int main(int argc, char* argv[])
{
// Open a BAM file:
char* bamFile = argv[1];
// char* bamIndex = argv[2];
BAMReader reader(bamFile); // , bamIndex);
reader.open();
assert(reader);
// Print out list of reference sequences, and their lengths:
cout << "Found " << reader.num_refs() << " reference sequences:" << endl;
for(int i=0; i<reader.num_refs(); ++i)
cout << setw(9) << reader.refs()[i] << " " << reader.lens()[i] << endl;
// Print out list of reads:
for (BAMReader::iterator i = reader.get_iterator(); i.good(); i.next()) {
BAMRead read = i.get();
cout << read.to_string();
for (Sequence::iterator s_iter = read.get_seq().get_iterator(); s_iter.good(); s_iter.next())
cout << s_iter.get(); // nuc from SEQ
cout << endl;
for (Cigar::iterator c_iter = read.get_cigar().get_iterator(); c_iter.good(); c_iter.next())
cout << c_iter.len() << ":" << c_iter.op() << "; ";
cout << endl;
// Don't use MD directly. Use BAMUtils.
BAMUtils utils(read);
cout << utils.get_qdna() << endl << utils.get_matcha() << endl << utils.get_tdna() << endl << endl;
}
}
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;
}