
// - Verify reference allele
// - Left align
// - Sort
// - Filter for block substitutions, write
FILE *output_vcf = NULL;
if (!output_vcf_filename.empty()) {
output_vcf = fopen(output_vcf_filename.c_str(), "w");
if (!output_vcf) {
fprintf(stderr,"ERROR: Cannot open %s for writing\n", output_vcf_filename.c_str());
return 1;
}
fprintf(output_vcf, "##fileformat=VCFv4.1\n");
if (allow_block_substitutions)
fprintf(output_vcf, "##allowBlockSubstitutions=true\n");
fprintf(output_vcf, "#CHROM\tPOS\tID\tREF\tALT\tQUAL\tFILTER\tINFO\n");
}
FILE *output_bed = NULL;
if (!output_bed_filename.empty()) {
output_bed = fopen(output_bed_filename.c_str(), "w");
if (!output_bed) {
fprintf(stderr,"ERROR: Cannot open %s for writing\n", output_bed_filename.c_str());
if (output_vcf)
fclose(output_vcf);
return 1;
}
if (allow_block_substitutions)
fprintf(output_bed, "track name=\"hotspot\" type=bedDetail allowBlockSubstitutions=true\n");
else
fprintf(output_bed, "track name=\"hotspot\" type=bedDetail\n");
}
for (int chr_idx = 0; chr_idx < (int)ref_index.size(); ++chr_idx) {
for (deque<Allele>::iterator A = alleles[chr_idx].begin(); A != alleles[chr_idx].end(); ++A) {
// check bed file
if (junc.contain(A->chr_idx, A->pos, (unsigned int) A->ref.size())) {
A->filtered = true;
A->line_status->filter_message_prefix = "hotspot BED line contain the complete overlapping region of two amplicon, the variant cannot be detected by tvc";
continue;
}
if (not junc.contained_in_ampl(A->chr_idx, A->pos, (unsigned int) A->ref.size())) {
A->filtered = true;
A->line_status->filter_message_prefix = "hotspot BED line is not contained in any amplicon, the variant cannot be detected by tvc";
continue;
}
// Invalid characters
bool valid = true;
for (const char *c = A->ref.c_str(); *c ; ++c)
if (*c != 'A' and *c != 'C' and *c != 'G' and *c != 'T')
valid = false;
for (const char *c = A->alt.c_str(); *c ; ++c)
if (*c != 'A' and *c != 'C' and *c != 'G' and *c != 'T')
valid = false;
if (not valid) {
A->filtered = true;
A->line_status->filter_message_prefix = "REF and/or ALT contain characters other than ACGT: ";
A->line_status->filter_message = "REF = " + A->ref + " ALT = " + A->alt;
continue;
}
// Filter REF == ALT
if (A->ref == A->alt) {
A->filtered = true;
A->line_status->filter_message_prefix = "REF and ALT alleles equal";
continue;
}
// Confirm reference allele.
string ref_expected;
for (int idx = 0; idx < (int) A->ref.size(); ++idx)
ref_expected += ref_index[chr_idx].base(A->pos + idx);
if (A->ref != ref_expected) {
A->filtered = true;
A->line_status->filter_message_prefix = "Provided REF allele does not match reference: ";
A->line_status->filter_message = "Expected " + ref_expected + ", found " + A->ref;
continue;
}
// Trim
int ref_start = 0;
int ref_end = A->ref.size();
int alt_end = A->alt.size();
// Option 1: trim all trailing bases;
//while(ref_end and alt_end and A->ref[ref_end-1] == A->alt[alt_end-1]) {
// --ref_end;
// --alt_end;
//}
// Option 2: trim all leading basees;
//while (ref_start < ref_end and ref_start < alt_end and A->ref[ref_start] == A->alt[ref_start])
// ++ref_start;
// Option 3: trim anchor base if vcf
if (!input_vcf_filename.empty()) {
if (ref_end and alt_end and (ref_end == 1 or alt_end == 1) and A->ref[0] == A->alt[0])
ref_start = 1;
}
A->pos += ref_start;
A->ref = A->ref.substr(ref_start, ref_end-ref_start);
A->alt = A->alt.substr(ref_start, alt_end-ref_start);
ref_end -= ref_start;
alt_end -= ref_start;
// Left align
if (left_alignment && A->custom_tags.find("BSTRAND") == A->custom_tags.end()) { // black list variant not to be left aligned.
string trailing;
int can_do = 0, need_do = 0;
int ref_end_orig= ref_end, alt_end_orig = alt_end;
while(ref_end and alt_end and A->ref[ref_end-1] == A->alt[alt_end-1]) {
ref_end--; alt_end--;
}
if (ref_end == 0 || alt_end == 0) {
can_do = need_do = 1; // indel type, ZZ
} else {
int tmp_start = ref_start;
int ref_end_0 = ref_end, alt_end_0 = alt_end; // end after remove trailing match ZZ
while (tmp_start < ref_end and tmp_start < alt_end and A->ref[tmp_start] == A->alt[tmp_start])
++tmp_start;
if (tmp_start == ref_end || tmp_start == alt_end) {
can_do = 1; need_do = 0; // indel but indel is not at the left. ZZ
} else {
ref_end--; alt_end--;
while(ref_end and alt_end and A->ref[ref_end-1] == A->alt[alt_end-1]) {
ref_end--; alt_end--;
}
if (ref_end == 0 || alt_end == 0) {
// complex with 1 bp MM at right end
can_do = need_do = 1;
if (ref_end + alt_end == 0) need_do = 0; // SNP
} else {
int tmp_start0 = tmp_start; // start after removing leading matches
tmp_start++;
while (tmp_start < ref_end_orig and tmp_start < alt_end_orig and A->ref[tmp_start] == A->alt[tmp_start])
tmp_start++;
if (tmp_start >= ref_end_0 || tmp_start >= alt_end_0 || ref_end <= tmp_start0 || alt_end <= tmp_start0) {
// 1MM plus indel in middle, by definition cannot move the indel left enough to change A->pos
can_do = 1; need_do = 0;
} // else real complex
}
}
}
if (!can_do or !need_do) {
// do nothing
// if !can_do need add some more DP
ref_end = ref_end_orig;
alt_end = alt_end_orig;
} else {
// left align the indel part, here either ref_end = 0 or alt_end = 0
int opos = A->pos;
while (A->pos > 0) {
char nuc = ref_index[chr_idx].base(A->pos-1);
if (ref_end > 0 and A->ref[ref_end-1] != nuc)
break;
if (alt_end > 0 and A->alt[alt_end-1] != nuc)
break;
A->ref = string(1,nuc) + A->ref;
A->alt = string(1,nuc) + A->alt;
A->pos--;
}
if (ref_end != ref_end_orig) {
// trailing part is aligned, the whole ref and alt need to be kept. ZZ
ref_end = A->ref.size();
alt_end = A->alt.size();
}
if (junc.contain(chr_idx, A->pos, ref_end) or not junc.contained_in_ampl(chr_idx, A->pos, ref_end)) {
// after left align the hotspot contain an overlap region, revert to the original ZZ
if (opos != A->pos) {
A->ref.erase(0, opos-A->pos);
A->alt.erase(0, opos-A->pos);
A->pos = opos;
ref_end = ref_end_orig;
alt_end = alt_end_orig;
}
}
}
}
A->ref.resize(ref_end);
A->alt.resize(alt_end);
// Filter block substitutions: take 1
if (ref_end > 0 and alt_end > 0 and ref_end != alt_end and not allow_block_substitutions and not filter_bypass) {
A->filtered = true;
A->line_status->filter_message_prefix = "Block substitutions not supported";
continue;
}
}
if (output_bed) {
// Sort - without anchor base
stable_sort(alleles[chr_idx].begin(), alleles[chr_idx].end(), compare_alleles);
// Write
for (deque<Allele>::iterator I = alleles[chr_idx].begin(); I != alleles[chr_idx].end(); ++I) {
if (I->filtered)
continue;
fprintf(output_bed, "%s\t%ld\t%ld\t%s\tREF=%s;OBS=%s",
ref_index[chr_idx].chr.c_str(), I->pos, I->pos + I->ref.size(), I->id.c_str(),
I->ref.c_str(), I->alt.c_str());
for (map<string,string>::iterator C = I->custom_tags.begin(); C != I->custom_tags.end(); ++C)
fprintf(output_bed, ";%s=%s", C->first.c_str(), C->second.c_str());
fprintf(output_bed, "\tNONE\n");
/*
if (I->pos)
fprintf(output_bed, "%s\t%ld\t%ld\t%s\t0\t+\tREF=%s;OBS=%s;ANCHOR=%c\tNONE\n",
ref_index[chr_idx].chr.c_str(), I->pos, I->pos + I->ref.size(), I->id.c_str(),
I->ref.c_str(), I->alt.c_str(), ref_index[chr_idx].base(I->pos-1));
else
fprintf(output_bed, "%s\t%ld\t%ld\t%s\t0\t+\tREF=%s;OBS=%s;ANCHOR=\tNONE\n",
ref_index[chr_idx].chr.c_str(), I->pos, I->pos + I->ref.size(), I->id.c_str(),
I->ref.c_str(), I->alt.c_str());
*/
}
}
if (output_vcf) {
// Add anchor base to indels
for (deque<Allele>::iterator I = alleles[chr_idx].begin(); I != alleles[chr_idx].end(); ++I) {
if (I->filtered)
continue;
if (not I->ref.empty() and not I->alt.empty())
continue;
if (I->pos == 0) {
I->filtered = true;
I->line_status->filter_message_prefix = "INDELs at chromosome start not supported";
continue;
}
I->pos--;
I->ref = string(1,ref_index[chr_idx].base(I->pos)) + I->ref;
I->alt = string(1,ref_index[chr_idx].base(I->pos)) + I->alt;
}
// Sort - with anchor base
stable_sort(alleles[chr_idx].begin(), alleles[chr_idx].end(), compare_alleles);
// Merge alleles, remove block substitutions, write
for (deque<Allele>::iterator A = alleles[chr_idx].begin(); A != alleles[chr_idx].end(); ) {
string max_ref;
deque<Allele>::iterator B = A;
for (; B != alleles[chr_idx].end() and B->pos == A->pos; ++B)
if (!B->filtered and max_ref.size() < B->ref.size())
max_ref = B->ref;
bool filtered = true;
map<string,set<string> > unique_alts_and_ids;
for (deque<Allele>::iterator I = A; I != B; ++I) {
if (I->filtered)
continue;
string new_alt = I->alt + max_ref.substr(I->ref.size());
if (new_alt.size() > 1 and max_ref.size() > 1 and new_alt.size() != max_ref.size() and not allow_block_substitutions and not filter_bypass) {
I->filtered = true;
I->line_status->filter_message_prefix = "Block substitutions not supported (post-merge)";
continue;
}
I->ref = max_ref;
I->alt = new_alt;
// Filter alleles with duplicate ALT + ID pairs
map<string,set<string> >::iterator alt_iter = unique_alts_and_ids.find(new_alt);
if (alt_iter != unique_alts_and_ids.end()) {
if (alt_iter->second.count(I->id) > 0) {
I->filtered = true;
I->line_status->filter_message_prefix = "Duplicate allele and ID";
continue;
}
}
unique_alts_and_ids[new_alt].insert(I->id);
filtered = false;
}
if (not filtered) {
fprintf(output_vcf, "%s\t%ld\t.\t%s\t",
ref_index[chr_idx].chr.c_str(), A->pos+1, max_ref.c_str());
bool comma = false;
map<string,map<string,string> > unique_alts_and_tags;
set<string> unique_tags;
set<string> unique_alt_alleles;
for (deque<Allele>::iterator I = A; I != B; ++I) {
if (I->filtered)
continue;
unique_alts_and_tags[I->alt].insert(I->custom_tags.begin(), I->custom_tags.end());
for (map<string,string>::iterator S = I->custom_tags.begin(); S != I->custom_tags.end(); ++S)
unique_tags.insert(S->first);
if (unique_alt_alleles.count(I->alt) > 0)
continue;
unique_alt_alleles.insert(I->alt);
if (comma)
fprintf(output_vcf, ",");
comma = true;
fprintf(output_vcf, "%s", I->alt.c_str());
}
/*
for (deque<Allele>::iterator I = A; I != B; ++I) {
if (I->filtered)
continue;
map<string,map<string,string> >::iterator Q = unique_alts_and_tags.find(I->alt);
if (comma)
fprintf(output_vcf, ",");
comma = true;
if (Q == unique_alts_and_tags.end()) {fprintf(output_vcf, "."); continue;}
fprintf(output_vcf, "%s", Q->first.c_str());
}
*/
fprintf(output_vcf, "\t.\t.\tOID=");
comma = false;
for (deque<Allele>::iterator I = A; I != B; ++I) {
if (I->filtered)
continue;
if (comma)
fprintf(output_vcf, ",");
comma = true;
fprintf(output_vcf, "%s", I->id.c_str());
}
fprintf(output_vcf, ";OPOS=");
comma = false;
for (deque<Allele>::iterator I = A; I != B; ++I) {
if (I->filtered)
continue;
if (comma)
fprintf(output_vcf, ",");
comma = true;
fprintf(output_vcf, "%ld", I->opos+1);
}
fprintf(output_vcf, ";OREF=");
comma = false;
for (deque<Allele>::iterator I = A; I != B; ++I) {
if (I->filtered)
continue;
if (comma)
fprintf(output_vcf, ",");
comma = true;
fprintf(output_vcf, "%s", I->oref.c_str());
}
fprintf(output_vcf, ";OALT=");
comma = false;
for (deque<Allele>::iterator I = A; I != B; ++I) {
if (I->filtered)
continue;
if (comma)
fprintf(output_vcf, ",");
comma = true;
fprintf(output_vcf, "%s", I->oalt.c_str());
}
fprintf(output_vcf, ";OMAPALT=");
comma = false;
for (deque<Allele>::iterator I = A; I != B; ++I) {
if (I->filtered)
continue;
if (comma)
fprintf(output_vcf, ",");
comma = true;
fprintf(output_vcf, "%s", I->alt.c_str());
}
for (set<string>::iterator S = unique_tags.begin(); S != unique_tags.end(); ++S) {
fprintf(output_vcf, ";%s=", S->c_str());
comma=false;
for (deque<Allele>::iterator I = A; I != B; ++I) {
if (I->filtered)
continue;
map<string,map<string,string> >::iterator Q = unique_alts_and_tags.find(I->alt);
if (comma)
fprintf(output_vcf, ",");
comma = true;
if (Q == unique_alts_and_tags.end()) {fprintf(output_vcf, "."); continue;}
map<string,string>::iterator W = Q->second.find(*S);
if (W == Q->second.end())
fprintf(output_vcf, ".");
else
fprintf(output_vcf, "%s", W->second.c_str());
}
}
// fprintf(output_vcf, ";%s=%s", S->first.c_str(), S->second.c_str());
fprintf(output_vcf, "\n");
}
A = B;
}
}
}
if (output_bed) {
fflush(output_bed);
fclose(output_bed);
}
if (output_vcf) {
fflush(output_vcf);
fclose(output_vcf);
}
int lines_ignored = 0;
for (deque<LineStatus>::iterator L = line_status.begin(); L != line_status.end(); ++L) {
if (L->filter_message_prefix) {
if (L->chr_idx >= 0)
printf("Line %d ignored: [%s:%ld %s] %s%s\n", L->line_number, ref_index[L->chr_idx].chr.c_str(), L->opos+1, L->id.c_str(),
L->filter_message_prefix, L->filter_message.c_str());
else
printf("Line %d ignored: %s%s\n", L->line_number, L->filter_message_prefix, L->filter_message.c_str());
lines_ignored++;
}
}
printf("Ignored %d out of %d lines\n", lines_ignored, (int)line_status.size());
munmap(ref, ref_stat.st_size);
close(ref_handle);
if (lines_ignored > 0 and strict_check) return 1;
return 0;
}
int main (int argc, const char *argv[])
{
time_t program_start_time;
time(&program_start_time);
Json::Value calibration_json(Json::objectValue);
DumpStartingStateOfProgram (argc,argv,program_start_time, calibration_json["Calibration"]);
//
// Step 1. Process command line options
//
OptArgs opts;
opts.ParseCmdLine(argc, argv);
// enable floating point exceptions during program execution
if (opts.GetFirstBoolean('-', "float-exceptions", true)) {
cout << "Calibration: Floating point exceptions enabled." << endl;
feenableexcept(FE_DIVBYZERO | FE_INVALID | FE_OVERFLOW);
} //*/
CalibrationContext calib_context;
if (not calib_context.InitializeFromOpts(opts)){
PrintHelp_CalModules();
}
HistogramCalibration master_histogram(opts, calib_context);
calib_context.hist_calibration_master = &master_histogram;
LinearCalibrationModel master_linear_model(opts, calib_context);
calib_context.linear_model_master = &master_linear_model;
opts.CheckNoLeftovers();
//
// Step 2. Execute threaded calibration
//
int calibration_thread_time = 0;
if (calib_context.successive_fit) {
// first train linear model
if (master_linear_model.DoTraining()) {
int l_thread_time = 0;
for (int i_iteration=0; i_iteration<calib_context.num_train_iterations; i_iteration++) {
cout << " -Training Iteration " << i_iteration+1;
l_thread_time = ExecuteThreadedCalibrationTraining(calib_context);
// Activate master linear model after every round of training
master_linear_model.CreateCalibrationModel(false); // make linear model
master_linear_model.SetModelGainsAndOffsets(); // expand for use in basecalling
calibration_thread_time += l_thread_time;
calib_context.bam_reader.Rewind(); // reset all files for another pass
cout << " Duration = " << l_thread_time << endl;
}
}
// Then apply it during polish model training
if (master_histogram.DoTraining()) {
calib_context.local_fit_linear_model = false;
calib_context.local_fit_polish_model = true;
calibration_thread_time += ExecuteThreadedCalibrationTraining(calib_context);
}
}
else {
// Single pass in which both models are fit jointly
calibration_thread_time=ExecuteThreadedCalibrationTraining(calib_context);
}
//
// Step 3. Create models, write output, and close modules
//
// Linear Model
if (master_linear_model.CreateCalibrationModel())
master_linear_model.ExportModelToJson(calibration_json["LinearModel"], "");
// HP histogram calibration
if (master_histogram.CreateCalibrationModel())
master_histogram.ExportModelToJson(calibration_json["HPHistogram"]);
// Transfer stuff from calibration context and close bam reader
calib_context.Close(calibration_json["Calibration"]);
time_t program_end_time;
time(&program_end_time);
calibration_json["Calibration"]["end_time"] = get_time_iso_string(program_end_time);
calibration_json["Calibration"]["total_duration"] = (Json::Int)difftime(program_end_time,program_start_time);
calibration_json["Calibration"]["calibration_duration"] = (Json::Int)calibration_thread_time;
SaveJson(calibration_json, calib_context.filename_json);
return EXIT_SUCCESS;
}
int main(int argc, const char *argv[]) {
OptArgs opts;
opts.ParseCmdLine(argc, argv);
int hpLength;
string statsOut;
string alignmentOut;
string pairedOut;
string flowsOut;
string summaryOut;
string samFile;
string qScoreCol;
string wellsFile;
string bfmaskFile;
string snrFile;
string binnedHpSigFile;
string flowErrFile;
string gcErrFile;
int gcWin;
string flowOrder;
string keySeq;
int numFlows;
bool help;
int qLength;
double colCenter;
double rowCenter;
int colSize;
int rowSize;
int sampleSize;
string wellsToUse;
string run1, run2;
opts.GetOption(run1, "", '-', "sff1");
opts.GetOption(run2, "", '-', "sff2");
opts.GetOption(wellsToUse, "", '-', "use-wells");
opts.GetOption(samFile, "", '-', "sam-parsed");
opts.GetOption(statsOut, "", '-', "stats-out");
opts.GetOption(flowsOut, "", '-', "flows-out");
opts.GetOption(alignmentOut, "", '-', "align-out");
opts.GetOption(summaryOut, "", '-', "summary-out");
opts.GetOption(pairedOut, "", '-', "paired-out");
opts.GetOption(numFlows, "40", '-', "num-flows");
opts.GetOption(hpLength, "6", '-', "max-hp");
opts.GetOption(qScoreCol, "q7Len", '-', "qscore-col");
opts.GetOption(qLength, "25", '-', "min-qlength");
opts.GetOption(help, "false", 'h', "help");
opts.GetOption(wellsFile, "", '-', "wells-file");
opts.GetOption(bfmaskFile, "", '-', "bfmask-file");
opts.GetOption(snrFile, "", '-', "snr-file");
opts.GetOption(binnedHpSigFile, "", '-', "binned-hp-sig-file");
opts.GetOption(flowErrFile, "", '-', "flow-err-file");
opts.GetOption(gcErrFile, "", '-', "gc-err-file");
opts.GetOption(flowOrder, "", '-', "flow-order");
opts.GetOption(keySeq, "", '-', "key-seq");
opts.GetOption(colCenter, "0.5", '-', "col-center");
opts.GetOption(rowCenter, "0.5", '-', "row-center");
opts.GetOption(colSize, "0", '-', "col-size");
opts.GetOption(rowSize, "0", '-', "row-size");
opts.GetOption(gcErrFile, "", '-', "gc-err-file");
opts.GetOption(gcWin, "40", '-', "gc-win");
opts.GetOption(sampleSize, "100000", '-', "sample-size");
if (help || samFile.empty() || statsOut.empty() || summaryOut.empty()) {
usage();
}
opts.CheckNoLeftovers();
// Some checks to make sure sensible bounds have been set
if(colCenter < 0 || colCenter > 1) {
cerr << "AnalyzeHPErrs - col-center must be in the range [0,1]" << endl;
exit(1);
}
if(rowCenter < 0 || rowCenter > 1) {
cerr << "AnalyzeHPErrs - row-center must be in the range [0,1]" << endl;
exit(1);
}
if(colSize < 0) {
cerr << "AnalyzeHPErrs - col-size cannot be negative." << endl;
exit(1);
}
if(rowSize < 0) {
cerr << "AnalyzeHPErrs - row-size cannot be negative." << endl;
exit(1);
}
// Determine rows & cols if a bfmask file was supplied
int nRow=0;
int nCol=0;
if(!bfmaskFile.empty()) {
if(GetRowColFromBfmask(bfmaskFile, &nRow, &nCol)) {
cerr << "AnalyzeHPErrs - problem determining rows & columns from bfmask file " << bfmaskFile << endl;
exit(1);
}
}
// Set up fds object
FlowDiffStats* fds;
if (!run1.empty()) {
SffDiffStats* sds = new SffDiffStats(hpLength, nCol, nRow, qScoreCol, run1, run2);
if (!pairedOut.empty())
sds->SetPairedOut(pairedOut);
fds = dynamic_cast<FlowDiffStats*>(sds);
}
else {
GenomeDiffStats* gds = new GenomeDiffStats(hpLength, nCol, nRow, qScoreCol);
if(alignmentOut != "") {
gds->SetAlignmentsOut(alignmentOut);
}
if (!flowsOut.empty()) {
gds->SetFlowsOut(flowsOut);
}
fds = dynamic_cast<FlowDiffStats*>(gds);
}
if (gcErrFile != "") {
fds->SetFlowGCOut(gcErrFile);
fds->SetGCWindowSize(gcWin);
}
if(keySeq != "") {
fds->SetKeySeq(keySeq);
}
if(flowOrder != "") {
fds->SetFlowOrder(flowOrder);
}
fds->SetStatsOut(statsOut);
if (!wellsToUse.empty()) {
std::vector<int> wells;
std::vector<bool> use;
ReadSetFromFile(wellsToUse, 0, wells);
use.resize(nRow * nCol, false);
int count = 0;
ReservoirSample<int> wellSample(sampleSize);
for (size_t i = 0; i < wells.size(); i++) {
wellSample.Add(wells[i]);
}
wells = wellSample.GetData();
for (size_t i = 0; i < wells.size(); i++) {
use[wells[i]] = true;
count++;
}
cout << "Read: " << count << " reads." << endl;
fds->SetWellToAnalyze(use);
}
// Set integer-value row & column bounds
int minRow=-1;
int maxRow=-1;
int minCol=-1;
int maxCol=-1;
if(colSize > 0 || rowSize > 0) {
if(bfmaskFile.empty()) {
cerr << "AnalyzeHPErrs - must specify bfmask file when restricting row or column ranges" << endl;
exit(1);
}
if(rowSize > 0) {
minRow = floor(nRow * rowCenter - rowSize / 2.0);
maxRow = minRow + rowSize;
minRow = std::max(0,minRow);
maxRow = std::min(nRow,maxRow);
}
if(colSize > 0) {
minCol = floor(nCol * colCenter - colSize / 2.0);
maxCol = minCol + colSize;
minCol = std::max(0,minCol);
maxCol = std::min(nCol,maxCol);
}
}
if (wellsFile != "") {
std::vector<int32_t> xSubset, ySubset;
fds->FillInSubset(samFile, qLength, minRow, maxRow, minCol, maxCol, xSubset, ySubset);
if(bfmaskFile.empty()) {
cerr << "AnalyzeHPErrs - must specify bfmask file when specifying wells file" << endl;
exit(1);
}
fds->SetWellsFile(wellsFile, nRow, nCol, numFlows, xSubset, ySubset);
}
if (snrFile != "") {
cout << "Opening snr file: " << snrFile << endl;
fds->SetSNROut(snrFile);
}
if (binnedHpSigFile != "") {
cout << "Opening binned HP signal file: " << binnedHpSigFile << endl;
fds->SetBinnedHpSigOut(binnedHpSigFile);
}
if (flowErrFile != "") {
cout << "Opening flow err file: " << flowErrFile << endl;
fds->SetFlowErrOut(flowErrFile);
}
ofstream summary;
summary.open(summaryOut.c_str());
cout << "Reading and analyzing alignments from: " << samFile << endl;
if(minCol > -1 || maxCol > -1)
cout << " Restricting to " << (maxCol-minCol) << " cols in the range [" << minCol << "," << maxCol << ")" << endl;
if(minRow > -1 || maxRow > -1)
cout << " Restricting to " << (maxRow-minRow) << " rows in the range [" << minRow << "," << maxRow << ")" << endl;
fds->SetAlignmentInFile(samFile);
fds->FilterAndCompare(numFlows, summary, qLength, minRow, maxRow, minCol, maxCol);
summary.close();
delete fds;
cout << "Done." << endl;
return 0;
}
int PrepareHotspots(int argc, const char *argv[])
{
OptArgs opts;
opts.ParseCmdLine(argc, argv);
string input_bed_filename = opts.GetFirstString ('b', "input-bed", "");
string input_vcf_filename = opts.GetFirstString ('v', "input-vcf", "");
string output_bed_filename = opts.GetFirstString ('d', "output-bed", "");
string output_vcf_filename = opts.GetFirstString ('o', "output-vcf", "");
string reference_filename = opts.GetFirstString ('r', "reference", "");
bool left_alignment = opts.GetFirstBoolean('a', "left-alignment", false);
bool filter_bypass = opts.GetFirstBoolean('f', "filter-bypass", false);
bool allow_block_substitutions = opts.GetFirstBoolean('s', "allow-block-substitutions", false);
opts.CheckNoLeftovers();
if((input_bed_filename.empty() == input_vcf_filename.empty()) or
(output_bed_filename.empty() and output_vcf_filename.empty()) or reference_filename.empty()) {
PrepareHotspotsHelp();
return 1;
}
// Populate chromosome list from reference.fai
// Use mmap to fetch the entire reference
int ref_handle = open(reference_filename.c_str(),O_RDONLY);
struct stat ref_stat;
fstat(ref_handle, &ref_stat);
char *ref = (char *)mmap(0, ref_stat.st_size, PROT_READ, MAP_SHARED, ref_handle, 0);
FILE *fai = fopen((reference_filename+".fai").c_str(), "r");
if (!fai) {
fprintf(stderr, "ERROR: Cannot open %s.fai\n", reference_filename.c_str());
return 1;
}
vector<Reference> ref_index;
map<string,int> ref_map;
char line[1024], chrom_name[1024];
while (fgets(line, 1024, fai) != NULL) {
Reference ref_entry;
long chr_start;
if (5 != sscanf(line, "%s\t%ld\t%ld\t%d\t%d", chrom_name, &ref_entry.size, &chr_start,
&ref_entry.bases_per_line, &ref_entry.bytes_per_line))
continue;
ref_entry.chr = chrom_name;
ref_entry.start = ref + chr_start;
ref_index.push_back(ref_entry);
ref_map[ref_entry.chr] = (int) ref_index.size() - 1;
}
fclose(fai);
// Load input BED or load input VCF, group by chromosome
deque<LineStatus> line_status;
vector<deque<Allele> > alleles(ref_index.size());
if (!input_bed_filename.empty()) {
FILE *input = fopen(input_bed_filename.c_str(),"r");
if (!input) {
fprintf(stderr,"ERROR: Cannot open %s\n", input_bed_filename.c_str());
return 1;
}
char line2[65536];
int line_number = 0;
bool line_overflow = false;
while (fgets(line2, 65536, input) != NULL) {
if (line2[0] and line2[strlen(line2)-1] != '\n' and strlen(line2) == 65535) {
line_overflow = true;
continue;
}
line_number++;
if (line_overflow) {
line_overflow = false;
line_status.push_back(LineStatus(line_number));
line_status.back().filter_message_prefix = "Malformed hotspot BED line: line length exceeds 64K";
continue;
}
if (strncmp(line2, "browser", 7) == 0)
continue;
if (strncmp(line2, "track", 5) == 0) {
if (string::npos != string(line2).find("allowBlockSubstitutions=true"))
allow_block_substitutions = true;
continue;
}
char *current_chr = strtok(line2, "\t\r\n");
char *current_start = strtok(NULL, "\t\r\n");
char *current_end = strtok(NULL, "\t\r\n");
char *current_id = strtok(NULL, "\t\r\n");
char *penultimate = strtok(NULL, "\t\r\n");
char *ultimate = strtok(NULL, "\t\r\n");
for (char *next = strtok(NULL, "\t\r\n"); next; next = strtok(NULL, "\t\r\n")) {
penultimate = ultimate;
ultimate = next;
}
if (!current_chr or !current_start or !current_end or !current_id or !penultimate or !ultimate) {
line_status.push_back(LineStatus(line_number));
line_status.back().filter_message_prefix = "Malformed hotspot BED line: expected at least 6 fields";
continue;
}
Allele allele;
string string_chr(current_chr);
if (ref_map.find(string_chr) != ref_map.end())
allele.chr_idx = ref_map[string_chr];
else if (ref_map.find("chr"+string_chr) != ref_map.end())
allele.chr_idx = ref_map["chr"+string_chr];
else if (string_chr == "MT" and ref_map.find("chrM") != ref_map.end())
allele.chr_idx = ref_map["chrM"];
else {
line_status.push_back(LineStatus(line_number));
line_status.back().filter_message_prefix = "Unknown chromosome name: ";
line_status.back().filter_message = string_chr;
continue;
}
allele.pos = strtol(current_start,NULL,10);
allele.id = current_id;
char *current_ref = NULL;
char *current_alt = NULL;
for (char *next = strtok(penultimate, ";"); next; next = strtok(NULL, ";")) {
if (strncmp(next,"REF=",4) == 0)
current_ref = next;
else if (strncmp(next,"OBS=",4) == 0)
current_alt = next;
}
if (!current_ref or !current_alt) {
line_status.push_back(LineStatus(line_number));
line_status.back().filter_message_prefix = "Malformed hotspot BED line: REF and OBS fields required in penultimate column";
continue;
}
for (char *pos = current_ref+4; *pos; ++pos)
allele.ref += toupper(*pos);
for (char *pos = current_alt+4; *pos; ++pos)
allele.alt += toupper(*pos);
allele.filtered = false;
line_status.push_back(LineStatus(line_number));
allele.line_status = &line_status.back();
allele.opos = allele.pos;
allele.oref = allele.ref;
allele.oalt = allele.alt;
alleles[allele.chr_idx].push_back(allele);
line_status.back().allele = &alleles[allele.chr_idx].back();
}
fclose(input);
}
if (!input_vcf_filename.empty()) {
FILE *input = fopen(input_vcf_filename.c_str(),"r");
if (!input) {
fprintf(stderr,"ERROR: Cannot open %s\n", input_vcf_filename.c_str());
return 1;
}
char line2[65536];
int line_number = 0;
bool line_overflow = false;
while (fgets(line2, 65536, input) != NULL) {
if (line2[0] and line2[strlen(line2)-1] != '\n' and strlen(line2) == 65535) {
line_overflow = true;
continue;
}
line_number++;
if (line_overflow) {
line_overflow = false;
line_status.push_back(LineStatus(line_number));
line_status.back().filter_message_prefix = "Malformed hotspot VCF line: line length exceeds 64K";
continue;
}
if (strncmp(line2, "##allowBlockSubstitutions=true", 30) == 0) {
allow_block_substitutions = true;
continue;
}
if (line2[0] == '#')
continue;
char *current_chr = strtok(line2, "\t\r\n");
char *current_start = strtok(NULL, "\t\r\n");
char *current_id = strtok(NULL, "\t\r\n");
char *current_ref = strtok(NULL, "\t\r\n");
char *current_alt = strtok(NULL, "\t\r\n");
if (!current_chr or !current_start or !current_id or !current_ref or !current_alt) {
line_status.push_back(LineStatus(line_number));
line_status.back().filter_message_prefix = "Malformed hotspot VCF line: expected at least 5 fields";
continue;
}
string string_chr(current_chr);
int chr_idx = 0;
if (ref_map.find(string_chr) != ref_map.end())
chr_idx = ref_map[string_chr];
else if (ref_map.find("chr"+string_chr) != ref_map.end())
chr_idx = ref_map["chr"+string_chr];
else if (string_chr == "MT" and ref_map.find("chrM") != ref_map.end())
chr_idx = ref_map["chrM"];
else {
line_status.push_back(LineStatus(line_number));
line_status.back().filter_message_prefix = "Unknown chromosome name: ";
line_status.back().filter_message = string_chr;
continue;
}
for (char *pos = current_ref; *pos; ++pos)
*pos = toupper(*pos);
for (char *pos = current_alt; *pos; ++pos)
*pos = toupper(*pos);
for (char *sub_alt = strtok(current_alt,","); sub_alt; sub_alt = strtok(NULL,",")) {
Allele allele;
allele.chr_idx = chr_idx;
allele.ref = current_ref;
allele.alt = sub_alt;
allele.pos = strtol(current_start,NULL,10)-1;
allele.id = current_id;
if (allele.id == ".")
allele.id = "hotspot";
allele.filtered = false;
line_status.push_back(LineStatus(line_number));
allele.line_status = &line_status.back();
allele.opos = allele.pos;
allele.oref = allele.ref;
allele.oalt = allele.alt;
alleles[allele.chr_idx].push_back(allele);
line_status.back().allele = &alleles[allele.chr_idx].back();
}
}
fclose(input);
}
// Process by chromosome:
// - Verify reference allele
// - Left align
// - Sort
// - Filter for block substitutions, write
FILE *output_vcf = NULL;
if (!output_vcf_filename.empty()) {
output_vcf = fopen(output_vcf_filename.c_str(), "w");
if (!output_vcf) {
fprintf(stderr,"ERROR: Cannot open %s for writing\n", output_vcf_filename.c_str());
return 1;
}
fprintf(output_vcf, "##fileformat=VCFv4.1\n");
if (allow_block_substitutions)
fprintf(output_vcf, "##allowBlockSubstitutions=true\n");
fprintf(output_vcf, "#CHROM\tPOS\tID\tREF\tALT\tQUAL\tFILTER\tINFO\n");
}
FILE *output_bed = NULL;
if (!output_bed_filename.empty()) {
output_bed = fopen(output_bed_filename.c_str(), "w");
if (!output_bed) {
fprintf(stderr,"ERROR: Cannot open %s for writing\n", output_bed_filename.c_str());
if (output_vcf)
fclose(output_vcf);
return 1;
}
if (allow_block_substitutions)
fprintf(output_bed, "track name=\"hotspot\" type=bedDetail allowBlockSubstitutions=true\n");
else
fprintf(output_bed, "track name=\"hotspot\" type=bedDetail\n");
}
for (int chr_idx = 0; chr_idx < (int)ref_index.size(); ++chr_idx) {
for (deque<Allele>::iterator A = alleles[chr_idx].begin(); A != alleles[chr_idx].end(); ++A) {
// Invalid characters
bool valid = true;
for (const char *c = A->ref.c_str(); *c ; ++c)
if (*c != 'A' and *c != 'C' and *c != 'G' and *c != 'T')
valid = false;
for (const char *c = A->alt.c_str(); *c ; ++c)
if (*c != 'A' and *c != 'C' and *c != 'G' and *c != 'T')
valid = false;
if (not valid) {
A->filtered = true;
A->line_status->filter_message_prefix = "REF and/or ALT contain characters other than ACGT: ";
A->line_status->filter_message = "REF = " + A->ref + " ALT = " + A->alt;
continue;
}
// Filter REF == ALT
if (A->ref == A->alt) {
A->filtered = true;
A->line_status->filter_message_prefix = "REF and ALT alleles equal";
continue;
}
// Confirm reference allele.
string ref_expected;
for (int idx = 0; idx < (int) A->ref.size(); ++idx)
ref_expected += ref_index[chr_idx].base(A->pos + idx);
if (A->ref != ref_expected) {
A->filtered = true;
A->line_status->filter_message_prefix = "Provided REF allele does not match reference: ";
A->line_status->filter_message = "Expected " + ref_expected + ", found " + A->ref;
continue;
}
// Trim
int ref_start = 0;
int ref_end = A->ref.size();
int alt_end = A->alt.size();
// Option 1: trim all trailing bases
//while(ref_end and alt_end and A->ref[ref_end-1] == A->alt[alt_end-1]) {
// --ref_end;
// --alt_end;
//}
// Option 2: trim all leading basees
//while (ref_start < ref_end and ref_start < alt_end and A->ref[ref_start] == A->alt[ref_start])
// ++ref_start;
// Option 3: trim anchor base if vcf
if (!input_vcf_filename.empty()) {
if (ref_end and alt_end and (ref_end == 1 or alt_end == 1) and A->ref[0] == A->alt[0])
ref_start = 1;
}
A->pos += ref_start;
A->ref = A->ref.substr(ref_start, ref_end-ref_start);
A->alt = A->alt.substr(ref_start, alt_end-ref_start);
ref_end -= ref_start;
alt_end -= ref_start;
// Left align
if (left_alignment) {
while (A->pos > 0) {
char nuc = ref_index[chr_idx].base(A->pos-1);
if (ref_end > 0 and A->ref[ref_end-1] != nuc)
break;
if (alt_end > 0 and A->alt[alt_end-1] != nuc)
break;
A->ref = string(1,nuc) + A->ref;
A->alt = string(1,nuc) + A->alt;
A->pos--;
}
}
A->ref.resize(ref_end);
A->alt.resize(alt_end);
// Filter block substitutions: take 1
if (ref_end > 0 and alt_end > 0 and ref_end != alt_end and not allow_block_substitutions and not filter_bypass) {
A->filtered = true;
A->line_status->filter_message_prefix = "Block substitutions not supported";
continue;
}
}
if (output_bed) {
// Sort - without anchor base
sort(alleles[chr_idx].begin(), alleles[chr_idx].end(), compare_alleles);
// Write
for (deque<Allele>::iterator I = alleles[chr_idx].begin(); I != alleles[chr_idx].end(); ++I) {
if (I->filtered)
continue;
if (I->pos)
fprintf(output_bed, "%s\t%ld\t%ld\t%s\t0\t+\tREF=%s;OBS=%s;ANCHOR=%c\tNONE\n",
ref_index[chr_idx].chr.c_str(), I->pos, I->pos + I->ref.size(), I->id.c_str(),
I->ref.c_str(), I->alt.c_str(), ref_index[chr_idx].base(I->pos-1));
else
fprintf(output_bed, "%s\t%ld\t%ld\t%s\t0\t+\tREF=%s;OBS=%s;ANCHOR=\tNONE\n",
ref_index[chr_idx].chr.c_str(), I->pos, I->pos + I->ref.size(), I->id.c_str(),
I->ref.c_str(), I->alt.c_str());
}
}
if (output_vcf) {
// Add anchor base to indels
for (deque<Allele>::iterator I = alleles[chr_idx].begin(); I != alleles[chr_idx].end(); ++I) {
if (I->filtered)
continue;
if (not I->ref.empty() and not I->alt.empty())
continue;
if (I->pos == 0) {
I->filtered = true;
I->line_status->filter_message_prefix = "INDELs at chromosome start not supported";
continue;
}
I->pos--;
I->ref = string(1,ref_index[chr_idx].base(I->pos)) + I->ref;
I->alt = string(1,ref_index[chr_idx].base(I->pos)) + I->alt;
}
// Sort - with anchor base
sort(alleles[chr_idx].begin(), alleles[chr_idx].end(), compare_alleles);
// Merge alleles, remove block substitutions, write
for (deque<Allele>::iterator A = alleles[chr_idx].begin(); A != alleles[chr_idx].end(); ) {
string max_ref;
deque<Allele>::iterator B = A;
for (; B != alleles[chr_idx].end() and B->pos == A->pos; ++B)
if (!B->filtered and max_ref.size() < B->ref.size())
max_ref = B->ref;
bool filtered = true;
for (deque<Allele>::iterator I = A; I != B; ++I) {
if (I->filtered)
continue;
string new_alt = I->alt + max_ref.substr(I->ref.size());
if (new_alt.size() > 1 and max_ref.size() > 1 and new_alt.size() != max_ref.size() and not allow_block_substitutions and not filter_bypass) {
I->filtered = true;
I->line_status->filter_message_prefix = "Block substitutions not supported (post-merge)";
continue;
}
I->ref = max_ref;
I->alt = new_alt;
filtered = false;
}
if (not filtered) {
fprintf(output_vcf, "%s\t%ld\t.\t%s\t",
ref_index[chr_idx].chr.c_str(), A->pos+1, max_ref.c_str());
bool comma = false;
set<string> unique_alt_alleles;
for (deque<Allele>::iterator I = A; I != B; ++I) {
if (I->filtered)
continue;
if (unique_alt_alleles.count(I->alt) > 0)
continue;
unique_alt_alleles.insert(I->alt);
if (comma)
fprintf(output_vcf, ",");
comma = true;
fprintf(output_vcf, "%s", I->alt.c_str());
}
fprintf(output_vcf, "\t.\t.\tOID=");
comma = false;
for (deque<Allele>::iterator I = A; I != B; ++I) {
if (I->filtered)
continue;
if (comma)
fprintf(output_vcf, ",");
comma = true;
fprintf(output_vcf, "%s", I->id.c_str());
}
fprintf(output_vcf, ";OPOS=");
comma = false;
for (deque<Allele>::iterator I = A; I != B; ++I) {
if (I->filtered)
continue;
if (comma)
fprintf(output_vcf, ",");
comma = true;
fprintf(output_vcf, "%ld", I->opos+1);
}
fprintf(output_vcf, ";OREF=");
comma = false;
for (deque<Allele>::iterator I = A; I != B; ++I) {
if (I->filtered)
continue;
if (comma)
fprintf(output_vcf, ",");
comma = true;
fprintf(output_vcf, "%s", I->oref.c_str());
}
fprintf(output_vcf, ";OALT=");
comma = false;
for (deque<Allele>::iterator I = A; I != B; ++I) {
if (I->filtered)
continue;
if (comma)
fprintf(output_vcf, ",");
comma = true;
fprintf(output_vcf, "%s", I->oalt.c_str());
}
fprintf(output_vcf, ";OMAPALT=");
comma = false;
for (deque<Allele>::iterator I = A; I != B; ++I) {
if (I->filtered)
continue;
if (comma)
fprintf(output_vcf, ",");
comma = true;
fprintf(output_vcf, "%s", I->alt.c_str());
}
fprintf(output_vcf, "\n");
}
A = B;
}
}
}
if (output_bed) {
fflush(output_bed);
fclose(output_bed);
}
if (output_vcf) {
fflush(output_vcf);
fclose(output_vcf);
}
int lines_ignored = 0;
for (deque<LineStatus>::iterator L = line_status.begin(); L != line_status.end(); ++L) {
if (L->filter_message_prefix) {
if (L->allele)
printf("Line %d ignored: [%s:%ld %s] %s%s\n", L->line_number, ref_index[L->allele->chr_idx].chr.c_str(), L->allele->opos+1, L->allele->id.c_str(),
L->filter_message_prefix, L->filter_message.c_str());
else
printf("Line %d ignored: %s%s\n", L->line_number, L->filter_message_prefix, L->filter_message.c_str());
lines_ignored++;
}
}
printf("Ignored %d out of %d lines\n", lines_ignored, (int)line_status.size());
munmap(ref, ref_stat.st_size);
close(ref_handle);
return 0;
}
int main(int argc, const char* argv[])
{
printf ("tvcvalidator %s-%s (%s) - Prototype tvc validation tool\n\n",
IonVersion::GetVersion().c_str(), IonVersion::GetRelease().c_str(), IonVersion::GetSvnRev().c_str());
if (argc == 1) {
VariantValidatorHelp();
return 1;
}
OptArgs opts;
opts.ParseCmdLine(argc, argv);
if (opts.GetFirstBoolean('v', "version", false)) {
return 0;
}
if (opts.GetFirstBoolean('h', "help", false)) {
VariantValidatorHelp();
return 0;
}
string input_vcf_filename = opts.GetFirstString ('i', "input-vcf", "");
string truth_filename = opts.GetFirstString ('t', "truth-file", "");
string truth_dir = opts.GetFirstString ('d', "truth-dir", "/results/plugins/validateVariantCaller/files");
// TODO: reference optional, only used to verify reference allele in input-vcf and truth files
//string reference_filename = opts.GetFirstString ('r', "reference", "");
opts.CheckNoLeftovers();
//
// Step 1. Load input VCF file into memory
//
if (input_vcf_filename.empty()) {
VariantValidatorHelp();
cerr << "ERROR: Input VCF file not specified " << endl;
return 1;
}
VariantCallerResults results_vcf;
results_vcf.load_vcf(input_vcf_filename);
printf("Loaded VCF %s with %d variant calls\n", input_vcf_filename.c_str(), (int)results_vcf.variants.size());
//
// Step 2. Parse truth files, compare them to the input vcf, and compute match scores
//
if (not truth_filename.empty()) {
ValidatorTruth truth;
truth.ReadTruthFile(truth_filename);
truth.CompareToCalls(results_vcf);
return 0;
}
truth_dir += "/*.bed";
glob_t glob_result;
glob(truth_dir.c_str(), GLOB_TILDE, NULL, &glob_result);
for(unsigned int i = 0; i < glob_result.gl_pathc; ++i) {
ValidatorTruth truth;
truth.ReadTruthFile(string(glob_result.gl_pathv[i]));
truth.CompareToCalls(results_vcf);
}
globfree(&glob_result);
return 0;
}
int main(int argc, const char *argv[])
{
OptArgs opts;
opts.ParseCmdLine(argc, argv);
string inFile, outFile;
bool help = false;
bool version = false;
double lower = -5.0;
double upper = 28.0;
opts.GetOption(inFile, "", 'i', "input-file");
opts.GetOption(outFile, "", 'o', "output-file");
opts.GetOption(lower, "-5.0", '-', "wells-convert-low");
opts.GetOption(upper, "28.0", '-', "wells-convert-high");
opts.GetOption(help, "false", 'h', "help");
opts.GetOption(version, "false", 'v', "version");
opts.CheckNoLeftovers();
if (version)
{
fprintf (stdout, "%s", IonVersion::GetFullVersion("RawWellsConverter").c_str());
exit(0);
}
if (inFile.empty() || help)
{
cout << "RawWellsConverter - Convert unsigned short type wells file to float type wells file, or vice versa." << endl
<< "options: " << endl
<< " -i,--input-file input wells file." << endl
<< " -o,--output-file output wells file." << endl
<< " ,--wells-convert-low lower bound for converting to unsigned short." << endl
<< " ,--wells-convert-high upper bound for converting to unsigned short." << endl
<< " -h,--help this message." << endl
<< "" << endl
<< "usage: " << endl
<< " RawWellsConverter -i input_path/1.wells -o output_path/1.wells " << endl;
exit(1);
}
struct stat sb;
if(stat(inFile.c_str(), &sb) != 0)
{
cerr << "RawWellsConverter ERROR: " << inFile << " does not exist." << endl;
exit (1);
}
if (outFile.empty())
{
outFile = inFile;
outFile += ".converted";
}
string cmd("cp ");
cmd += inFile;
cmd += " ";
cmd += outFile;
int ret0 = system(cmd.c_str());
hid_t root = H5Fopen(outFile.c_str(), H5F_ACC_RDWR, H5P_DEFAULT);
if(root < 0)
{
cerr << "RawWellsConverter ERROR: Fail to open " << outFile << endl;
exit(1);
}
H5G_info_t group_info;
group_info.nlinks = 0;
if(H5Gget_info(root, &group_info) < 0)
{
H5Fclose(root);
cerr << "RawWellsConverter ERROR: Fail H5Gget_info." << endl;
exit(1);
}
char name[10];
string sName;
bool bWells = false;
bool bCopies = false;
for(unsigned int i = 0; i < group_info.nlinks; ++i)
{
int size = H5Gget_objname_by_idx(root, i, NULL, 0);
if(H5Gget_objname_by_idx(root, i, name, size + 1) < 0)
{
H5Fclose(root);
cerr << "RawWellsConverter ERROR: Fail H5Gget_objname_by_idx." << endl;
exit(1);
}
else
{
sName = name;
if(sName == "wells")
{
bWells = true;
}
if(sName == "wells_copies")
{
bCopies = true;
}
}
}
if(!bWells)
{
H5Fclose(root);
cerr << "RawWellsConverter ERROR: There is no dataset wells." << endl;
exit(1);
}
hid_t dsWells = H5Dopen2(root, "wells", H5P_DEFAULT);
if(dsWells < 0)
{
H5Fclose(root);
cerr << "RawWellsConverter ERROR: Fail H5Dopen2 wells." << endl;
exit(1);
}
bool saveAsUShort = false;
if(H5Aexists(dsWells, "convert_low") > 0)
{
hid_t attrLower = H5Aopen(dsWells, "convert_low", H5T_NATIVE_FLOAT );
H5Aread(attrLower, H5T_NATIVE_FLOAT, &lower);
saveAsUShort = true;
H5Aclose(attrLower);
}
if(H5Aexists(dsWells, "convert_high") > 0)
{
hid_t attrUpper = H5Aopen(dsWells, "convert_high", H5T_NATIVE_FLOAT);
H5Aread(attrUpper, H5T_NATIVE_FLOAT, &upper);
saveAsUShort = true;
H5Aclose(attrUpper);
}
hid_t dataSpace = H5Dget_space(dsWells);
if(dataSpace < 0)
{
H5Dclose(dsWells);
H5Fclose(root);
cerr << "RawWellsConverter ERROR: Fail H5Dget_space wells." << endl;
exit(1);
}
hssize_t dsSize = H5Sget_simple_extent_npoints(dataSpace);
if(dsSize < 1)
{
H5Sclose(dataSpace);
H5Dclose(dsWells);
H5Fclose(root);
cerr << "RawWellsConverter ERROR: Wrong size of dataset wells - " << dsSize << endl;
exit(1);
}
int nRows = 0;
int nCols = 0;
int nFlows = 0;
int rank = H5Sget_simple_extent_ndims(dataSpace);
if(rank != 3)
{
bCopies = false;
}
else
{
hsize_t dims_out[3];
int status_n = H5Sget_simple_extent_dims(dataSpace, dims_out, NULL);
if(status_n < 0)
{
bCopies = false;
}
else
{
nRows = dims_out[0];
nCols = dims_out[1];
nFlows = dims_out[2];
}
}
float* fPtr = new float[dsSize];
unsigned short* usPtr = new unsigned short[dsSize];
if(fPtr == NULL || usPtr == NULL)
{
H5Sclose(dataSpace);
H5Dclose(dsWells);
H5Fclose(root);
cerr << "RawWellsConverter ERROR: Fail to allocate fPtr or usPtr." << endl;
exit(1);
}
hid_t dcpl = H5Dget_create_plist(dsWells);
if(dcpl < 0)
{
H5Sclose(dataSpace);
H5Dclose(dsWells);
H5Fclose(root);
cerr << "RawWellsConverter ERROR: Fail H5Dget_create_plist." << endl;
exit(1);
}
hid_t dapl = H5Dget_access_plist(dsWells);
if(dapl < 0)
{
H5Pclose(dcpl);
H5Sclose(dataSpace);
H5Dclose(dsWells);
H5Fclose(root);
cerr << "RawWellsConverter ERROR: Fail H5Dget_access_plist." << endl;
exit(1);
}
if(saveAsUShort)
{
cout << "RawWellsConverter: converting unsigned short wells file - " << inFile << " to float wells file - " << outFile << " with boundary (" << lower << "," << upper << ")" << endl;
herr_t ret = H5Dread(dsWells, H5T_NATIVE_USHORT, H5S_ALL, H5S_ALL, H5P_DEFAULT, usPtr);
H5Dclose(dsWells);
if(ret < 0)
{
delete [] fPtr;
delete [] usPtr;
H5Sclose(dataSpace);
H5Pclose(dcpl);
H5Pclose(dapl);
H5Fclose(root);
cerr << "RawWellsConverter ERROR: Fail to read dataset wells." << endl;
exit(1);
}
float factor = 65535.0 / (upper - lower);
float* fPtr2 = fPtr;
unsigned short* usPtr2 = usPtr;
for(unsigned int i = 0; i < dsSize; ++i, ++fPtr2, ++usPtr2)
{
(*fPtr2) = (float)(*usPtr2) / factor + lower;
}
delete [] usPtr;
if(bCopies)
{
vector<float> copies(nRows * nCols, 1.0);
hid_t dsCopies = H5Dopen2(root, "wells_copies", H5P_DEFAULT);
if(dsCopies < 0)
{
cerr << "RawWellsConverter WARNING: 1.wells files does not have wells_copies." << endl;
}
else
{
hid_t dataSpace2 = H5Dget_space(dsCopies);
if(dataSpace2 < 0)
{
H5Dclose(dsCopies);
cerr << "RawWellsConverter WARNING: fail to H5Dget_space for dataset wells_copies." << endl;
}
else
{
hssize_t dsSize2 = H5Sget_simple_extent_npoints(dataSpace2);
H5Sclose(dataSpace2);
if(dsSize2 != (hssize_t)(nRows * nCols))
{
H5Dclose(dsCopies);
cerr << "RawWellsConverter WARNING: dataset wells_copies size is " << dsSize2 << ", it is different from nRows * nCols = " << nRows * nCols << endl;
}
else
{
herr_t ret = H5Dread(dsCopies, H5T_NATIVE_FLOAT, H5S_ALL, H5S_ALL, H5P_DEFAULT, &copies[0]);
H5Dclose(dsCopies);
if(ret < 0)
{
copies.resize(nRows * nCols, 1.0);
cerr << "RawWellsConverter WARNING: failto load dataset wells_copies." << endl;
}
}
}
}
uint64_t fptrCount = 0;
uint64_t copyCount = 0;
for(int row = 0; row < nRows; ++row)
{
for(int col = 0; col < nCols; ++col)
{
for(int flow = 0; flow < nFlows; ++flow)
{
if(copies[copyCount] > 0)
{
fPtr[fptrCount] *= copies[copyCount];
}
else
{
fPtr[fptrCount] = -1.0;
}
++fptrCount;
}
++copyCount;
}
}
}
H5Ldelete(root, "wells", H5P_DEFAULT);
hid_t dsWells2 = H5Dcreate2 (root, "wells", H5T_NATIVE_FLOAT, dataSpace, H5P_DEFAULT, dcpl, dapl);
if(dsWells2 < 0)
{
delete [] fPtr;
H5Sclose(dataSpace);
H5Pclose(dcpl);
H5Pclose(dapl);
H5Fclose(root);
cerr << "RawWellsConverter ERROR: Fail to create dataset wells." << endl;
exit(1);
}
ret = H5Dwrite(dsWells2, H5T_NATIVE_FLOAT, H5S_ALL, H5S_ALL, H5P_DEFAULT, fPtr);
delete [] fPtr;
H5Dclose(dsWells2);
H5Sclose(dataSpace);
H5Pclose(dcpl);
H5Pclose(dapl);
H5Fclose(root);
if(ret < 0)
{
cerr << "RawWellsConverter ERROR: Fail to write dataset wells." << endl;
exit(1);
}
}
else
{
cout << "RawWellsConverter: converting float wells file - " << inFile << " to unsigned short wells file - " << outFile << " with boundary (" << lower << "," << upper << ")" << endl;
herr_t ret = H5Dread(dsWells, H5T_NATIVE_FLOAT, H5S_ALL, H5S_ALL, H5P_DEFAULT, fPtr);
H5Dclose(dsWells);
if(ret < 0)
{
delete [] fPtr;
delete [] usPtr;
H5Sclose(dataSpace);
H5Pclose(dcpl);
H5Pclose(dapl);
H5Fclose(root);
cerr << "RawWellsConverter ERROR: Fail to read dataset wells." << endl;
exit(1);
}
float factor = 65535.0 / (upper - lower);
float* fPtr2 = fPtr;
unsigned short* usPtr2 = usPtr;
for(unsigned int i = 0; i < dsSize; ++i, ++fPtr2, ++usPtr2)
{
if(*fPtr2 < lower)
{
(*usPtr2) = 0;
}
else if(*fPtr2 > upper)
{
(*usPtr2) = 65535;
}
else
{
(*usPtr2) = (unsigned short)((*fPtr2 - lower) * factor);
}
}
delete [] fPtr;
H5Ldelete(root, "wells", H5P_DEFAULT);
hid_t dsWells2 = H5Dcreate2 (root, "wells", H5T_NATIVE_USHORT, dataSpace, H5P_DEFAULT, dcpl, dapl);
if(dsWells2 < 0)
{
delete [] usPtr;
H5Sclose(dataSpace);
H5Pclose(dcpl);
H5Pclose(dapl);
H5Fclose(root);
cerr << "RawWellsConverter ERROR: Fail to create dataset wells." << endl;
exit(1);
}
ret = H5Dwrite(dsWells2, H5T_NATIVE_USHORT, H5S_ALL, H5S_ALL, H5P_DEFAULT, usPtr);
delete [] usPtr;
if(dsWells2 < 0)
{
H5Dclose(dsWells2);
H5Sclose(dataSpace);
H5Pclose(dcpl);
H5Pclose(dapl);
H5Fclose(root);
cerr << "RawWellsConverter ERROR: Fail to write dataset wells." << endl;
exit(1);
}
float lower2 = (float)lower;
float upper2 = (float)upper;
hsize_t dimsa[1];
dimsa[0] = 1;
hid_t dataspacea = H5Screate_simple(1, dimsa, NULL);
hid_t attrLower = H5Acreate(dsWells2, "convert_low", H5T_NATIVE_FLOAT, dataspacea, H5P_DEFAULT, H5P_DEFAULT );
H5Awrite(attrLower, H5T_NATIVE_FLOAT, &lower2);
H5Aclose(attrLower);
hid_t attrUpper = H5Acreate(dsWells2, "convert_high", H5T_NATIVE_FLOAT, dataspacea, H5P_DEFAULT, H5P_DEFAULT );
H5Awrite(attrUpper, H5T_NATIVE_FLOAT, &upper2);
H5Aclose(attrUpper);
H5Sclose(dataspacea);
H5Dclose(dsWells2);
H5Sclose(dataSpace);
H5Pclose(dcpl);
H5Pclose(dapl);
H5Fclose(root);
}
return 0;
}
int main(int argc, const char *argv[])
{
#ifdef _DEBUG
atexit(memstatus);
dbgmemInit();
#endif /* _DEBUG */
printf ("%s - %s-%s (%s)\n", argv[0], IonVersion::GetVersion().c_str(), IonVersion::GetRelease().c_str(), IonVersion::GetSvnRev().c_str());
string bamInputFilename;
string fastaInputFilename;
string jsonOutputFilename;
bool help;
OptArgs opts;
opts.ParseCmdLine(argc, argv);
opts.GetOption(bamInputFilename, "", '-', "bam");
opts.GetOption(fastaInputFilename, "", '-', "ref");
opts.GetOption(jsonOutputFilename, "TFStats.json", '-', "output-json");
opts.GetOption(help, "false", 'h', "help");
opts.CheckNoLeftovers();
if (help || bamInputFilename.empty() || fastaInputFilename.empty())
return showHelp();
// Parse BAM header
BAMReader bamReader(bamInputFilename);
bamReader.open();
bam_header_t *header = (bam_header_t *)bamReader.get_header_ptr();
int numFlows = 0;
string flowOrder;
string key;
if (header->l_text >= 3) {
if (header->dict == 0)
header->dict = sam_header_parse2(header->text);
int nEntries = 0;
char **tmp = sam_header2list(header->dict, "RG", "FO", &nEntries);
if (nEntries) {
flowOrder = tmp[0];
numFlows = flowOrder.length();
}
if (tmp)
free(tmp);
nEntries = 0;
tmp = sam_header2list(header->dict, "RG", "KS", &nEntries);
if (nEntries) {
key = tmp[0];
}
if (tmp)
free(tmp);
}
if (numFlows <= 0) {
fprintf(stderr, "[TFMapper] Could not retrieve flow order from FO BAM tag. SFF-specific tags absent?\n");
exit(1);
}
if (key.empty()) {
fprintf(stderr, "[TFMapper] Could not retrieve key sequence from KS BAM tag. SFF-specific tags absent?\n");
exit(1);
}
//printf("Retrieved flow order from bam: %s (%d)\n", flowOrder.c_str(), numFlows);
//printf("Retrieved key from bam: %s\n", key.c_str());
// Retrieve test fragment sequences
vector<string> referenceSequences;
PopulateReferenceSequences(referenceSequences, fastaInputFilename, header->n_targets, header->target_name, string(""));
// Process the BAM reads and generate metrics
int numTFs = header->n_targets;
vector<int> TFCount(numTFs,0);
MetricGeneratorQualityHistograms metricGeneratorQualityHistograms[numTFs];
MetricGeneratorHPAccuracy metricGeneratorHPAccuracy[numTFs];
MetricGeneratorSNR metricGeneratorSNR[numTFs];
MetricGeneratorAvgIonogram metricGeneratorAvgIonogram[numTFs];
for (BAMReader::iterator i = bamReader.get_iterator(); i.good(); i.next()) {
BAMRead bamRead = i.get();
int bestTF = bamRead.get_tid();
if (bestTF < 0)
continue;
BAMUtils bamUtil(bamRead);
TFCount[bestTF]++;
// Extract flowspace signal from FZ BAM tag
uint16_t *bam_flowgram = NULL;
uint8_t *fz = bam_aux_get(bamRead.get_bam_ptr(), "FZ");
if (fz != NULL) {
if (fz[0] == (uint8_t)'B' && fz[1] == (uint8_t)'S' && *((uint32_t *)(fz+2)) == (uint32_t)numFlows)
bam_flowgram = (uint16_t *)(fz+6);
}
if (bam_flowgram == NULL) {
fprintf(stderr, "[TFMapper] Could not retrieve flow signal from FZ BAM tag. SFF-specific tags absent?\n");
exit(1);
}
// Use alignments to generate "synchronized" flowspace reference and read ionograms
// TODO: Do proper flowspace alignment
string genome = key + bamUtil.get_tdna();
string calls = key + bamUtil.get_qdna();
int numBases = min(genome.length(),calls.length());
vector<int> refIonogram(numFlows, 0);
vector<int> readIonogram(numFlows, 0);
int numFlowsRead = 0;
int numFlowsRef = 0;
char gC = flowOrder[0];
int gBC = 0;
for (int iBase = 0; (iBase < numBases) && (numFlowsRead < numFlows) && (numFlowsRef < numFlows); iBase++) {
// Conversion for reads (independent of reference)
if (calls[iBase] != '-') {
while ((calls[iBase] != flowOrder[numFlowsRead]) && (numFlowsRead < numFlows))
numFlowsRead++;
if (numFlowsRead < numFlows)
readIonogram[numFlowsRead]++;
}
if (genome[iBase] != '-') {
if (genome[iBase] != gC) {
// Since a new homopolymer begins, need to drop off the old one
while ((gC != flowOrder[numFlowsRef]) && (numFlowsRef < numFlows)) {
numFlowsRef++;
if (numFlowsRef < numFlows)
refIonogram[numFlowsRef] = 0;
}
if (numFlowsRef < numFlows)
refIonogram[numFlowsRef] = gBC;
gC = genome[iBase];
gBC = 0;
}
gBC++;
if (genome[iBase] == calls[iBase])
numFlowsRef = numFlowsRead;
}
}
int validFlows = min(numFlowsRef, numFlowsRead);
metricGeneratorSNR[bestTF].AddElement(bam_flowgram ,key.c_str(), flowOrder);
metricGeneratorAvgIonogram[bestTF].AddElement(bam_flowgram, numFlows);
metricGeneratorQualityHistograms[bestTF].AddElement(bamUtil.get_phred_len(10),bamUtil.get_phred_len(17));
for (int iFlow = 0; iFlow < validFlows-20; iFlow++)
metricGeneratorHPAccuracy[bestTF].AddElement(refIonogram[iFlow],readIonogram[iFlow]);
}
// Save stats to a json file
Json::Value outputJson(Json::objectValue);
for(int i = 0; i < numTFs; i++) {
if (TFCount[i] < minTFCount)
continue;
Json::Value currentTFJson(Json::objectValue);
currentTFJson["TF Name"] = header->target_name[i];
currentTFJson["TF Seq"] = referenceSequences[i];
currentTFJson["Num"] = TFCount[i];
currentTFJson["Top Reads"] = Json::Value(Json::arrayValue); // Obsolete
metricGeneratorSNR[i].PrintSNR(currentTFJson);
metricGeneratorHPAccuracy[i].PrintHPAccuracy(currentTFJson);
metricGeneratorQualityHistograms[i].PrintMetrics(currentTFJson);
metricGeneratorAvgIonogram[i].PrintIonograms(currentTFJson);
outputJson[header->target_name[i]] = currentTFJson;
}
bamReader.close(); // Closing invalidates the header pointers
if (!jsonOutputFilename.empty()) {
ofstream out(jsonOutputFilename.c_str(), ios::out);
if (out.good())
out << outputJson.toStyledString();
}
return 0;
}
