int main(int argc, char **argv)
{
if (argc < 4)
errx(1,
"usage\t:%s <bam> <split out> <discord out> (optional #threads)",
argv[0]);
char *bam_file_name = argv[1];
char *split_file_name = argv[2];
char *disc_file_name = argv[3];
int threads = 2;
if (argc == 5) {
threads = atoi(argv[4]);
}
samFile *disc = sam_open(disc_file_name, "wb");
samFile *split = sam_open(split_file_name, "wb");
samFile *in = sam_open(bam_file_name, "rb");
if(in == NULL)
errx(1, "Unable to open BAM/SAM file.");
// TODO: handle cram.
if (threads > 1) {
bgzf_mt(in->fp.bgzf, threads, 256);
}
hts_idx_t *idx = sam_index_load(in, bam_file_name);
if(idx == NULL)
errx(1,"Unable to open BAM/SAM index.");
bam_hdr_t *hdr = sam_hdr_read(in);
int r = sam_hdr_write(disc, hdr);
r = sam_hdr_write(split, hdr);
bam1_t *aln = bam_init1();
int ret;
while(ret = sam_read1(in, hdr, aln) >= 0) {
if (((aln->core.flag) & 1294) == 0)
r = sam_write1(disc, hdr, aln);
uint8_t *sa = bam_aux_get(aln, "SA");
if (sa != 0) {
char *sa_tag = strdup(bam_aux2Z(sa));
if ( count_tags(sa_tag) == 1) {
char *chrm, strand, *cigar;
uint32_t pos;
split_sa_tag(sa_tag,
&chrm,
&pos,
&strand,
&cigar);
struct line sa, al;
calcOffsets(cigar,
pos,
strand,
&sa);
sa.chrm = chrm;
sa.strand = strand;
calcAlnOffsets(bam_get_cigar(aln),
aln->core.n_cigar,
aln->core.pos,
bam_is_rev(aln) ? '-' : '+',
&al);
al.chrm = hdr->target_name[aln->core.tid];
al.strand = bam_is_rev(aln) ? '-' : '+';
struct line *left = &al, *right = &sa;
if (left->SQO > right->SQO) {
left = &sa;
right = &al;
}
int overlap = MAX(1 + MIN(left->EQO, right->EQO) -
MAX(left->SQO, right->SQO), 0);
int alen1 = 1 + left->EQO - left->SQO;
int alen2 = 1 + right->EQO - right->SQO;
int mno = MIN(alen1-overlap, alen2-overlap);
if (mno < MIN_NON_OVERLAP)
continue;
if ( (strcmp(left->chrm, right->chrm) == 0) &&
(left->strand == right->strand) ) {
int leftDiag, rightDiag, insSize;
if (left->strand == '-') {
leftDiag = left->rapos - left->sclip;
rightDiag = (right->rapos + right->raLen) -
(right->sclip + right->qaLen);
insSize = rightDiag - leftDiag;
} else {
leftDiag = (left->rapos + left->raLen) -
(left->sclip + left->qaLen);
rightDiag = right->rapos - right->sclip;
insSize = leftDiag - rightDiag;
}
int desert = right->SQO - left->EQO - 1;
if ((abs(insSize) < MIN_INDEL_SIZE) ||
((desert > 0) && (
(desert - (int)MAX(0, insSize)) >
MAX_UNMAPPED_BASES)))
continue;
}
char *qname = bam_get_qname(aln);
if ((aln->core.flag & 64) == 64)
qname[0] = 'A';
else
qname[0] = 'B';
r = sam_write1(split, hdr, aln);
}
free(sa_tag);
}
}
bam_destroy1(aln);
hts_idx_destroy(idx);
bam_hdr_destroy(hdr);
sam_close(in);
sam_close(disc);
sam_close(split);
if(ret < -1) {
errx(1, "lumpy_filter: error reading bam: %s\n", bam_file_name);
}
}
int extract_main(int argc, char *argv[]) {
char *opref = NULL, *oname, *p;
int c, i;
Config config;
//Defaults
config.keepCpG = 1;
config.keepCHG = 0;
config.keepCHH = 0;
config.minMapq = 10;
config.minPhred = 5;
config.keepDupes = 0;
config.keepSingleton = 0, config.keepDiscordant = 0;
config.merge = 0;
config.maxDepth = 2000;
config.fai = NULL;
config.fp = NULL;
config.bai = NULL;
config.reg = NULL;
config.bedName = NULL;
config.bed = NULL;
config.fraction = 0;
config.counts = 0;
config.logit = 0;
for(i=0; i<16; i++) config.bounds[i] = 0;
static struct option lopts[] = {
{"opref", 1, NULL, 'o'},
{"fraction", 0, NULL, 'f'},
{"counts", 0, NULL, 'c'},
{"logit", 0, NULL, 'm'},
{"noCpG", 0, NULL, 1},
{"CHG", 0, NULL, 2},
{"CHH", 0, NULL, 3},
{"keepDupes", 0, NULL, 4},
{"keepSingleton",0, NULL, 5},
{"keepDiscordant",0,NULL, 6},
{"OT", 1, NULL, 7},
{"OB", 1, NULL, 8},
{"CTOT", 1, NULL, 9},
{"CTOB", 1, NULL, 10},
{"mergeContext", 0, NULL, 11},
{"help", 0, NULL, 'h'},
{0, 0, NULL, 0}
};
while((c = getopt_long(argc, argv, "q:p:r:l:o:D:f:c:m:", lopts,NULL)) >=0) {
switch(c) {
case 'h' :
extract_usage();
return 0;
case 'o' :
opref = strdup(optarg);
break;
case 'D' :
config.maxDepth = atoi(optarg);
break;
case 'r':
config.reg = strdup(optarg);
break;
case 'l' :
config.bedName = optarg;
break;
case 1 :
config.keepCpG = 0;
break;
case 2 :
config.keepCHG = 1;
break;
case 3 :
config.keepCHH = 1;
break;
case 4 :
config.keepDupes = 1;
break;
case 5 :
config.keepSingleton = 1;
break;
case 6 :
config.keepDiscordant = 1;
break;
case 7 :
parseBounds(optarg, config.bounds, 0);
break;
case 8 :
parseBounds(optarg, config.bounds, 1);
break;
case 9 :
parseBounds(optarg, config.bounds, 2);
break;
case 10 :
parseBounds(optarg, config.bounds, 3);
break;
case 11 :
config.merge = 1;
break;
case 'q' :
config.minMapq = atoi(optarg);
break;
case 'p' :
config.minPhred = atoi(optarg);
break;
case 'm' :
config.logit = 1;
break;
case 'f' :
config.fraction = 1;
break;
case 'c' :
config.counts = 1;
break;
case '?' :
default :
fprintf(stderr, "Invalid option '%c'\n", c);
extract_usage();
return 1;
}
}
if(argc == 1) {
extract_usage();
return 0;
}
if(argc-optind != 2) {
fprintf(stderr, "You must supply a reference genome in fasta format and an input BAM file!!!\n");
extract_usage();
return -1;
}
//Are the options reasonable?
if(config.minPhred < 1) {
fprintf(stderr, "-p %i is invalid. resetting to 1, which is the lowest possible value.\n", config.minPhred);
config.minPhred = 1;
}
if(config.minMapq < 0) {
fprintf(stderr, "-q %i is invalid. Resetting to 0, which is the lowest possible value.\n", config.minMapq);
config.minMapq = 0;
}
if(config.fraction+config.counts+config.logit > 1) {
fprintf(stderr, "More than one of --fraction, --counts, and --logit were specified. These are mutually exclusive.\n");
extract_usage();
return 1;
}
//Has more than one output format been requested?
if(config.fraction + config.counts + config.logit > 1) {
fprintf(stderr, "You may specify AT MOST one of -c/--counts, -f/--fraction, or -m/--logit.\n");
return -6;
}
//Is there still a metric to output?
if(!(config.keepCpG + config.keepCHG + config.keepCHH)) {
fprintf(stderr, "You haven't specified any metrics to output!\nEither don't use the --noCpG option or specify --CHG and/or --CHH.\n");
return -1;
}
//Open the files
if((config.fai = fai_load(argv[optind])) == NULL) {
fprintf(stderr, "Couldn't open the index for %s!\n", argv[optind]);
extract_usage();
return -2;
}
if((config.fp = hts_open(argv[optind+1], "rb")) == NULL) {
fprintf(stderr, "Couldn't open %s for reading!\n", argv[optind+1]);
return -4;
}
if((config.bai = sam_index_load(config.fp, argv[optind+1])) == NULL) {
fprintf(stderr, "Couldn't load the index for %s, will attempt to build it.\n", argv[optind+1]);
if(bam_index_build(argv[optind+1], 0) < 0) {
fprintf(stderr, "Couldn't build the index for %s! File corrupted?\n", argv[optind+1]);
return -5;
}
if((config.bai = sam_index_load(config.fp, argv[optind+1])) == NULL) {
fprintf(stderr, "Still couldn't load the index, quiting.\n");
return -5;
}
}
//Output files
config.output_fp = malloc(sizeof(FILE *) * 3);
assert(config.output_fp);
if(opref == NULL) {
opref = strdup(argv[optind+1]);
assert(opref);
p = strrchr(opref, '.');
if(p != NULL) *p = '\0';
fprintf(stderr, "writing to prefix:'%s'\n", opref);
}
if(config.fraction) {
oname = malloc(sizeof(char) * (strlen(opref)+19));
} else if(config.counts) {
oname = malloc(sizeof(char) * (strlen(opref)+21));
} else if(config.logit) {
oname = malloc(sizeof(char) * (strlen(opref)+20));
} else {
oname = malloc(sizeof(char) * (strlen(opref)+14));
}
assert(oname);
if(config.keepCpG) {
if(config.fraction) {
sprintf(oname, "%s_CpG.meth.bedGraph", opref);
} else if(config.counts) {
sprintf(oname, "%s_CpG.counts.bedGraph", opref);
} else if(config.logit) {
sprintf(oname, "%s_CpG.logit.bedGraph", opref);
} else {
sprintf(oname, "%s_CpG.bedGraph", opref);
}
config.output_fp[0] = fopen(oname, "w");
if(config.output_fp[0] == NULL) {
fprintf(stderr, "Couldn't open the output CpG metrics file for writing! Insufficient permissions?\n");
return -3;
}
printHeader(config.output_fp[0], "CpG", opref, config);
}
if(config.keepCHG) {
if(config.fraction) {
sprintf(oname, "%s_CHG.meth.bedGraph", opref);
} else if(config.counts) {
sprintf(oname, "%s_CHG.counts.bedGraph", opref);
} else if(config.logit) {
sprintf(oname, "%s_CHG.logit.bedGraph", opref);
} else {
sprintf(oname, "%s_CHG.bedGraph", opref);
}
config.output_fp[1] = fopen(oname, "w");
if(config.output_fp[1] == NULL) {
fprintf(stderr, "Couldn't open the output CHG metrics file for writing! Insufficient permissions?\n");
return -3;
}
printHeader(config.output_fp[1], "CHG", opref, config);
}
if(config.keepCHH) {
if(config.fraction) {
sprintf(oname, "%s_CHH.meth.bedGraph", opref);
} else if(config.counts) {
sprintf(oname, "%s_CHH.counts.bedGraph", opref);
} else if(config.logit) {
sprintf(oname, "%s_CHH.logit.bedGraph", opref);
} else {
sprintf(oname, "%s_CHH.bedGraph", opref);
}
config.output_fp[2] = fopen(oname, "w");
if(config.output_fp[2] == NULL) {
fprintf(stderr, "Couldn't open the output CHH metrics file for writing! Insufficient permissions?\n");
return -3;
}
printHeader(config.output_fp[2], "CHH", opref, config);
}
//Run the pileup
extractCalls(&config);
//Close things up
hts_close(config.fp);
fai_destroy(config.fai);
if(config.keepCpG) fclose(config.output_fp[0]);
if(config.keepCHG) fclose(config.output_fp[1]);
if(config.keepCHH) fclose(config.output_fp[2]);
hts_idx_destroy(config.bai);
free(opref);
if(config.reg) free(config.reg);
if(config.bed) destroyBED(config.bed);
free(oname);
free(config.output_fp);
return 0;
}
void train_one_round(const Fast5Map& name_map, size_t round)
{
const PoreModelMap& current_models = PoreModelSet::get_models(opt::trained_model_type);
// Initialize the training summary stats for each kmer for each model
ModelTrainingMap model_training_data;
for(auto current_model_iter = current_models.begin(); current_model_iter != current_models.end(); current_model_iter++) {
// one summary entry per kmer in the model
std::vector<StateSummary> summaries(current_model_iter->second.get_num_states());
model_training_data[current_model_iter->first] = summaries;
}
// Open the BAM and iterate over reads
// load bam file
htsFile* bam_fh = sam_open(opt::bam_file.c_str(), "r");
assert(bam_fh != NULL);
// load bam index file
std::string index_filename = opt::bam_file + ".bai";
hts_idx_t* bam_idx = bam_index_load(index_filename.c_str());
assert(bam_idx != NULL);
// read the bam header
bam_hdr_t* hdr = sam_hdr_read(bam_fh);
// load reference fai file
faidx_t *fai = fai_load(opt::genome_file.c_str());
hts_itr_t* itr;
// If processing a region of the genome, only emit events aligned to this window
int clip_start = -1;
int clip_end = -1;
if(opt::region.empty()) {
// TODO: is this valid?
itr = sam_itr_queryi(bam_idx, HTS_IDX_START, 0, 0);
} else {
fprintf(stderr, "Region: %s\n", opt::region.c_str());
itr = sam_itr_querys(bam_idx, hdr, opt::region.c_str());
hts_parse_reg(opt::region.c_str(), &clip_start, &clip_end);
}
#ifndef H5_HAVE_THREADSAFE
if(opt::num_threads > 1) {
fprintf(stderr, "You enabled multi-threading but you do not have a threadsafe HDF5\n");
fprintf(stderr, "Please recompile nanopolish's built-in libhdf5 or run with -t 1\n");
exit(1);
}
#endif
// Initialize iteration
std::vector<bam1_t*> records(opt::batch_size, NULL);
for(size_t i = 0; i < records.size(); ++i) {
records[i] = bam_init1();
}
int result;
size_t num_reads_realigned = 0;
size_t num_records_buffered = 0;
Progress progress("[methyltrain]");
do {
assert(num_records_buffered < records.size());
// read a record into the next slot in the buffer
result = sam_itr_next(bam_fh, itr, records[num_records_buffered]);
num_records_buffered += result >= 0;
// realign if we've hit the max buffer size or reached the end of file
if(num_records_buffered == records.size() || result < 0) {
#pragma omp parallel for
for(size_t i = 0; i < num_records_buffered; ++i) {
bam1_t* record = records[i];
size_t read_idx = num_reads_realigned + i;
if( (record->core.flag & BAM_FUNMAP) == 0) {
add_aligned_events(name_map, fai, hdr, record, read_idx, clip_start, clip_end, round, model_training_data);
}
}
num_reads_realigned += num_records_buffered;
num_records_buffered = 0;
}
if(opt::progress) {
fprintf(stderr, "Realigned %zu reads in %.1lfs\r", num_reads_realigned, progress.get_elapsed_seconds());
}
} while(result >= 0);
assert(num_records_buffered == 0);
progress.end();
// open the summary file
std::stringstream summary_fn;
summary_fn << "methyltrain" << opt::out_suffix << ".summary";
FILE* summary_fp = fopen(summary_fn.str().c_str(), "w");
fprintf(summary_fp, "model_short_name\tkmer\tnum_matches\tnum_skips\t"
"num_stays\tnum_events_for_training\twas_trained\t"
"trained_level_mean\ttrained_level_stdv\n");
// open the tsv file with the raw training data
std::stringstream training_fn;
training_fn << "methyltrain" << opt::out_suffix << ".round" << round << ".events.tsv";
std::ofstream training_ofs(training_fn.str());
// write out a header for the training data
StateTrainingData::write_header(training_ofs);
// iterate over models: template, complement_pop1, complement_pop2
for(auto model_training_iter = model_training_data.begin();
model_training_iter != model_training_data.end(); model_training_iter++) {
// Initialize the trained model from the input model
auto current_model_iter = current_models.find(model_training_iter->first);
assert(current_model_iter != current_models.end());
std::string model_name = model_training_iter->first;
std::string model_short_name = current_model_iter->second.metadata.get_short_name();
// Initialize the new model from the current model
PoreModel updated_model = current_model_iter->second;
uint32_t k = updated_model.k;
const std::vector<StateSummary>& summaries = model_training_iter->second;
// Generate the complete set of kmers
std::string gen_kmer(k, 'A');
std::vector<std::string> all_kmers;
for(size_t ki = 0; ki < summaries.size(); ++ki) {
all_kmers.push_back(gen_kmer);
mtrain_alphabet->lexicographic_next(gen_kmer);
}
assert(gen_kmer == std::string(k, 'A'));
assert(all_kmers.front() == std::string(k, 'A'));
assert(all_kmers.back() == std::string(k, 'T'));
// Update means for each kmer
#pragma omp parallel for
for(size_t ki = 0; ki < summaries.size(); ++ki) {
assert(ki < all_kmers.size());
std::string kmer = all_kmers[ki];
// write the observed values to a tsv file
#pragma omp critical
{
for(size_t ei = 0; ei < summaries[ki].events.size(); ++ei) {
summaries[ki].events[ei].write_tsv(training_ofs, model_short_name, kmer);
}
}
bool is_m_kmer = kmer.find('M') != std::string::npos;
bool update_kmer = opt::training_target == TT_ALL_KMERS ||
(is_m_kmer && opt::training_target == TT_METHYLATED_KMERS) ||
(!is_m_kmer && opt::training_target == TT_UNMETHYLATED_KMERS);
bool trained = false;
// only train if there are a sufficient number of events for this kmer
if(update_kmer && summaries[ki].events.size() >= opt::min_number_of_events_to_train) {
// train a mixture model where a minority of k-mers aren't methylated
ParamMixture mixture;
float incomplete_methylation_rate = 0.05f;
std::string um_kmer = mtrain_alphabet->unmethylate(kmer);
size_t um_ki = mtrain_alphabet->kmer_rank(um_kmer.c_str(), k);
// Initialize the training parameters. If this is a kmer containing
// a methylation site we train a two component mixture, otherwise
// just fit a gaussian
float major_weight = is_m_kmer ? 1 - incomplete_methylation_rate : 1.0f;
mixture.log_weights.push_back(log(major_weight));
mixture.params.push_back(current_model_iter->second.get_parameters(ki));
if(is_m_kmer) {
// add second unmethylated component
mixture.log_weights.push_back(std::log(incomplete_methylation_rate));
mixture.params.push_back(current_model_iter->second.get_parameters(um_ki));
}
if(opt::verbose > 1) {
fprintf(stderr, "INIT__MIX %s\t%s\t[%.2lf %.2lf %.2lf]\t[%.2lf %.2lf %.2lf]\n", model_training_iter->first.c_str(), kmer.c_str(),
std::exp(mixture.log_weights[0]), mixture.params[0].level_mean, mixture.params[0].level_stdv,
std::exp(mixture.log_weights[1]), mixture.params[1].level_mean, mixture.params[1].level_stdv);
}
ParamMixture trained_mixture = train_gaussian_mixture(summaries[ki].events, mixture);
if(opt::verbose > 1) {
fprintf(stderr, "TRAIN_MIX %s\t%s\t[%.2lf %.2lf %.2lf]\t[%.2lf %.2lf %.2lf]\n", model_training_iter->first.c_str(), kmer.c_str(),
std::exp(trained_mixture.log_weights[0]), trained_mixture.params[0].level_mean, trained_mixture.params[0].level_stdv,
std::exp(trained_mixture.log_weights[1]), trained_mixture.params[1].level_mean, trained_mixture.params[1].level_stdv);
}
#pragma omp critical
updated_model.states[ki] = trained_mixture.params[0];
if (model_stdv()) {
ParamMixture ig_mixture;
// weights
ig_mixture.log_weights = trained_mixture.log_weights;
// states
ig_mixture.params.emplace_back(trained_mixture.params[0]);
if(is_m_kmer) {
ig_mixture.params.emplace_back(current_model_iter->second.get_parameters(um_ki));
}
// run training
auto trained_ig_mixture = train_invgaussian_mixture(summaries[ki].events, ig_mixture);
LOG("methyltrain", debug)
<< "IG_INIT__MIX " << model_training_iter->first.c_str() << " " << kmer.c_str() << " ["
<< std::fixed << std::setprecision(5) << ig_mixture.params[0].sd_mean << " "
<< ig_mixture.params[1].sd_mean << "]" << std::endl
<< "IG_TRAIN_MIX " << model_training_iter->first.c_str() << " " << kmer.c_str() << " ["
<< trained_ig_mixture.params[0].sd_mean << " "
<< trained_ig_mixture.params[1].sd_mean << "]" << std::endl;
// update state
#pragma omp critical
{
updated_model.states[ki] = trained_ig_mixture.params[0];
}
}
trained = true;
}
#pragma omp critical
{
fprintf(summary_fp, "%s\t%s\t%d\t%d\t%d\t%zu\t%d\t%.2lf\t%.2lf\n",
model_short_name.c_str(), kmer.c_str(),
summaries[ki].num_matches, summaries[ki].num_skips, summaries[ki].num_stays,
summaries[ki].events.size(), trained, updated_model.states[ki].level_mean, updated_model.states[ki].level_stdv);
}
// add the updated model into the collection (or replace what is already there)
PoreModelSet::insert_model(opt::trained_model_type, updated_model);
}
}
// cleanup records
for(size_t i = 0; i < records.size(); ++i) {
bam_destroy1(records[i]);
}
// cleanup
sam_itr_destroy(itr);
bam_hdr_destroy(hdr);
fai_destroy(fai);
sam_close(bam_fh);
hts_idx_destroy(bam_idx);
fclose(summary_fp);
}
/*
* Performs pileup
* @param conf configuration for this pileup
* @param n number of files specified in fn
* @param fn filenames
*/
static int mpileup(mplp_conf_t *conf, int n, char **fn)
{
extern void *bcf_call_add_rg(void *rghash, const char *hdtext, const char *list);
extern void bcf_call_del_rghash(void *rghash);
mplp_aux_t **data;
int i, tid, pos, *n_plp, beg0 = 0, end0 = INT_MAX, ref_len, max_depth, max_indel_depth;
const bam_pileup1_t **plp;
mplp_ref_t mp_ref = MPLP_REF_INIT;
bam_mplp_t iter;
bam_hdr_t *h = NULL; /* header of first file in input list */
char *ref;
void *rghash = NULL;
FILE *pileup_fp = NULL;
bcf_callaux_t *bca = NULL;
bcf_callret1_t *bcr = NULL;
bcf_call_t bc;
htsFile *bcf_fp = NULL;
bcf_hdr_t *bcf_hdr = NULL;
bam_sample_t *sm = NULL;
kstring_t buf;
mplp_pileup_t gplp;
memset(&gplp, 0, sizeof(mplp_pileup_t));
memset(&buf, 0, sizeof(kstring_t));
memset(&bc, 0, sizeof(bcf_call_t));
data = calloc(n, sizeof(mplp_aux_t*));
plp = calloc(n, sizeof(bam_pileup1_t*));
n_plp = calloc(n, sizeof(int));
sm = bam_smpl_init();
if (n == 0) {
fprintf(pysam_stderr,"[%s] no input file/data given\n", __func__);
exit(EXIT_FAILURE);
}
// read the header of each file in the list and initialize data
for (i = 0; i < n; ++i) {
bam_hdr_t *h_tmp;
data[i] = calloc(1, sizeof(mplp_aux_t));
data[i]->fp = sam_open_format(fn[i], "rb", &conf->ga.in);
if ( !data[i]->fp )
{
fprintf(pysam_stderr, "[%s] failed to open %s: %s\n", __func__, fn[i], strerror(errno));
exit(EXIT_FAILURE);
}
if (hts_set_opt(data[i]->fp, CRAM_OPT_DECODE_MD, 0)) {
fprintf(pysam_stderr, "Failed to set CRAM_OPT_DECODE_MD value\n");
exit(EXIT_FAILURE);
}
if (conf->fai_fname && hts_set_fai_filename(data[i]->fp, conf->fai_fname) != 0) {
fprintf(pysam_stderr, "[%s] failed to process %s: %s\n",
__func__, conf->fai_fname, strerror(errno));
exit(EXIT_FAILURE);
}
data[i]->conf = conf;
data[i]->ref = &mp_ref;
h_tmp = sam_hdr_read(data[i]->fp);
if ( !h_tmp ) {
fprintf(pysam_stderr,"[%s] fail to read the header of %s\n", __func__, fn[i]);
exit(EXIT_FAILURE);
}
bam_smpl_add(sm, fn[i], (conf->flag&MPLP_IGNORE_RG)? 0 : h_tmp->text);
// Collect read group IDs with PL (platform) listed in pl_list (note: fragile, strstr search)
rghash = bcf_call_add_rg(rghash, h_tmp->text, conf->pl_list);
if (conf->reg) {
hts_idx_t *idx = sam_index_load(data[i]->fp, fn[i]);
if (idx == NULL) {
fprintf(pysam_stderr, "[%s] fail to load index for %s\n", __func__, fn[i]);
exit(EXIT_FAILURE);
}
if ( (data[i]->iter=sam_itr_querys(idx, h_tmp, conf->reg)) == 0) {
fprintf(pysam_stderr, "[E::%s] fail to parse region '%s' with %s\n", __func__, conf->reg, fn[i]);
exit(EXIT_FAILURE);
}
if (i == 0) beg0 = data[i]->iter->beg, end0 = data[i]->iter->end;
hts_idx_destroy(idx);
}
else
data[i]->iter = NULL;
if (i == 0) h = data[i]->h = h_tmp; // save the header of the first file
else {
// FIXME: check consistency between h and h_tmp
bam_hdr_destroy(h_tmp);
// we store only the first file's header; it's (alleged to be)
// compatible with the i-th file's target_name lookup needs
data[i]->h = h;
}
}
// allocate data storage proportionate to number of samples being studied sm->n
gplp.n = sm->n;
gplp.n_plp = calloc(sm->n, sizeof(int));
gplp.m_plp = calloc(sm->n, sizeof(int));
gplp.plp = calloc(sm->n, sizeof(bam_pileup1_t*));
fprintf(pysam_stderr, "[%s] %d samples in %d input files\n", __func__, sm->n, n);
// write the VCF header
if (conf->flag & MPLP_BCF)
{
const char *mode;
if ( conf->flag & MPLP_VCF )
mode = (conf->flag&MPLP_NO_COMP)? "wu" : "wz"; // uncompressed VCF or compressed VCF
else
mode = (conf->flag&MPLP_NO_COMP)? "wub" : "wb"; // uncompressed BCF or compressed BCF
bcf_fp = bcf_open(conf->output_fname? conf->output_fname : "-", mode);
if (bcf_fp == NULL) {
fprintf(pysam_stderr, "[%s] failed to write to %s: %s\n", __func__, conf->output_fname? conf->output_fname : "standard output", strerror(errno));
exit(EXIT_FAILURE);
}
// BCF header creation
bcf_hdr = bcf_hdr_init("w");
kstring_t str = {0,0,NULL};
ksprintf(&str, "##samtoolsVersion=%s+htslib-%s\n",samtools_version(),hts_version());
bcf_hdr_append(bcf_hdr, str.s);
str.l = 0;
ksprintf(&str, "##samtoolsCommand=samtools mpileup");
for (i=1; i<conf->argc; i++) ksprintf(&str, " %s", conf->argv[i]);
kputc('\n', &str);
bcf_hdr_append(bcf_hdr, str.s);
if (conf->fai_fname)
{
str.l = 0;
ksprintf(&str, "##reference=file://%s\n", conf->fai_fname);
bcf_hdr_append(bcf_hdr, str.s);
}
// Translate BAM @SQ tags to BCF ##contig tags
// todo: use/write new BAM header manipulation routines, fill also UR, M5
for (i=0; i<h->n_targets; i++)
{
str.l = 0;
ksprintf(&str, "##contig=<ID=%s,length=%d>", h->target_name[i], h->target_len[i]);
bcf_hdr_append(bcf_hdr, str.s);
}
free(str.s);
bcf_hdr_append(bcf_hdr,"##ALT=<ID=*,Description=\"Represents allele(s) other than observed.\">");
bcf_hdr_append(bcf_hdr,"##INFO=<ID=INDEL,Number=0,Type=Flag,Description=\"Indicates that the variant is an INDEL.\">");
bcf_hdr_append(bcf_hdr,"##INFO=<ID=IDV,Number=1,Type=Integer,Description=\"Maximum number of reads supporting an indel\">");
bcf_hdr_append(bcf_hdr,"##INFO=<ID=IMF,Number=1,Type=Float,Description=\"Maximum fraction of reads supporting an indel\">");
bcf_hdr_append(bcf_hdr,"##INFO=<ID=DP,Number=1,Type=Integer,Description=\"Raw read depth\">");
bcf_hdr_append(bcf_hdr,"##INFO=<ID=VDB,Number=1,Type=Float,Description=\"Variant Distance Bias for filtering splice-site artefacts in RNA-seq data (bigger is better)\",Version=\"3\">");
bcf_hdr_append(bcf_hdr,"##INFO=<ID=RPB,Number=1,Type=Float,Description=\"Mann-Whitney U test of Read Position Bias (bigger is better)\">");
bcf_hdr_append(bcf_hdr,"##INFO=<ID=MQB,Number=1,Type=Float,Description=\"Mann-Whitney U test of Mapping Quality Bias (bigger is better)\">");
bcf_hdr_append(bcf_hdr,"##INFO=<ID=BQB,Number=1,Type=Float,Description=\"Mann-Whitney U test of Base Quality Bias (bigger is better)\">");
bcf_hdr_append(bcf_hdr,"##INFO=<ID=MQSB,Number=1,Type=Float,Description=\"Mann-Whitney U test of Mapping Quality vs Strand Bias (bigger is better)\">");
#if CDF_MWU_TESTS
bcf_hdr_append(bcf_hdr,"##INFO=<ID=RPB2,Number=1,Type=Float,Description=\"Mann-Whitney U test of Read Position Bias [CDF] (bigger is better)\">");
bcf_hdr_append(bcf_hdr,"##INFO=<ID=MQB2,Number=1,Type=Float,Description=\"Mann-Whitney U test of Mapping Quality Bias [CDF] (bigger is better)\">");
bcf_hdr_append(bcf_hdr,"##INFO=<ID=BQB2,Number=1,Type=Float,Description=\"Mann-Whitney U test of Base Quality Bias [CDF] (bigger is better)\">");
bcf_hdr_append(bcf_hdr,"##INFO=<ID=MQSB2,Number=1,Type=Float,Description=\"Mann-Whitney U test of Mapping Quality vs Strand Bias [CDF] (bigger is better)\">");
#endif
bcf_hdr_append(bcf_hdr,"##INFO=<ID=SGB,Number=1,Type=Float,Description=\"Segregation based metric.\">");
bcf_hdr_append(bcf_hdr,"##INFO=<ID=MQ0F,Number=1,Type=Float,Description=\"Fraction of MQ0 reads (smaller is better)\">");
bcf_hdr_append(bcf_hdr,"##INFO=<ID=I16,Number=16,Type=Float,Description=\"Auxiliary tag used for calling, see description of bcf_callret1_t in bam2bcf.h\">");
bcf_hdr_append(bcf_hdr,"##INFO=<ID=QS,Number=R,Type=Float,Description=\"Auxiliary tag used for calling\">");
bcf_hdr_append(bcf_hdr,"##FORMAT=<ID=PL,Number=G,Type=Integer,Description=\"List of Phred-scaled genotype likelihoods\">");
if ( conf->fmt_flag&B2B_FMT_DP )
bcf_hdr_append(bcf_hdr,"##FORMAT=<ID=DP,Number=1,Type=Integer,Description=\"Number of high-quality bases\">");
if ( conf->fmt_flag&B2B_FMT_DV )
bcf_hdr_append(bcf_hdr,"##FORMAT=<ID=DV,Number=1,Type=Integer,Description=\"Number of high-quality non-reference bases\">");
if ( conf->fmt_flag&B2B_FMT_DPR )
bcf_hdr_append(bcf_hdr,"##FORMAT=<ID=DPR,Number=R,Type=Integer,Description=\"Number of high-quality bases observed for each allele\">");
if ( conf->fmt_flag&B2B_INFO_DPR )
bcf_hdr_append(bcf_hdr,"##INFO=<ID=DPR,Number=R,Type=Integer,Description=\"Number of high-quality bases observed for each allele\">");
if ( conf->fmt_flag&B2B_FMT_DP4 )
bcf_hdr_append(bcf_hdr,"##FORMAT=<ID=DP4,Number=4,Type=Integer,Description=\"Number of high-quality ref-fwd, ref-reverse, alt-fwd and alt-reverse bases\">");
if ( conf->fmt_flag&B2B_FMT_SP )
bcf_hdr_append(bcf_hdr,"##FORMAT=<ID=SP,Number=1,Type=Integer,Description=\"Phred-scaled strand bias P-value\">");
if ( conf->fmt_flag&B2B_FMT_AD )
bcf_hdr_append(bcf_hdr,"##FORMAT=<ID=AD,Number=R,Type=Integer,Description=\"Allelic depths\">");
if ( conf->fmt_flag&B2B_FMT_ADF )
bcf_hdr_append(bcf_hdr,"##FORMAT=<ID=ADF,Number=R,Type=Integer,Description=\"Allelic depths on the forward strand\">");
if ( conf->fmt_flag&B2B_FMT_ADR )
bcf_hdr_append(bcf_hdr,"##FORMAT=<ID=ADR,Number=R,Type=Integer,Description=\"Allelic depths on the reverse strand\">");
if ( conf->fmt_flag&B2B_INFO_AD )
bcf_hdr_append(bcf_hdr,"##INFO=<ID=AD,Number=R,Type=Integer,Description=\"Total allelic depths\">");
if ( conf->fmt_flag&B2B_INFO_ADF )
bcf_hdr_append(bcf_hdr,"##INFO=<ID=ADF,Number=R,Type=Integer,Description=\"Total allelic depths on the forward strand\">");
if ( conf->fmt_flag&B2B_INFO_ADR )
bcf_hdr_append(bcf_hdr,"##INFO=<ID=ADR,Number=R,Type=Integer,Description=\"Total allelic depths on the reverse strand\">");
for (i=0; i<sm->n; i++)
bcf_hdr_add_sample(bcf_hdr, sm->smpl[i]);
bcf_hdr_add_sample(bcf_hdr, NULL);
bcf_hdr_write(bcf_fp, bcf_hdr);
// End of BCF header creation
// Initialise the calling algorithm
bca = bcf_call_init(-1., conf->min_baseQ);
bcr = calloc(sm->n, sizeof(bcf_callret1_t));
bca->rghash = rghash;
bca->openQ = conf->openQ, bca->extQ = conf->extQ, bca->tandemQ = conf->tandemQ;
bca->min_frac = conf->min_frac;
bca->min_support = conf->min_support;
bca->per_sample_flt = conf->flag & MPLP_PER_SAMPLE;
bc.bcf_hdr = bcf_hdr;
bc.n = sm->n;
bc.PL = malloc(15 * sm->n * sizeof(*bc.PL));
if (conf->fmt_flag)
{
assert( sizeof(float)==sizeof(int32_t) );
bc.DP4 = malloc(sm->n * sizeof(int32_t) * 4);
bc.fmt_arr = malloc(sm->n * sizeof(float)); // all fmt_flag fields
if ( conf->fmt_flag&(B2B_INFO_DPR|B2B_FMT_DPR|B2B_INFO_AD|B2B_INFO_ADF|B2B_INFO_ADR|B2B_FMT_AD|B2B_FMT_ADF|B2B_FMT_ADR) )
{
// first B2B_MAX_ALLELES fields for total numbers, the rest per-sample
bc.ADR = (int32_t*) malloc((sm->n+1)*B2B_MAX_ALLELES*sizeof(int32_t));
bc.ADF = (int32_t*) malloc((sm->n+1)*B2B_MAX_ALLELES*sizeof(int32_t));
for (i=0; i<sm->n; i++)
{
bcr[i].ADR = bc.ADR + (i+1)*B2B_MAX_ALLELES;
bcr[i].ADF = bc.ADF + (i+1)*B2B_MAX_ALLELES;
}
}
}
}
else {
pileup_fp = conf->output_fname? fopen(conf->output_fname, "w") : pysam_stdout;
if (pileup_fp == NULL) {
fprintf(pysam_stderr, "[%s] failed to write to %s: %s\n", __func__, conf->output_fname, strerror(errno));
exit(EXIT_FAILURE);
}
}
// init pileup
iter = bam_mplp_init(n, mplp_func, (void**)data);
if ( conf->flag & MPLP_SMART_OVERLAPS ) bam_mplp_init_overlaps(iter);
max_depth = conf->max_depth;
if (max_depth * sm->n > 1<<20)
fprintf(pysam_stderr, "(%s) Max depth is above 1M. Potential memory hog!\n", __func__);
if (max_depth * sm->n < 8000) {
max_depth = 8000 / sm->n;
fprintf(pysam_stderr, "<%s> Set max per-file depth to %d\n", __func__, max_depth);
}
max_indel_depth = conf->max_indel_depth * sm->n;
bam_mplp_set_maxcnt(iter, max_depth);
bcf1_t *bcf_rec = bcf_init1();
int ret;
// begin pileup
while ( (ret=bam_mplp_auto(iter, &tid, &pos, n_plp, plp)) > 0) {
if (conf->reg && (pos < beg0 || pos >= end0)) continue; // out of the region requested
if (conf->bed && tid >= 0 && !bed_overlap(conf->bed, h->target_name[tid], pos, pos+1)) continue;
mplp_get_ref(data[0], tid, &ref, &ref_len);
//printf("tid=%d len=%d ref=%p/%s\n", tid, ref_len, ref, ref);
if (conf->flag & MPLP_BCF) {
int total_depth, _ref0, ref16;
for (i = total_depth = 0; i < n; ++i) total_depth += n_plp[i];
group_smpl(&gplp, sm, &buf, n, fn, n_plp, plp, conf->flag & MPLP_IGNORE_RG);
_ref0 = (ref && pos < ref_len)? ref[pos] : 'N';
ref16 = seq_nt16_table[_ref0];
bcf_callaux_clean(bca, &bc);
for (i = 0; i < gplp.n; ++i)
bcf_call_glfgen(gplp.n_plp[i], gplp.plp[i], ref16, bca, bcr + i);
bc.tid = tid; bc.pos = pos;
bcf_call_combine(gplp.n, bcr, bca, ref16, &bc);
bcf_clear1(bcf_rec);
bcf_call2bcf(&bc, bcf_rec, bcr, conf->fmt_flag, 0, 0);
bcf_write1(bcf_fp, bcf_hdr, bcf_rec);
// call indels; todo: subsampling with total_depth>max_indel_depth instead of ignoring?
if (!(conf->flag&MPLP_NO_INDEL) && total_depth < max_indel_depth && bcf_call_gap_prep(gplp.n, gplp.n_plp, gplp.plp, pos, bca, ref, rghash) >= 0)
{
bcf_callaux_clean(bca, &bc);
for (i = 0; i < gplp.n; ++i)
bcf_call_glfgen(gplp.n_plp[i], gplp.plp[i], -1, bca, bcr + i);
if (bcf_call_combine(gplp.n, bcr, bca, -1, &bc) >= 0) {
bcf_clear1(bcf_rec);
bcf_call2bcf(&bc, bcf_rec, bcr, conf->fmt_flag, bca, ref);
bcf_write1(bcf_fp, bcf_hdr, bcf_rec);
}
}
} else {
fprintf(pileup_fp, "%s\t%d\t%c", h->target_name[tid], pos + 1, (ref && pos < ref_len)? ref[pos] : 'N');
for (i = 0; i < n; ++i) {
int j, cnt;
for (j = cnt = 0; j < n_plp[i]; ++j) {
const bam_pileup1_t *p = plp[i] + j;
int c = p->qpos < p->b->core.l_qseq
? bam_get_qual(p->b)[p->qpos]
: 0;
if (c >= conf->min_baseQ) ++cnt;
}
fprintf(pileup_fp, "\t%d\t", cnt);
if (n_plp[i] == 0) {
fputs("*\t*", pileup_fp);
if (conf->flag & MPLP_PRINT_MAPQ) fputs("\t*", pileup_fp);
if (conf->flag & MPLP_PRINT_POS) fputs("\t*", pileup_fp);
} else {
for (j = 0; j < n_plp[i]; ++j) {
const bam_pileup1_t *p = plp[i] + j;
int c = p->qpos < p->b->core.l_qseq
? bam_get_qual(p->b)[p->qpos]
: 0;
if (c >= conf->min_baseQ)
pileup_seq(pileup_fp, plp[i] + j, pos, ref_len, ref);
}
putc('\t', pileup_fp);
for (j = 0; j < n_plp[i]; ++j) {
const bam_pileup1_t *p = plp[i] + j;
int c = p->qpos < p->b->core.l_qseq
? bam_get_qual(p->b)[p->qpos]
: 0;
if (c >= conf->min_baseQ) {
c = c + 33 < 126? c + 33 : 126;
putc(c, pileup_fp);
}
}
if (conf->flag & MPLP_PRINT_MAPQ) {
putc('\t', pileup_fp);
for (j = 0; j < n_plp[i]; ++j) {
const bam_pileup1_t *p = plp[i] + j;
int c = bam_get_qual(p->b)[p->qpos];
if ( c < conf->min_baseQ ) continue;
c = plp[i][j].b->core.qual + 33;
if (c > 126) c = 126;
putc(c, pileup_fp);
}
}
if (conf->flag & MPLP_PRINT_POS) {
putc('\t', pileup_fp);
int last = 0;
for (j = 0; j < n_plp[i]; ++j) {
const bam_pileup1_t *p = plp[i] + j;
int c = bam_get_qual(p->b)[p->qpos];
if ( c < conf->min_baseQ ) continue;
if (last++) putc(',', pileup_fp);
fprintf(pileup_fp, "%d", plp[i][j].qpos + 1); // FIXME: fprintf(pysam_stdout, ) is very slow...
}
}
}
}
putc('\n', pileup_fp);
}
}
// clean up
free(bc.tmp.s);
bcf_destroy1(bcf_rec);
if (bcf_fp)
{
hts_close(bcf_fp);
bcf_hdr_destroy(bcf_hdr);
bcf_call_destroy(bca);
free(bc.PL);
free(bc.DP4);
free(bc.ADR);
free(bc.ADF);
free(bc.fmt_arr);
free(bcr);
}
if (pileup_fp && conf->output_fname) fclose(pileup_fp);
bam_smpl_destroy(sm); free(buf.s);
for (i = 0; i < gplp.n; ++i) free(gplp.plp[i]);
free(gplp.plp); free(gplp.n_plp); free(gplp.m_plp);
bcf_call_del_rghash(rghash);
bam_mplp_destroy(iter);
bam_hdr_destroy(h);
for (i = 0; i < n; ++i) {
sam_close(data[i]->fp);
if (data[i]->iter) hts_itr_destroy(data[i]->iter);
free(data[i]);
}
free(data); free(plp); free(n_plp);
free(mp_ref.ref[0]);
free(mp_ref.ref[1]);
return ret;
}
int scorereads_main(int argc, char** argv)
{
parse_scorereads_options(argc, argv);
omp_set_num_threads(opt::num_threads);
Fast5Map name_map(opt::reads_file);
ModelMap models;
if (!opt::models_fofn.empty())
models = read_models_fofn(opt::models_fofn);
// Open the BAM and iterate over reads
// load bam file
htsFile* bam_fh = sam_open(opt::bam_file.c_str(), "r");
assert(bam_fh != NULL);
// load bam index file
std::string index_filename = opt::bam_file + ".bai";
hts_idx_t* bam_idx = bam_index_load(index_filename.c_str());
assert(bam_idx != NULL);
// read the bam header
bam_hdr_t* hdr = sam_hdr_read(bam_fh);
// load reference fai file
faidx_t *fai = fai_load(opt::genome_file.c_str());
hts_itr_t* itr;
// If processing a region of the genome, only emit events aligned to this window
int clip_start = -1;
int clip_end = -1;
if(opt::region.empty()) {
// TODO: is this valid?
itr = sam_itr_queryi(bam_idx, HTS_IDX_START, 0, 0);
} else {
fprintf(stderr, "Region: %s\n", opt::region.c_str());
itr = sam_itr_querys(bam_idx, hdr, opt::region.c_str());
hts_parse_reg(opt::region.c_str(), &clip_start, &clip_end);
}
#ifndef H5_HAVE_THREADSAFE
if(opt::num_threads > 1) {
fprintf(stderr, "You enabled multi-threading but you do not have a threadsafe HDF5\n");
fprintf(stderr, "Please recompile nanopolish's built-in libhdf5 or run with -t 1\n");
exit(1);
}
#endif
// Initialize iteration
std::vector<bam1_t*> records(opt::batch_size, NULL);
for(size_t i = 0; i < records.size(); ++i) {
records[i] = bam_init1();
}
int result;
size_t num_reads_realigned = 0;
size_t num_records_buffered = 0;
do {
assert(num_records_buffered < records.size());
// read a record into the next slot in the buffer
result = sam_itr_next(bam_fh, itr, records[num_records_buffered]);
num_records_buffered += result >= 0;
// realign if we've hit the max buffer size or reached the end of file
if(num_records_buffered == records.size() || result < 0) {
#pragma omp parallel for schedule(dynamic)
for(size_t i = 0; i < num_records_buffered; ++i) {
bam1_t* record = records[i];
size_t read_idx = num_reads_realigned + i;
if( (record->core.flag & BAM_FUNMAP) == 0) {
//load read
std::string read_name = bam_get_qname(record);
std::string fast5_path = name_map.get_path(read_name);
SquiggleRead sr(read_name, fast5_path);
// TODO: early exit when have processed all of the reads in readnames
if (!opt::readnames.empty() &&
std::find(opt::readnames.begin(), opt::readnames.end(), read_name) == opt::readnames.end() )
continue;
for(size_t strand_idx = 0; strand_idx < NUM_STRANDS; ++strand_idx) {
std::vector<EventAlignment> ao = alignment_from_read(sr, strand_idx, read_idx,
models, fai, hdr,
record, clip_start, clip_end);
if (ao.size() == 0)
continue;
// Update pore model based on alignment
if ( opt::calibrate )
recalibrate_model(sr, strand_idx, ao, false);
double score = model_score(sr, strand_idx, fai, ao, 500);
if (score > 0)
continue;
#pragma omp critical(print)
std::cout << read_name << " " << ( strand_idx ? "complement" : "template" )
<< " " << sr.pore_model[strand_idx].name << " " << score << std::endl;
}
}
}
num_reads_realigned += num_records_buffered;
num_records_buffered = 0;
}
} while(result >= 0);
// cleanup records
for(size_t i = 0; i < records.size(); ++i) {
bam_destroy1(records[i]);
}
// cleanup
sam_itr_destroy(itr);
bam_hdr_destroy(hdr);
fai_destroy(fai);
sam_close(bam_fh);
hts_idx_destroy(bam_idx);
return 0;
}
void bam_access_closehts(){
if(fholder->idx) hts_idx_destroy(fholder->idx);
if(fholder->in) hts_close(fholder->in);
if(fholder) free(fholder);
return;
}
int main_bedcov(int argc, char *argv[])
{
gzFile fp;
kstring_t str;
kstream_t *ks;
hts_idx_t **idx;
aux_t **aux;
int *n_plp, dret, i, n, c, min_mapQ = 0;
int64_t *cnt;
const bam_pileup1_t **plp;
int usage = 0;
sam_global_args ga = SAM_GLOBAL_ARGS_INIT;
static const struct option lopts[] = {
SAM_OPT_GLOBAL_OPTIONS('-', 0, '-', '-', 0),
{ NULL, 0, NULL, 0 }
};
while ((c = getopt_long(argc, argv, "Q:", lopts, NULL)) >= 0) {
switch (c) {
case 'Q': min_mapQ = atoi(optarg); break;
default: if (parse_sam_global_opt(c, optarg, lopts, &ga) == 0) break;
/* else fall-through */
case '?': usage = 1; break;
}
if (usage) break;
}
if (usage || optind + 2 > argc) {
fprintf(stderr, "Usage: samtools bedcov [options] <in.bed> <in1.bam> [...]\n\n");
fprintf(stderr, "Options:\n");
fprintf(stderr, " -Q <int> mapping quality threshold [0]\n");
sam_global_opt_help(stderr, "-.--.");
return 1;
}
memset(&str, 0, sizeof(kstring_t));
n = argc - optind - 1;
aux = calloc(n, sizeof(aux_t*));
idx = calloc(n, sizeof(hts_idx_t*));
for (i = 0; i < n; ++i) {
aux[i] = calloc(1, sizeof(aux_t));
aux[i]->min_mapQ = min_mapQ;
aux[i]->fp = sam_open_format(argv[i+optind+1], "r", &ga.in);
if (aux[i]->fp)
idx[i] = sam_index_load(aux[i]->fp, argv[i+optind+1]);
if (aux[i]->fp == 0 || idx[i] == 0) {
fprintf(stderr, "ERROR: fail to open index BAM file '%s'\n", argv[i+optind+1]);
return 2;
}
// TODO bgzf_set_cache_size(aux[i]->fp, 20);
aux[i]->header = sam_hdr_read(aux[i]->fp);
if (aux[i]->header == NULL) {
fprintf(stderr, "ERROR: failed to read header for '%s'\n",
argv[i+optind+1]);
return 2;
}
}
cnt = calloc(n, 8);
fp = gzopen(argv[optind], "rb");
ks = ks_init(fp);
n_plp = calloc(n, sizeof(int));
plp = calloc(n, sizeof(bam_pileup1_t*));
while (ks_getuntil(ks, KS_SEP_LINE, &str, &dret) >= 0) {
char *p, *q;
int tid, beg, end, pos;
bam_mplp_t mplp;
for (p = q = str.s; *p && *p != '\t'; ++p);
if (*p != '\t') goto bed_error;
*p = 0; tid = bam_name2id(aux[0]->header, q); *p = '\t';
if (tid < 0) goto bed_error;
for (q = p = p + 1; isdigit(*p); ++p);
if (*p != '\t') goto bed_error;
*p = 0; beg = atoi(q); *p = '\t';
for (q = p = p + 1; isdigit(*p); ++p);
if (*p == '\t' || *p == 0) {
int c = *p;
*p = 0; end = atoi(q); *p = c;
} else goto bed_error;
for (i = 0; i < n; ++i) {
if (aux[i]->iter) hts_itr_destroy(aux[i]->iter);
aux[i]->iter = sam_itr_queryi(idx[i], tid, beg, end);
}
mplp = bam_mplp_init(n, read_bam, (void**)aux);
bam_mplp_set_maxcnt(mplp, 64000);
memset(cnt, 0, 8 * n);
while (bam_mplp_auto(mplp, &tid, &pos, n_plp, plp) > 0)
if (pos >= beg && pos < end)
for (i = 0; i < n; ++i) cnt[i] += n_plp[i];
for (i = 0; i < n; ++i) {
kputc('\t', &str);
kputl(cnt[i], &str);
}
puts(str.s);
bam_mplp_destroy(mplp);
continue;
bed_error:
fprintf(stderr, "Errors in BED line '%s'\n", str.s);
}
free(n_plp); free(plp);
ks_destroy(ks);
gzclose(fp);
free(cnt);
for (i = 0; i < n; ++i) {
if (aux[i]->iter) hts_itr_destroy(aux[i]->iter);
hts_idx_destroy(idx[i]);
bam_hdr_destroy(aux[i]->header);
sam_close(aux[i]->fp);
free(aux[i]);
}
free(aux); free(idx);
free(str.s);
sam_global_args_free(&ga);
return 0;
}
static int query_regions(args_t *args, char *fname, char **regs, int nregs)
{
int i;
htsFile *fp = hts_open(fname,"r");
if ( !fp ) error("Could not read %s\n", fname);
enum htsExactFormat format = hts_get_format(fp)->format;
regidx_t *reg_idx = NULL;
if ( args->targets_fname )
{
reg_idx = regidx_init(args->targets_fname, NULL, NULL, 0, NULL);
if ( !reg_idx ) error("Could not read %s\n", args->targets_fname);
}
if ( format == bcf )
{
htsFile *out = hts_open("-","w");
if ( !out ) error("Could not open stdout\n", fname);
hts_idx_t *idx = bcf_index_load(fname);
if ( !idx ) error("Could not load .csi index of %s\n", fname);
bcf_hdr_t *hdr = bcf_hdr_read(fp);
if ( !hdr ) error("Could not read the header: %s\n", fname);
if ( args->print_header )
if ( bcf_hdr_write(out,hdr)!=0 ) error("Failed to write to %s\n", fname);
if ( !args->header_only )
{
bcf1_t *rec = bcf_init();
for (i=0; i<nregs; i++)
{
hts_itr_t *itr = bcf_itr_querys(idx,hdr,regs[i]);
while ( bcf_itr_next(fp, itr, rec) >=0 )
{
if ( reg_idx && !regidx_overlap(reg_idx, bcf_seqname(hdr,rec),rec->pos,rec->pos+rec->rlen-1, NULL) ) continue;
if ( bcf_write(out,hdr,rec)!=0 ) error("Failed to write to %s\n", fname);
}
tbx_itr_destroy(itr);
}
bcf_destroy(rec);
}
if ( hts_close(out) ) error("hts_close returned non-zero status for stdout\n");
bcf_hdr_destroy(hdr);
hts_idx_destroy(idx);
}
else if ( format==vcf || format==sam || format==unknown_format )
{
tbx_t *tbx = tbx_index_load(fname);
if ( !tbx ) error("Could not load .tbi/.csi index of %s\n", fname);
kstring_t str = {0,0,0};
if ( args->print_header )
{
while ( hts_getline(fp, KS_SEP_LINE, &str) >= 0 )
{
if ( !str.l || str.s[0]!=tbx->conf.meta_char ) break;
puts(str.s);
}
}
if ( !args->header_only )
{
int nseq;
const char **seq = NULL;
if ( reg_idx ) seq = tbx_seqnames(tbx, &nseq);
for (i=0; i<nregs; i++)
{
hts_itr_t *itr = tbx_itr_querys(tbx, regs[i]);
if ( !itr ) continue;
while (tbx_itr_next(fp, tbx, itr, &str) >= 0)
{
if ( reg_idx && !regidx_overlap(reg_idx,seq[itr->curr_tid],itr->curr_beg,itr->curr_end-1, NULL) ) continue;
puts(str.s);
}
tbx_itr_destroy(itr);
}
free(seq);
}
free(str.s);
tbx_destroy(tbx);
}
else if ( format==bam )
error("Please use \"samtools view\" for querying BAM files.\n");
if ( reg_idx ) regidx_destroy(reg_idx);
if ( hts_close(fp) ) error("hts_close returned non-zero status: %s\n", fname);
for (i=0; i<nregs; i++) free(regs[i]);
free(regs);
return 0;
}
int convert(int argc, char **argv)
{
if (argc < 2) return convert_help();
int c;
char *in=NULL, *out=NULL, *bim=NULL, *vid=NULL, *tmp_dir=NULL, *ped=NULL;
uint32_t num_fields, num_records, col = 2;
int i_is_set = 0,
o_is_set = 0,
f_is_set = 0,
b_is_set = 0,
v_is_set = 0,
t_is_set = 0,
p_is_set = 0,
r_is_set = 0;
while((c = getopt (argc, argv, "hi:o:f:r:b:v:t:p:c:")) != -1) {
switch (c) {
case 'c':
col = atoi(optarg);
break;
case 'p':
p_is_set = 1;
ped = optarg;
break;
case 't':
t_is_set = 1;
tmp_dir = optarg;
break;
case 'v':
v_is_set = 1;
vid = optarg;
break;
case 'b':
b_is_set = 1;
bim = optarg;
break;
case 'i':
i_is_set = 1;
in = optarg;
break;
case 'o':
o_is_set = 1;
out = optarg;
break;
case 'f':
f_is_set = 1;
num_fields = atoi(optarg);
break;
case 'r':
r_is_set = 1;
num_records = atoi(optarg);
break;
case 'h':
convert_help();
return 1;
case '?':
if ( (optopt == 'i') ||
(optopt == 'f') ||
(optopt == 'r') ||
(optopt == 't') ||
(optopt == 's') ||
(optopt == 'p') ||
(optopt == 'c') ||
(optopt == 'o') )
fprintf (stderr, "Option -%c requires an argument.\n",
optopt);
else if (isprint (optopt))
fprintf (stderr, "Unknown option `-%c'.\n", optopt);
else
fprintf (stderr, "Unknown option character `\\x%x'.\n", optopt);
default:
convert_help();
return 1;
}
}
char *type = argv[0];
if (i_is_set == 0) {
printf("Input file is not set\n");
return convert_help();
}
if (strcmp(type, "bcf") == 0) {
if ( (f_is_set == 0) || (r_is_set == 0) ) {
fprintf(stderr,"Attempting to autodetect num of records "
"and fields from %s\n", in);
//Try and auto detect the sizes, need the index
tbx_t *tbx = NULL;
hts_idx_t *idx = NULL;
htsFile *fp = hts_open(in,"rb");
if ( !fp ) {
fprintf(stderr,"Could not read %s\n", in);
return 1;
}
bcf_hdr_t *hdr = bcf_hdr_read(fp);
if ( !hdr ) {
fprintf(stderr,"Could not read the header: %s\n", in);
return 1;
}
if (hts_get_format(fp)->format==vcf) {
tbx = tbx_index_load(in);
if ( !tbx ) {
fprintf(stderr,"Could not load TBI index: %s\n", in);
return 1;
}
} else if ( hts_get_format(fp)->format==bcf ) {
idx = bcf_index_load(in);
if ( !idx ) {
fprintf(stderr,"Could not load CSI index: %s\n", in);
return 1;
}
} else {
fprintf(stderr,
"Could not detect the file type as VCF or BCF: %s\n",
in);
return 1;
}
num_fields = hdr->n[BCF_DT_SAMPLE];
num_records = 0;
const char **seq;
int nseq;
seq = tbx ? tbx_seqnames(tbx, &nseq) :
bcf_index_seqnames(idx, hdr, &nseq);
int i;
uint32_t sum = 0;
for (i = 0; i < nseq; ++i) {
uint64_t records, v;
hts_idx_get_stat(tbx ? tbx->idx: idx, i, &records, &v);
num_records += records;
}
fprintf(stderr, "Number of records:%u\tNumber of fields:%u\n",
num_records, num_fields);
free(seq);
hts_close(fp);
bcf_hdr_destroy(hdr);
if (idx)
hts_idx_destroy(idx);
if (tbx)
tbx_destroy(tbx);
}
if (o_is_set == 0) {
out = (char*)malloc(strlen(in) + 5); // 5 for ext and \0
strcpy(out,in);
strcat(out, ".gqt");
}
if (b_is_set == 0) {
bim = (char*)malloc(strlen(in) + 5); // 5 for ext and \0
strcpy(bim,in);
strcat(bim, ".bim");
}
if (v_is_set == 0) {
vid = (char*)malloc(strlen(in) + 5); // 5 for ext and \0
strcpy(vid,in);
strcat(vid, ".vid");
}
if (t_is_set == 0) {
tmp_dir = (char*)malloc(3*sizeof(char)); // "./\0"
strcpy(tmp_dir,"./");
}
int r = bcf_wahbm(in, out, bim, vid, tmp_dir, num_fields, num_records);
return r;
}
if (strcmp(type, "ped") == 0) {
if (o_is_set == 0) {
if (p_is_set == 1) {
out = (char*)malloc(strlen(ped) + 4); // 4 for ext and \0
strcpy(out,ped);
strcat(out, ".db");
} else {
out = (char*)malloc(strlen(in) + 4); // 4 for ext and \0
strcpy(out,in);
strcat(out, ".db");
}
}
fprintf(stderr, "Creating sample database %s\n", out);
return ped_ped(in, ped, col, out);
}
return convert_help();
}
