void get_dqstats(
const bam_pileup1_t* buf,
int n_reads,
int ref_base,
uint32_t wanted_bases,
dqstats_t *dqs
)
{
int i, j;
int base;
memset(dqs, 0, sizeof(dqstats_t));
for (i = 0; i < n_reads; ++i) {
if (buf[i].is_del || buf[i].b->core.flag&BAM_FUNMAP)
continue;
++dqs->total_depth;
dqs->total_mean_mapQ += buf[i].b->core.qual;
base = bam1_seqi(bam1_seq(buf[i].b), buf[i].qpos);
if (base == ref_base)
++dqs->dp4[bam1_strand(buf[i].b)];
else
++dqs->dp4[2+bam1_strand(buf[i].b)];
for (j = 0; j < 4; ++j) {
int value = 1 << j;
if ((base & value) == base) {
++dqs->base_occ[j];
if (value & wanted_bases) {
dqs->mean_baseQ[j] += bam1_qual(buf[i].b)[buf[i].qpos];
dqs->mean_mapQ[j] += buf[i].b->core.qual;
}
}
}
}
for (i = 0; i < 4; ++i) {
if (dqs->base_occ[i] > 0) {
dqs->mean_baseQ[i] = dqs->mean_baseQ[i]/(double)dqs->base_occ[i] + .499;
dqs->mean_mapQ[i] = dqs->mean_mapQ[i]/(double)dqs->base_occ[i] + .499;
}
}
if (dqs->total_depth > 0)
dqs->total_mean_mapQ = dqs->total_mean_mapQ / (double)dqs->total_depth + .499;
}
static int fetch_disc_read_callback(const bam1_t* alignment, void* data) {
// MEI_data* mei_data = static_cast<MEI_data*>(data);
std::pair<MEI_data*, UserDefinedSettings*>* env = static_cast<std::pair<MEI_data*, UserDefinedSettings*>*>(data);
MEI_data* mei_data = env->first;
UserDefinedSettings* userSettings = env->second;
if (!(alignment->core.flag & BAM_FUNMAP || alignment->core.flag & BAM_FMUNMAP) && // Both ends are mapped.
!is_concordant(alignment, mei_data->current_insert_size) && // Ends map discordantly.
// Extra check for (very) large mapping distance. This is done beside the check for read
// discordance to speed up computation by ignoring signals from small structural variants.
(alignment->core.tid != alignment->core.mtid ||
abs(alignment->core.pos - alignment->core.mpos) > userSettings->MIN_DD_MAP_DISTANCE)) {
// Save alignment as simple_read object.
std::string read_name = enrich_read_name(bam1_qname(alignment), alignment->core.flag & BAM_FREAD1);
char strand = bam1_strand(alignment)? Minus : Plus;
char mate_strand = bam1_mstrand(alignment)? Minus : Plus;
std::string read_group;
get_read_group(alignment, read_group);
std::string sample_name;
get_sample_name(read_group, mei_data->sample_names, sample_name);
simple_read* read = new simple_read(read_name, alignment->core.tid, alignment->core.pos, strand, sample_name,
get_sequence(bam1_seq(alignment), alignment->core.l_qseq),
alignment->core.mtid, alignment->core.mpos, mate_strand);
mei_data->discordant_reads.push_back(read);
}
return 0;
}
static inline int bam1_lt(const bam1_p a, const bam1_p b)
{
if (g_is_by_qname) {
int t = strnum_cmp(bam1_qname(a), bam1_qname(b));
return (t < 0 || (t == 0 && (a->core.flag&0xc0) < (b->core.flag&0xc0)));
} else return (((uint64_t)a->core.tid<<32|(a->core.pos+1)<<1|bam1_strand(a)) < ((uint64_t)b->core.tid<<32|(b->core.pos+1)<<1|bam1_strand(b)));
}
static inline void pileup_seq(const bam_pileup1_t *p, int pos, int ref_len, const char *ref)
{
int j;
if (p->is_head) {
putchar('^');
putchar(p->b->core.qual > 93? 126 : p->b->core.qual + 33);
}
if (!p->is_del) {
int c = bam_nt16_rev_table[bam1_seqi(bam1_seq(p->b), p->qpos)];
if (ref) {
int rb = pos < ref_len? ref[pos] : 'N';
if (c == '=' || bam_nt16_table[c] == bam_nt16_table[rb]) c = bam1_strand(p->b)? ',' : '.';
else c = bam1_strand(p->b)? tolower(c) : toupper(c);
} else {
if (c == '=') c = bam1_strand(p->b)? ',' : '.';
else c = bam1_strand(p->b)? tolower(c) : toupper(c);
}
putchar(c);
} else putchar(p->is_refskip? (bam1_strand(p->b)? '<' : '>') : '*');
if (p->indel > 0) {
putchar('+'); printw(p->indel, stdout);
for (j = 1; j <= p->indel; ++j) {
int c = bam_nt16_rev_table[bam1_seqi(bam1_seq(p->b), p->qpos + j)];
putchar(bam1_strand(p->b)? tolower(c) : toupper(c));
}
} else if (p->indel < 0) {
printw(p->indel, stdout);
for (j = 1; j <= -p->indel; ++j) {
int c = (ref && (int)pos+j < ref_len)? ref[pos+j] : 'N';
putchar(bam1_strand(p->b)? tolower(c) : toupper(c));
}
}
if (p->is_tail) putchar('$');
}
static bwa_seq_t *bwa_read_bam(bwa_seqio_t *bs, int n_needed, int *n, int is_comp, int trim_qual)
{
bwa_seq_t *seqs, *p;
int n_seqs, l, i;
long n_trimmed = 0, n_tot = 0;
bam1_t *b;
int res;
b = bam_init1();
n_seqs = 0;
seqs = (bwa_seq_t*)calloc(n_needed, sizeof(bwa_seq_t));
while ((res = bam_read1(bs->fp, b)) >= 0) {
uint8_t *s, *q;
int go = 0;
if ((bs->which & 1) && (b->core.flag & BAM_FREAD1)) go = 1;
if ((bs->which & 2) && (b->core.flag & BAM_FREAD2)) go = 1;
if ((bs->which & 4) && !(b->core.flag& BAM_FREAD1) && !(b->core.flag& BAM_FREAD2))go = 1;
if (go == 0) continue;
l = b->core.l_qseq;
p = &seqs[n_seqs++];
p->tid = -1; // no assigned to a thread
p->qual = 0;
p->full_len = p->clip_len = p->len = l;
n_tot += p->full_len;
s = bam1_seq(b); q = bam1_qual(b);
p->seq = (ubyte_t*)calloc(p->len + 1, 1);
p->qual = (ubyte_t*)calloc(p->len + 1, 1);
for (i = 0; i != p->full_len; ++i) {
p->seq[i] = bam_nt16_nt4_table[(int)bam1_seqi(s, i)];
p->qual[i] = q[i] + 33 < 126? q[i] + 33 : 126;
}
if (bam1_strand(b)) { // then reverse
seq_reverse(p->len, p->seq, 1);
seq_reverse(p->len, p->qual, 0);
}
if (trim_qual >= 1) n_trimmed += bwa_trim_read(trim_qual, p);
p->rseq = (ubyte_t*)calloc(p->full_len, 1);
memcpy(p->rseq, p->seq, p->len);
seq_reverse(p->len, p->seq, 0); // *IMPORTANT*: will be reversed back in bwa_refine_gapped()
seq_reverse(p->len, p->rseq, is_comp);
p->name = strdup((const char*)bam1_qname(b));
if (n_seqs == n_needed) break;
}
if (res < 0 && res != -1) err_fatal_simple("Error reading bam file");
*n = n_seqs;
if (n_seqs && trim_qual >= 1)
fprintf(stderr, "[bwa_read_seq] %.1f%% bases are trimmed.\n", 100.0f * n_trimmed/n_tot);
if (n_seqs == 0) {
free(seqs);
bam_destroy1(b);
return 0;
}
bam_destroy1(b);
return seqs;
}
void hash_reads( table* T, const char* reads_fn, interval_stack* is )
{
samfile_t* reads_f = samopen( reads_fn, "rb", NULL );
if( reads_f == NULL ) {
failf( "Can't open bam file '%s'.", reads_fn );
}
bam_index_t* reads_index = bam_index_load( reads_fn );
if( reads_index == NULL ) {
failf( "Can't open bam index '%s.bai'.", reads_fn );
}
bam_init_header_hash( reads_f->header );
table_create( T, reads_f->header->n_targets );
T->seq_names = (char**)malloc( sizeof(char*) * reads_f->header->n_targets );
size_t k;
for( k = 0; k < reads_f->header->n_targets; k++ ) {
T->seq_names[k] = strdup(reads_f->header->target_name[k]);
}
log_puts( LOG_MSG, "hashing reads ... \n" );
log_indent();
bam_iter_t read_iter;
bam1_t* read = bam_init1();
int tid;
interval_stack::iterator i;
for( i = is->begin(); i != is->end(); i++ ) {
tid = bam_get_tid( reads_f->header, i->seqname );
if( tid < 0 ) continue;
read_iter = bam_iter_query( reads_index, tid,
i->start, i->end );
while( bam_iter_read( reads_f->x.bam, read_iter, read ) >= 0 ) {
if( bam1_strand(read) == i->strand ) {
table_inc( T, read );
}
}
bam_iter_destroy(read_iter);
}
bam_destroy1(read);
log_unindent();
log_printf( LOG_MSG, "done. (%zu unique reads hashed)\n", T->m );
bam_index_destroy(reads_index);
samclose(reads_f);
}
static char *_bamqual(const bam1_t * bam, BAM_DATA bd)
{
const uint32_t len = bam->core.l_qseq;
const unsigned char *bamq = bam1_qual(bam);
char *s = Calloc(len + 1, char);
for (uint32_t i = 0; i < len; ++i)
s[i] = bamq[i] + 33;
if (bd->reverseComplement && (bam1_strand(bam) == 1))
_reverse(s, len);
s[len] = '\0';
return s;
}
/*!
@abstract Get the color quality of the color encoding the previous and current base
@param b pointer to an alignment
@param i The i-th position, 0-based
@return color quality
@discussion Returns 0 no color information is found.
*/
char bam_aux_getCQi(bam1_t *b, int i)
{
uint8_t *c = bam_aux_get(b, "CQ");
char *cq = NULL;
// return the base if the tag was not found
if(0 == c) return 0;
cq = bam_aux2Z(c);
// adjust for strandedness
if(bam1_strand(b)) i = strlen(cq) - 1 - i;
return cq[i];
}
/*!
@abstract Get the color encoding the previous and current base
@param b pointer to an alignment
@param i The i-th position, 0-based
@return color
@discussion Returns 0 no color information is found.
*/
char bam_aux_getCSi(bam1_t *b, int i)
{
uint8_t *c = bam_aux_get(b, "CS");
char *cs = NULL;
// return the base if the tag was not found
if(0 == c) return 0;
cs = bam_aux2Z(c);
// adjust for strandedness and leading adaptor
if(bam1_strand(b)) i = strlen(cs) - 1 - i;
else i++;
return cs[i];
}
static char *_bamseq(const bam1_t * bam, BAM_DATA bd)
{
static const char key[] = {
'-', 'A', 'C', 'M', 'G', 'R', 'S', 'V',
'T', 'W', 'Y', 'H', 'K', 'D', 'B', 'N'
};
const uint32_t len = bam->core.l_qseq;
const unsigned char *seq = bam1_seq(bam);
char *s = Calloc(len + 1, char);
for (uint32_t i = 0; i < len; ++i)
s[i] = key[bam1_seqi(seq, i)];
if (bd->reverseComplement && (bam1_strand(bam) == 1))
_reverseComplement(s, len);
s[len] = '\0';
return s;
}
// Returns true for a given read whether it is concordantly mapped together with its mate.
bool is_concordant(const bam1_t* read, unsigned int insert_size) {
if (read->core.flag & BAM_FUNMAP || read->core.flag & BAM_FMUNMAP) {
// At least one of the pair is unmapped.
return false;
}
if (read->core.tid != read->core.mtid) {
// Reads mapping to different chromosomes.
return false;
}
if (bam1_strand(read) == bam1_mstrand(read)) {
// Reads mapping to the same strand.
return false;
}
// Return true if insert size is as expected. This definition of concordance is
// equivalent to read-pair construction elsewhere in Pindel.
return (unsigned int) abs(read->core.isize) < read->core.l_qseq + 2 * insert_size;
}
/*!
@abstract Get the color error profile at the give position
@param b pointer to an alignment
@return the original color if the color was an error, '-' (dash) otherwise
@discussion Returns 0 no color information is found.
*/
char bam_aux_getCEi(bam1_t *b, int i)
{
int cs_i;
uint8_t *c = bam_aux_get(b, "CS");
char *cs = NULL;
char prev_b, cur_b;
char cur_color, cor_color;
// return the base if the tag was not found
if(0 == c) return 0;
cs = bam_aux2Z(c);
// adjust for strandedness and leading adaptor
if(bam1_strand(b)) { //reverse strand
cs_i = strlen(cs) - 1 - i;
// get current color
cur_color = cs[cs_i];
// get previous base. Note: must rc adaptor
prev_b = (cs_i == 1) ? "TGCAN"[(int)bam_aux_nt2int(cs[0])] : bam_nt16_rev_table[bam1_seqi(bam1_seq(b), i+1)];
// get current base
cur_b = bam_nt16_rev_table[bam1_seqi(bam1_seq(b), i)];
}
else {
cs_i=i+1;
// get current color
cur_color = cs[cs_i];
// get previous base
prev_b = (0 == i) ? cs[0] : bam_nt16_rev_table[bam1_seqi(bam1_seq(b), i-1)];
// get current base
cur_b = bam_nt16_rev_table[bam1_seqi(bam1_seq(b), i)];
}
// corrected color
cor_color = bam_aux_ntnt2cs(prev_b, cur_b);
if(cur_color == cor_color) {
return '-';
}
else {
return cur_color;
}
}
// Mostly stolen from bwa_read_bam.
void bam1_to_seq(bam1_t *raw, bwa_seq_t *p, int is_comp, int trim_qual)
{
// long n_trimmed = 0;
uint8_t *s, *q;
int i, l = raw->core.l_qseq;
p->tid = -1; // no assigned to a thread
p->qual = 0;
p->full_len = p->clip_len = p->len = l;
// n_tot += p->full_len;
s = bam1_seq(raw);
q = bam1_qual(raw);
p->seq = (ubyte_t*)calloc(p->len + 1, 1);
p->qual = (ubyte_t*)calloc(p->len + 1, 1);
for (i = 0; i != p->full_len; ++i) {
p->seq[i] = bam_nt16_nt4_table[(int)bam1_seqi(s, i)];
p->qual[i] = q[i] + 33 < 126? q[i] + 33 : 126;
}
if (bam1_strand(raw)) { // then reverse
seq_reverse(p->len, p->seq, 1);
seq_reverse(p->len, p->qual, 0);
}
if (trim_qual >= 1) /* n_trimmed += */ bwa_trim_read(trim_qual, p);
p->rseq = (ubyte_t*)calloc(p->full_len, 1);
memcpy(p->rseq, p->seq, p->len);
seq_reverse(p->len, p->seq, 0); // *IMPORTANT*: will be reversed back in bwa_refine_gapped()
seq_reverse(p->len, p->rseq, is_comp);
p->max_entries = 0 ;
// We don't set a name, it's contained in the original record
// anyway.
// p->name = strdup((const char*)bam1_qname(raw));
// No place to put the tally right now.
// if (n_seqs && trim_qual >= 1)
// fprintf(stderr, "[bwa_read_seq] %.1f%% bases are trimmed.\n", 100.0f * n_trimmed/n_tot);
}
// TODO soft-clipping
bam1_t *sw_align(graph_t *g, bam1_t *b, node_t *n, sw_heap_t *heap, char *rg_id, int32_t offset, cov_cutoffs_t *cutoffs, uint8_t correct_bases, uint8_t use_qualities, int32_t max_total_coverage, int32_t max_heap_size)
{
char *colors = NULL;
char *color_qualities = NULL;
char base, qual;
uint8_t space = SRMA_SPACE_NT;
uint8_t strand;
int32_t i, j, aln_start;
int32_t num_start_nodes_added=0;
int32_t sw_node_i=-1, sw_node_best_i=-1, sw_node_cur_i=-1, sw_node_next_i=-1;
int32_t soft_clip_start_l = 0, soft_clip_end_l = 0;
strand = bam1_strand(b);
// soft-clipping
if(1 == strand) { //reverse
// going from 3'->5'
soft_clip_start_l = sw_align_get_soft_clip(b, 1);
soft_clip_end_l = sw_align_get_soft_clip(b, 0);
}
else {
// going from 5'->3'
soft_clip_start_l = sw_align_get_soft_clip(b, 0);
soft_clip_end_l = sw_align_get_soft_clip(b, 1);
}
// FOR NOW
if(0 < soft_clip_start_l || 0 < soft_clip_end_l) {
return b;
}
// Check color space
colors = sw_align_get_cs(b);
if(NULL == colors) {
space = SRMA_SPACE_NT;
}
else {
space = SRMA_SPACE_CS;
color_qualities = sw_align_get_cq(b);
// Some aligners include a quality value for the adapter. A quality value
// IMHO should not be given for an unobserved (assumed) peice of data. Trim
// the first quality in this case
if(strlen(colors) == strlen(color_qualities)) { // ignore leading quality
color_qualities++;
}
if(0 < soft_clip_start_l || 0 < soft_clip_end_l) {
srma_error(__func__, "Soft clipping not supported for color space", Exit, OutOfRange);
}
}
// remove mate info
b->core.flag &= ~(BAM_FPROPER_PAIR | BAM_FMREVERSE | BAM_FMUNMAP);
b->core.mtid = -1;
b->core.mpos = -1;
b->core.isize = 0;
// re-type heap
heap->type = (1 == strand) ? SRMA_SW_HEAP_MAX : SRMA_SW_HEAP_MIN;
// bound with original alignment
sw_node_best_i = sw_align_bound(g, b, n, heap, strand, colors, color_qualities, space, cutoffs, use_qualities, max_total_coverage, max_heap_size);
if(0 <= sw_node_best_i) {
/*
sw_heap_reset(heap); // reset the heap, keep old nodes
fprintf(stderr, "BOUNDED score=%d coverage_sum=%hu\n",
heap->nodes[sw_node_best_i].score,
heap->nodes[sw_node_best_i].coverage_sum); // DEBUG
*/
}
else {
//fprintf(stderr, "NOT BOUNDED\n"); // DEBUG
// nodes do not need to be preserved
sw_heap_clear(heap);
}
//return b; // HERE DEBUG HERE BUG
// add start nodes
if(strand) {
if(SRMA_SPACE_CS == space) {
base = nt2int_table[(int)colors[1]];
qual = color_qualities[0];
}
else {
base = nt4bit_to_int[bam1_seqi(bam1_seq(b), b->core.l_qseq-1)];
qual = bam1_qual(b)[b->core.l_qseq-1] + 33;
}
aln_start = bam_calend(&b->core, bam1_cigar(b));
for(i=aln_start+offset;aln_start-offset<=i;i--) {
int32_t pos = graph_get_node_list_index_at_or_before(g, i);
node_list_t *list = graph_get_node_list(g, pos);
if(1 != pos && NULL != list) {
for(j=0;j<list->length;j++) {
node_t *node = list->nodes[j];
int32_t pass = pass_filters1(g, node, cutoffs, max_total_coverage);
if(0 == pass) {
sw_node_i = sw_heap_get_node_i(heap);
sw_node_init(&heap->nodes[sw_node_i], NULL, node, node->coverage, base, qual, use_qualities, space);
sw_heap_add_i(heap, sw_node_i);
}
else if(pass < 0) {
sw_heap_clear(heap); // clear heap
return b;
}
if(node->position < i) {
i = node->position;
}
num_start_nodes_added++;
}
}
}
}
else {
if(SRMA_SPACE_CS == space) {
base = nt2int_table[(int)colors[1]];
qual = color_qualities[0];
}
else {
base = nt4bit_to_int[bam1_seqi(bam1_seq(b), 0)];
qual = bam1_qual(b)[0] + 33;
}
aln_start = b->core.pos;
for(i=aln_start-offset;i<=aln_start+offset;i++) {
int32_t pos = graph_get_node_list_index_at_or_after(g, i);
node_list_t *list = graph_get_node_list(g, pos);
if(0 != pos && NULL != list) {
for(j=0;j<list->length;j++) {
node_t *node = list->nodes[j];
int32_t pass = pass_filters1(g, node, cutoffs, max_total_coverage);
if(0 == pass) {
sw_node_i = sw_heap_get_node_i(heap);
sw_node_init(&heap->nodes[sw_node_i], NULL, node, node->coverage, base, qual, use_qualities, space);
sw_heap_add_i(heap, sw_node_i);
}
else if(pass < 0) {
sw_heap_clear(heap); // clear heap
return b;
}
if(node->position < i) {
i = node->position;
}
num_start_nodes_added++;
}
}
}
}
if(0 == num_start_nodes_added) {
srma_error(__func__, "Did not add any start nodes", Exit, OutOfRange);
}
sw_node_cur_i = sw_heap_poll_i(heap);
while(0 <= sw_node_cur_i) {
if(max_heap_size < heap->queue_end - heap->queue_start + 1) {
// too many to consider
sw_heap_clear(heap); // clear heap
return b;
}
sw_node_next_i = sw_heap_peek_i(heap);
assert(0 <= sw_node_cur_i); // DEBUG
while(NODE_INSERTION != __node_type(heap->nodes[sw_node_cur_i].node)
&& 0 <= sw_node_next_i
&& 0 == sw_node_compare(&heap->nodes[sw_node_cur_i], &heap->nodes[sw_node_next_i], heap->type)) {
if(heap->nodes[sw_node_cur_i].score < heap->nodes[sw_node_next_i].score ||
(heap->nodes[sw_node_cur_i].score == heap->nodes[sw_node_next_i].score &&
heap->nodes[sw_node_cur_i].coverage_sum < heap->nodes[sw_node_next_i].coverage_sum)) {
sw_node_cur_i = sw_heap_poll_i(heap);
}
else {
// ignore the next node
sw_heap_poll_i(heap);
}
sw_node_next_i = sw_heap_peek_i(heap);
}
sw_node_next_i = -1;
if(heap->nodes[sw_node_cur_i].read_offset == b->core.l_qseq-1) { // found, keep best
if(sw_node_best_i < 0 ||
heap->nodes[sw_node_best_i].score < heap->nodes[sw_node_cur_i].score ||
(heap->nodes[sw_node_best_i].score == heap->nodes[sw_node_cur_i].score &&
heap->nodes[sw_node_best_i].coverage_sum < heap->nodes[sw_node_cur_i].coverage_sum)) {
//fprintf(stderr, "FOUND BEST\n"); // DEBUG
sw_node_best_i = sw_node_cur_i;
}
}
else if(0 <= sw_node_best_i &&
heap->nodes[sw_node_cur_i].score < heap->nodes[sw_node_best_i].score) {
// ignore, under the assumption that scores can only
// become more negative.
}
else {
edge_list_t *list = NULL;
if(1 == strand) { // reverse
list = heap->nodes[sw_node_cur_i].node->prev;
}
else {
list = heap->nodes[sw_node_cur_i].node->next;
}
{ // get the base and quality
if(SRMA_SPACE_CS == space) {
base = nt2int_table[(int)colors[1 + (heap->nodes[sw_node_cur_i].read_offset+1)]];
qual = color_qualities[heap->nodes[sw_node_cur_i].read_offset+1];
}
else {
if(strand) {
base = nt4bit_to_int[bam1_seqi(bam1_seq(b), b->core.l_qseq-1-heap->nodes[sw_node_cur_i].read_offset-1)];
qual = bam1_qual(b)[b->core.l_qseq-1-heap->nodes[sw_node_cur_i].read_offset-1] + 33;
}
else {
base = nt4bit_to_int[bam1_seqi(bam1_seq(b), (heap->nodes[sw_node_cur_i].read_offset+1))];
qual = bam1_qual(b)[(heap->nodes[sw_node_cur_i].read_offset+1)] + 33;
}
}
}
/*
node_t *node = heap->nodes[sw_node_cur_i].node;
fprintf(stderr, "NODE %d:%d offset=%d coverage=%d base=%d\n",
node->contig, node->position, node->offset, node->coverage, node->base);
fprintf(stderr, "SW_NODE read_offset=%d score=%d coverage_sum=%d start_position=%d space=%d\n",
heap->nodes[sw_node_cur_i].read_offset, heap->nodes[sw_node_cur_i].score, heap->nodes[sw_node_cur_i].coverage_sum, heap->nodes[sw_node_cur_i].start_position, space);
*/
for(i=0;i<list->length;i++) {
node_t *node_cur = list->nodes[i];
uint16_t coverage_cur = list->coverages[i];
int32_t pass = pass_filters(g, node_cur, coverage_cur, cutoffs, max_total_coverage);
if(0 == pass) {
// add to the heap
sw_node_i = sw_heap_get_node_i(heap);
// DEBUG
assert(0 <= sw_node_cur_i);
assert(0 <= heap->nodes[sw_node_cur_i].read_offset);
sw_node_init(&heap->nodes[sw_node_i], &heap->nodes[sw_node_cur_i], node_cur, coverage_cur, base, qual, use_qualities, space);
sw_heap_add_i(heap, sw_node_i);
}
else if(pass < 0) {
sw_heap_clear(heap); // clear heap
return b;
}
}
}
// get the next node
sw_node_cur_i = sw_heap_poll_i(heap);
}
/*
fprintf(stderr, "sw_node_best_i=%d\n", sw_node_best_i); // DEBUG
if(0 <= sw_node_best_i) {
fprintf(stderr, "END score=%d coverage_sum=%hu\n",
heap->nodes[sw_node_best_i].score,
heap->nodes[sw_node_best_i].coverage_sum); // DEBUG
}
*/
// update SAM/BAM
b = sw_align_update_bam(b, rg_id, heap, sw_node_best_i, space, colors, color_qualities, strand, correct_bases);
sw_heap_clear(heap); // clear heap
return b;
}
/**
* @brief Major quality check point for each read
*
* @param rm Empty read_metrics_t to be updated with the results
* @param temp_read The read to be assessed
* @param user_args User arguments to be considered during assessment
* @param bresults Block wide results of the parsing
* @param lpos Current position
* @return void
* @details Major quality check point for each read
* @todo nothing
*/
void quality_check(read_metrics_t *rm,bam1_t *temp_read,user_arguments_t *user_args,seq_block_t *bresults,int lpos){
static int pos_dupcounter=0,neg_dupcounter=0;
rm->skip=0;
rm->read_length=0;
rm->genomic_end= bam_calend(&temp_read->core,bam1_cigar(temp_read));
/* Determine read length */
if(bam1_pair(temp_read)){
++bresults->paired;
if (bam1_ppair(temp_read))++bresults->ppairs;
}
++bresults->total_reads;
if(temp_read->core.qual < user_args->TMAPQ || bam1_unmapped(temp_read)){
++bresults->lowqual;
rm->skip=1;
return;
}
if(user_args->UNIQUE && bam1_multimap(temp_read)){
rm->skip=1;
return;
}
if(!user_args->PAIRED){
rm->revcomp=bam1_strand(temp_read);
rm->read_length=bam_cigar2qlen(&temp_read->core,bam1_cigar(temp_read));
} else if (bam1_ppair(temp_read) && !bam1_notprimary(temp_read)){
rm->revcomp=bam1_revpair(temp_read);
if(!user_args->READTHROUGH){rm->read_length=bam_cigar2qlen(&temp_read->core,bam1_cigar(temp_read));//sets the read length only!!
}else if(temp_read->core.isize!=0 ){
if((bam1_firstr(temp_read)&&!bam1_revpair(temp_read))||(bam1_secondr(temp_read)&&bam1_mrevpair(temp_read))){
rm->read_length=temp_read->core.isize;
} else {
rm->skip=1;
return;
}
} else{
warning("ISIZE not set in SAM/BAM file. Re-run without using the readthrough_pairs option\n");
rm->skip=-4;
return;
}
} else{
rm->skip=1;
return;
}
if(!rm->read_length){
rm->read_length=temp_read->core.l_qseq;
if(!rm->read_length){
warning("Read length neither found in core.isize=%d, core.l_qseq=%d or cigar=%d!\n",temp_read->core.isize,temp_read->core.l_qseq,bam1_cigar(temp_read));
rm->skip=-4;
return;
}
}
/* END */
if(user_args->STRANDED!=0){
if((user_args->STRANDED==-1 && !rm->revcomp) || (user_args->STRANDED==1 && rm->revcomp)){
rm->skip=1;return;
}
}
if(user_args->COLLAPSE>0){
if(lpos==temp_read->core.pos){
if(!rm->revcomp)++pos_dupcounter;
else ++neg_dupcounter;
if(pos_dupcounter>=user_args->COLLAPSE || neg_dupcounter>=user_args->COLLAPSE){
++bresults->collapsed;
rm->skip=1;
return;
}
}else{
pos_dupcounter=0;
neg_dupcounter=0;
}
}
if(!rm->skip){
rm->revcomp ? ++bresults->neg_strand : ++bresults->pos_strand;
++bresults->filtered_reads;
bresults->mapmass+=rm->read_length;
}
}
int bam_merge_core2(int by_qname, const char *out, const char *headers, int n, char * const *fn, int flag, const char *reg, int level)
#endif
{
bamFile fpout, *fp;
heap1_t *heap;
bam_header_t *hout = 0;
bam_header_t *hheaders = NULL;
int i, j, *RG_len = 0;
uint64_t idx = 0;
char **RG = 0, mode[8];
bam_iter_t *iter = 0;
if (headers) {
tamFile fpheaders = sam_open(headers);
if (fpheaders == 0) {
const char *message = strerror(errno);
fprintf(stderr, "[bam_merge_core] cannot open '%s': %s\n", headers, message);
return -1;
}
hheaders = sam_header_read(fpheaders);
sam_close(fpheaders);
}
g_is_by_qname = by_qname;
fp = (bamFile*)calloc(n, sizeof(bamFile));
heap = (heap1_t*)calloc(n, sizeof(heap1_t));
iter = (bam_iter_t*)calloc(n, sizeof(bam_iter_t));
// prepare RG tag
if (flag & MERGE_RG) {
RG = (char**)calloc(n, sizeof(void*));
RG_len = (int*)calloc(n, sizeof(int));
for (i = 0; i != n; ++i) {
int l = strlen(fn[i]);
const char *s = fn[i];
if (l > 4 && strcmp(s + l - 4, ".bam") == 0) l -= 4;
for (j = l - 1; j >= 0; --j) if (s[j] == '/') break;
++j; l -= j;
RG[i] = calloc(l + 1, 1);
RG_len[i] = l;
strncpy(RG[i], s + j, l);
}
}
// read the first
for (i = 0; i != n; ++i) {
bam_header_t *hin;
fp[i] = bam_open(fn[i], "r");
if (fp[i] == 0) {
int j;
fprintf(stderr, "[bam_merge_core] fail to open file %s\n", fn[i]);
for (j = 0; j < i; ++j) bam_close(fp[j]);
free(fp); free(heap);
// FIXME: possible memory leak
return -1;
}
hin = bam_header_read(fp[i]);
if (i == 0) { // the first BAM
hout = hin;
} else { // validate multiple baf
int min_n_targets = hout->n_targets;
if (hin->n_targets < min_n_targets) min_n_targets = hin->n_targets;
for (j = 0; j < min_n_targets; ++j)
if (strcmp(hout->target_name[j], hin->target_name[j]) != 0) {
fprintf(stderr, "[bam_merge_core] different target sequence name: '%s' != '%s' in file '%s'\n",
hout->target_name[j], hin->target_name[j], fn[i]);
return -1;
}
// If this input file has additional target reference sequences,
// add them to the headers to be output
if (hin->n_targets > hout->n_targets) {
swap_header_targets(hout, hin);
// FIXME Possibly we should also create @SQ text headers
// for the newly added reference sequences
}
bam_header_destroy(hin);
}
}
if (hheaders) {
// If the text headers to be swapped in include any @SQ headers,
// check that they are consistent with the existing binary list
// of reference information.
if (hheaders->n_targets > 0) {
if (hout->n_targets != hheaders->n_targets) {
fprintf(stderr, "[bam_merge_core] number of @SQ headers in '%s' differs from number of target sequences\n", headers);
if (!reg) return -1;
}
for (j = 0; j < hout->n_targets; ++j)
if (strcmp(hout->target_name[j], hheaders->target_name[j]) != 0) {
fprintf(stderr, "[bam_merge_core] @SQ header '%s' in '%s' differs from target sequence\n", hheaders->target_name[j], headers);
if (!reg) return -1;
}
}
swap_header_text(hout, hheaders);
bam_header_destroy(hheaders);
}
if (reg) {
int tid, beg, end;
if (bam_parse_region(hout, reg, &tid, &beg, &end) < 0) {
fprintf(stderr, "[%s] Malformated region string or undefined reference name\n", __func__);
return -1;
}
for (i = 0; i < n; ++i) {
bam_index_t *idx;
idx = bam_index_load(fn[i]);
iter[i] = bam_iter_query(idx, tid, beg, end);
bam_index_destroy(idx);
}
}
for (i = 0; i < n; ++i) {
heap1_t *h = heap + i;
h->i = i;
h->b = (bam1_t*)calloc(1, sizeof(bam1_t));
if (bam_iter_read(fp[i], iter[i], h->b) >= 0) {
h->pos = ((uint64_t)h->b->core.tid<<32) | (uint32_t)((int32_t)h->b->core.pos+1)<<1 | bam1_strand(h->b);
h->idx = idx++;
}
else h->pos = HEAP_EMPTY;
}
if (flag & MERGE_UNCOMP) level = 0;
else if (flag & MERGE_LEVEL1) level = 1;
strcpy(mode, "w");
if (level >= 0) sprintf(mode + 1, "%d", level < 9? level : 9);
if ((fpout = strcmp(out, "-")? bam_open(out, "w") : bam_dopen(fileno(stdout), "w")) == 0) {
fprintf(stderr, "[%s] fail to create the output file.\n", __func__);
return -1;
}
bam_header_write(fpout, hout);
bam_header_destroy(hout);
#ifndef _PBGZF_USE
if (!(flag & MERGE_UNCOMP)) bgzf_mt(fpout, n_threads, 256);
#endif
ks_heapmake(heap, n, heap);
while (heap->pos != HEAP_EMPTY) {
bam1_t *b = heap->b;
if (flag & MERGE_RG) {
uint8_t *rg = bam_aux_get(b, "RG");
if (rg) bam_aux_del(b, rg);
bam_aux_append(b, "RG", 'Z', RG_len[heap->i] + 1, (uint8_t*)RG[heap->i]);
}
bam_write1_core(fpout, &b->core, b->data_len, b->data);
if ((j = bam_iter_read(fp[heap->i], iter[heap->i], b)) >= 0) {
heap->pos = ((uint64_t)b->core.tid<<32) | (uint32_t)((int)b->core.pos+1)<<1 | bam1_strand(b);
heap->idx = idx++;
} else if (j == -1) {
heap->pos = HEAP_EMPTY;
free(heap->b->data); free(heap->b);
heap->b = 0;
} else fprintf(stderr, "[bam_merge_core] '%s' is truncated. Continue anyway.\n", fn[heap->i]);
ks_heapadjust(heap, 0, n, heap);
}
if (flag & MERGE_RG) {
for (i = 0; i != n; ++i) free(RG[i]);
free(RG); free(RG_len);
}
for (i = 0; i != n; ++i) {
bam_iter_destroy(iter[i]);
bam_close(fp[i]);
}
bam_close(fpout);
free(fp); free(heap); free(iter);
return 0;
}
int bam1_strand_(bam1_t *b) { return bam1_strand(b); }
/* Counts probability of non-match count along the read after
* subtracting error prob at that position (using the original
* orientation). used_pos is an array of ints indicating whether
* position was used or not (trimmed, clipped etc). alnerrprof and
* used_pos must be of at least length b->core.l_qseq. Note: will add
* to alnerrprof and used_pos, i.e. arrays should be initialized to 0 if
* you don't want aggregate values.
*
* WARNING code duplication with count_cigar_ops but merging the two
* functions is messy.
*/
void
calc_read_alnerrprof(double *alnerrprof, unsigned long int *used_pos,
const bam1_t *b, const char *ref)
{
/* modelled after bam.c:bam_calend(), bam_format1_core() and
* pysam's aligned_pairs (./pysam/csamtools.pyx)
*/
uint32_t *cigar = bam1_cigar(b);
uint32_t k, i;
const bam1_core_t *c = &b->core;
#if 0
int32_t qlen = (int32_t) bam_cigar2qlen(c, cigar); /* read length */
#else
int qlen = b->core.l_qseq; /* read length */
#endif
uint32_t pos = c->pos; /* pos on genome */
uint32_t qpos = 0; /* pos on read/query */
uint32_t qpos_org = bam1_strand(b) ? qlen-qpos-1 : qpos;/* original qpos before mapping as possible reverse */
/* loop over cigar to get aligned bases
*
* read: bam_format1_core(NULL, b, BAM_OFDEC);
*/
for (k=0; k < c->n_cigar; ++k) { /* n_cigar: number of cigar operations */
int op = cigar[k] & BAM_CIGAR_MASK; /* the cigar operation */
uint32_t l = cigar[k] >> BAM_CIGAR_SHIFT;
/* following conditionals could be collapsed to much shorter
* code, but we keep them as they were in pysam's
* aligned_pairs to make later handling of indels easier
*/
if (op == BAM_CMATCH || op == BAM_CDIFF) {
for (i=pos; i<pos+l; i++) {
assert(qpos < qlen);
/* case agnostic */
char ref_nt = ref[i];
char read_nt = bam_nt16_rev_table[bam1_seqi(bam1_seq(b), qpos)];
int bq = bam1_qual(b)[qpos];
#if 0
printf("[M]MATCH qpos,i,ref,read = %d,%d,%c,%c\n", qpos, i, ref_nt, read_nt);
#endif
if (ref_nt != 'N') {
if (ref_nt != read_nt || op == BAM_CDIFF) {
alnerrprof[qpos_org] += (1.0 - PHREDQUAL_TO_PROB(bq));
} /* otherwise leave at 0.0 but count anyway */
used_pos[qpos_org] += 1;
}
qpos += 1;
qpos_org = bam1_strand(b) ? qlen-qpos-1 : qpos;
}
pos += l;
} else if (op == BAM_CINS) {
for (i=pos; i<pos+l; i++) {
assert(qpos < qlen);
alnerrprof[qpos] += (1.0 - PHREDQUAL_TO_PROB(INDEL_QUAL_DEFAULT));
used_pos[qpos] += 1;
#if 0
printf("INS qpos,i = %d,None\n", qpos);
#endif
qpos += 1;
qpos_org = bam1_strand(b) ? qlen-qpos-1 : qpos;
}
} else if (op == BAM_CDEL || op == BAM_CREF_SKIP) {
for (i=pos; i<pos+l; i++) {
#if 0
printf("DEL qpos,i = None,%d\n", i);
#endif
if (op == BAM_CDEL) {
alnerrprof[qpos] += (1.0 - PHREDQUAL_TO_PROB(INDEL_QUAL_DEFAULT));
used_pos[qpos] += 1;
}
}
pos += l;
/* deletion: don't increase qpos */
} else if (op == BAM_CSOFT_CLIP) {
#if 0
printf("SOFT CLIP qpos = %d\n", qpos);
#endif
qpos += l;
qpos_org = bam1_strand(b) ? qlen-qpos-1 : qpos;
} else if (op != BAM_CHARD_CLIP) {
LOG_WARN("Unknown op %d in cigar %s\n", op, cigar_str_from_bam(b));
}
} /* for k */
assert(pos == bam_calend(&b->core, bam1_cigar(b))); /* FIXME correct assert? what if hard clipped? */
if (qpos != qlen) {
LOG_FIXME("got qpos=%d and qlen=%d for cigar %s l_qseq %d\n", qpos, qlen, cigar_str_from_bam(b), b->core.l_qseq);
}
assert(qpos == qlen); /* FIXME correct assert? What if hard clipped? */
#if 0
fprintf(stderr, "%s:", __FUNCTION__);
for (i=0; i< b->core.l_qseq; i++) {
fprintf(stderr, " %g/%d", alnerrprof[i], used_pos[i]);
}
fprintf(stderr, "\n");
#endif
}
node_t *graph_add_sam(graph_t *g, bam1_t *b, ref_t *ref, int32_t use_threads)
{
bam_aln_t *aln = NULL;
int32_t aln_start, aln_index, ref_index, aln_ref_index;
int32_t i;
node_t *prev_node=NULL, *cur_node=NULL, *ret_node=NULL;
uint8_t type, strand;
aln_start = b->core.pos+1;
aln_ref_index = b->core.tid;
aln = bam_aln_init(b, ref);
strand = bam1_strand(b);
// --- SYNC ON ---
if(1 == use_threads) pthread_mutex_lock(&graph_mutex); // synchronize start
if(aln_start < g->position_start) {
int32_t diff = g->position_start - aln_start;
graph_nodes_realloc(g, g->position_end - aln_start + 1); // alloc more memory if needed
// shift up
for(i=g->position_end-g->position_start;0<=i;i--) {
// swap
node_list_t *list = g->nodes[i+diff];
g->nodes[i+diff] = g->nodes[i];
g->nodes[i] = list;
}
g->position_start = aln_start;
}
if(1 == g->is_empty) {
for(i=0;i<g->position_end - g->position_start + 1;i++) {
node_list_clear(g->nodes[i]);
assert(0 == g->nodes[i]->length); // DEBUG
}
g->position_start = aln_start;
if(ALN_GAP == aln->ref[0]) {
g->position_start--;
}
g->position_end = g->position_start;
g->contig = aln_ref_index + 1;
g->is_empty = 0;
}
if(1 == use_threads) pthread_mutex_unlock(&graph_mutex); // synchronize end
// --- SYNC OFF ---
for(aln_index=0,ref_index=-1;aln_index<aln->length;aln_index++,prev_node=cur_node) {
// Skip over a deletion
while(ALN_GAP == aln->read[aln_index]) {
aln_index++;
ref_index++;
}
if(aln->read[aln_index] == aln->ref[aln_index]) { // match
type = NODE_MATCH;
}
else if(aln->ref[aln_index] == ALN_GAP) { // insertion
type = NODE_INSERTION;
}
else { // mismatch
type = NODE_MISMATCH;
}
if(NULL == prev_node || NODE_INSERTION != __node_type(prev_node)) { // previous was an insertion, already on the position
ref_index++;
}
cur_node = graph_add_node(g,
node_init(aln->read[aln_index], type, g->contig, aln_start + ref_index, prev_node),
prev_node,
use_threads);
if(NULL == prev_node && 0 == strand) { // first node and forward strand
ret_node = cur_node;
}
}
if(1 == strand) {
ret_node = cur_node;
}
bam_aln_free(aln);
return ret_node;
}
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, tid0 = -1, beg0 = 0, end0 = 1u<<29, ref_len, ref_tid = -1, max_depth, max_indel_depth;
const bam_pileup1_t **plp;
bam_mplp_t iter;
bam_header_t *h = 0;
char *ref;
void *rghash = 0;
bcf_callaux_t *bca = 0;
bcf_callret1_t *bcr = 0;
bcf_call_t bc;
bcf_t *bp = 0;
bcf_hdr_t *bh = 0;
bam_sample_t *sm = 0;
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(void*));
plp = calloc(n, sizeof(void*));
n_plp = calloc(n, sizeof(int*));
sm = bam_smpl_init();
// read the header and initialize data
for (i = 0; i < n; ++i) {
bam_header_t *h_tmp;
data[i] = calloc(1, sizeof(mplp_aux_t));
data[i]->fp = strcmp(fn[i], "-") == 0? bam_dopen(fileno(stdin), "r") : bam_open(fn[i], "r");
data[i]->conf = conf;
h_tmp = bam_header_read(data[i]->fp);
data[i]->h = i? h : h_tmp; // for i==0, "h" has not been set yet
bam_smpl_add(sm, fn[i], (conf->flag&MPLP_IGNORE_RG)? 0 : h_tmp->text);
rghash = bcf_call_add_rg(rghash, h_tmp->text, conf->pl_list);
if (conf->reg) {
int beg, end;
bam_index_t *idx;
idx = bam_index_load(fn[i]);
if (idx == 0) {
fprintf(stderr, "[%s] fail to load index for %d-th input.\n", __func__, i+1);
exit(1);
}
if (bam_parse_region(h_tmp, conf->reg, &tid, &beg, &end) < 0) {
fprintf(stderr, "[%s] malformatted region or wrong seqname for %d-th input.\n", __func__, i+1);
exit(1);
}
if (i == 0) tid0 = tid, beg0 = beg, end0 = end;
data[i]->iter = bam_iter_query(idx, tid, beg, end);
bam_index_destroy(idx);
}
if (i == 0) h = h_tmp;
else {
// FIXME: to check consistency
bam_header_destroy(h_tmp);
}
}
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(void*));
fprintf(stderr, "[%s] %d samples in %d input files\n", __func__, sm->n, n);
// write the VCF header
if (conf->flag & MPLP_GLF) {
kstring_t s;
bh = calloc(1, sizeof(bcf_hdr_t));
s.l = s.m = 0; s.s = 0;
bp = bcf_open("-", (conf->flag&MPLP_NO_COMP)? "wu" : "w");
for (i = 0; i < h->n_targets; ++i) {
kputs(h->target_name[i], &s);
kputc('\0', &s);
}
bh->l_nm = s.l;
bh->name = malloc(s.l);
memcpy(bh->name, s.s, s.l);
s.l = 0;
for (i = 0; i < sm->n; ++i) {
kputs(sm->smpl[i], &s); kputc('\0', &s);
}
bh->l_smpl = s.l;
bh->sname = malloc(s.l);
memcpy(bh->sname, s.s, s.l);
bh->txt = malloc(strlen(BAM_VERSION) + 64);
bh->l_txt = 1 + sprintf(bh->txt, "##samtoolsVersion=%s\n", BAM_VERSION);
free(s.s);
bcf_hdr_sync(bh);
bcf_hdr_write(bp, bh);
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;
}
if (tid0 >= 0 && conf->fai) { // region is set
ref = faidx_fetch_seq(conf->fai, h->target_name[tid0], 0, 0x7fffffff, &ref_len);
ref_tid = tid0;
for (i = 0; i < n; ++i) data[i]->ref = ref, data[i]->ref_id = tid0;
} else ref_tid = -1, ref = 0;
iter = bam_mplp_init(n, mplp_func, (void**)data);
max_depth = conf->max_depth;
if (max_depth * sm->n > 1<<20)
fprintf(stderr, "(%s) Max depth is above 1M. Potential memory hog!\n", __func__);
if (max_depth * sm->n < 8000) {
max_depth = 8000 / sm->n;
fprintf(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);
int storeSize = 100;
int delStore[2][100] = {{0},{0}};
typedef char * mstring;
while (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;
if (tid != ref_tid) {
free(ref); ref = 0;
if (conf->fai) ref = faidx_fetch_seq(conf->fai, h->target_name[tid], 0, 0x7fffffff, &ref_len);
for (i = 0; i < n; ++i) data[i]->ref = ref, data[i]->ref_id = tid;
ref_tid = tid;
}
if (conf->flag & MPLP_GLF) {
int total_depth, _ref0, ref16;
bcf1_t *b = calloc(1, sizeof(bcf1_t));
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 = bam_nt16_table[_ref0];
for (i = 0; i < gplp.n; ++i)
bcf_call_glfgen(gplp.n_plp[i], gplp.plp[i], ref16, bca, bcr + i);
bcf_call_combine(gplp.n, bcr, ref16, &bc);
bcf_call2bcf(tid, pos, &bc, b, (conf->flag&(MPLP_FMT_DP|MPLP_FMT_SP))? bcr : 0,
(conf->flag&MPLP_FMT_SP), 0, 0);
bcf_write(bp, bh, b);
bcf_destroy(b);
// call indels
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) {
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, -1, &bc) >= 0) {
b = calloc(1, sizeof(bcf1_t));
bcf_call2bcf(tid, pos, &bc, b, (conf->flag&(MPLP_FMT_DP|MPLP_FMT_SP))? bcr : 0,
(conf->flag&MPLP_FMT_SP), bca, ref);
bcf_write(bp, bh, b);
bcf_destroy(b);
}
}
} else {
printf("%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;
printf("\t%d\t", n_plp[i]);
if (n_plp[i] == 0) {
printf("*\t*"); // FIXME: printf() is very slow...
if (conf->flag & MPLP_PRINT_POS) printf("\t*");
} else {
//MDW start
//for each position in the pileup column
int charLen = 16;
int countChars[ charLen ][2];
int countiChars[ charLen ][2];
int countGap[2]={0,0};
//double qvTotal=0;
int numStruck=0;
int numGood=0;
int tti;
int ttj;
mstring insAllele[100];
int insAlleleCnt[100];
int sf=0;
int flag=0;
//typedef char * string;
char insStr0[10000];
int iCnt0=0;
char insStr1[10000];
int iCnt1=0;
char delStr0[10000];
int dCnt0=0;
char delStr1[10000];
int dCnt1=0;
float qposP[10000];
int qposCnt=0;
//initialize with zeros
for(tti=0;tti<charLen;tti++){
countChars[tti][0]=0;
countChars[tti][1]=0;
}
// define repeat length here; look back up to 10 prior positions
// start one position away.
int replC=0; //
for(tti=1;tti<=15;tti++){
// check for greater than zero
if(toupper(ref[pos-1])==toupper(ref[pos-tti])){
replC++;
}else{ // breaks the chain at first non identical to current position not strict homopolymer
break;
}
}
int reprC=0; //
for(tti=1;tti<=15;tti++){
// check for greater than zero
if(toupper(ref[pos+1])==toupper(ref[pos+tti])){
reprC++;
}else{ // breaks the chain at first non identical to current position not strict homopolymer
break;
}
}
int repT = replC;
if(replC < reprC){
repT=reprC;
}
for (j = 0; j < n_plp[i]; ++j){
const bam_pileup1_t *p = plp[i] + j;
/*
SAME LOGIC AS pileup_seq()
*/
if(p->is_refskip){ // never count intron gaps in numStruck
continue;
}
if(p->is_del){ // skip deletion gap, after first position which is the first aligned char
continue;
}
if( p->b->core.qual < conf->min_mqToCount || // mapping quality
conf->maxrepC < (repT) || // max homopolymer run, this will not
(!p->is_del && bam1_qual(p->b)[p->qpos] < conf->min_baseQ) || // base quality for matches
p->alignedQPosBeg <= (conf->trimEnd ) || p->alignedQPosEnd <= (conf->trimEnd ) || // trimEnd is 1-based
p->zf == 1 || // fusion tag
p->ih > conf->maxIH || // max hit index
(p->nmd > conf->maxNM) || // max mismatch
(conf->flagFilter == 1 && !(p->b->core.flag&BAM_FPROPER_PAIR)) || // optionally keep only proper pairs
(conf->flagFilter == 2 && p->b->core.flag&BAM_FSECONDARY) || // optionally strike secondary
(conf->flagFilter == 3 && p->b->core.flag&BAM_FDUP) || // optionally strike dup
(conf->flagFilter == 4 && (p->b->core.flag&BAM_FDUP || p->b->core.flag&BAM_FSECONDARY)) || // optionally strike secondary or dup
(conf->flagFilter == 5 && (p->b->core.flag&BAM_FDUP || p->b->core.flag&BAM_FSECONDARY || p->b->core.flag&BAM_FQCFAIL || !(p->b->core.flag&BAM_FPROPER_PAIR) )) // optionally strike secondary, dup and QCfail
){
numStruck++;
continue;
}
//printf("repT=%d: %d %c %c %c %c \n",repT,p->indel,ref[pos],ref[pos-1],ref[pos-2],ref[pos-3]);
if(!p->is_del && p->indel==0){
countChars[ bam1_seqi(bam1_seq(p->b), p->qpos) ][ bam1_strand(p->b) ] ++;
numGood++;
}else if(p->is_refskip){
countGap[ bam1_strand(p->b) ]++;
}
if(p->indel<0){
numGood++;
if(bam1_strand(p->b) ==0){
for(tti=1;tti<= -p->indel; tti++) {
// current spot, starting at 0 in store, because indel<0 refers to next position
delStr0[dCnt0] = ref[pos+tti];
dCnt0++;
}
delStr0[dCnt0] = ',';
dCnt0++;
}else{
for(tti=1;tti<= -p->indel; tti++) {
// current spot, starting at 0 in store, because indel<0 refers to next position
delStr1[dCnt1] = ref[pos+tti];
dCnt1++;
}
delStr1[dCnt1] = ',';
dCnt1++;
}
}else if(p->indel>0){
numGood++;
if(bam1_strand(p->b) ==0){
for(tti=1;tti<= p->indel; tti++) {
// current spot, starting at 0 in store, because indel<0 refers to next position
insStr0[iCnt0] = bam_nt16_rev_table[bam1_seqi(bam1_seq(p->b), p->qpos + tti)];
iCnt0++;
}
insStr0[iCnt0] = ',';
iCnt0++;
}else{
for(tti=1;tti<= p->indel; tti++) {
// current spot, starting at 0 in store, because indel<0 refers to next position
insStr1[iCnt1] = bam_nt16_rev_table[bam1_seqi(bam1_seq(p->b), p->qpos + tti)];
iCnt1++;
}
insStr1[iCnt1] = ',';
iCnt1++;
}
}
//calculate position of variant within aligned read - no soft clips
if( toupper(ref[pos]) != toupper(bam_nt16_rev_table[bam1_seqi(bam1_seq(p->b), p->qpos)]) || p->indel>0 || p->indel<0 ){
//distance to end; calculate distance to end of aligned read. removes soft clips.
int distToEnd = (p->alignedQPosBeg < p->alignedQPosEnd) ? p->alignedQPosBeg : p->alignedQPosEnd;
qposP[qposCnt] = distToEnd;
qposCnt++;
// printf("id=%s, pos=%d",bam1_qname(p->b),distToEnd);
}
}
//
//print A,C,G,T, by +/-
printf("\t%d\t%d\t%d\t%d\t%d\t%d\t%d\t%d\t%d\t%d", countChars[1][0],countChars[1][1],
countChars[2][0],countChars[2][1],
countChars[4][0],countChars[4][1],
countChars[8][0],countChars[8][1],
countChars[7][0],countChars[7][1]);
putchar('\t');
for(tti=0;tti<dCnt0;tti++){
putchar(delStr0[tti]);
}
putchar('\t');
for(tti=0;tti<dCnt1;tti++){
putchar(delStr1[tti]);
}
putchar('\t');
for(tti=0;tti<iCnt0;tti++){
putchar(insStr0[tti]);
}
putchar('\t');
for(tti=0;tti<iCnt1;tti++){
putchar(insStr1[tti]);
}
printf("\t%d\t%d",numGood,numStruck);
// get non-ref qpos variation
float medqpos = -1;
float medAbsDev = -1;
if(qposCnt>0){
medqpos = median(qposCnt,qposP);
float absDev[qposCnt];
for(tti=0;tti<qposCnt;tti++){
absDev[tti] = abs(medqpos - qposP[tti]);
}
medAbsDev = median(qposCnt-1,absDev);
}
printf("\t%f",medAbsDev);
///END MDW
}
}
putchar('\n');
}
}
bcf_close(bp);
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);
bcf_hdr_destroy(bh); bcf_call_destroy(bca); free(bc.PL); free(bcr);
bam_mplp_destroy(iter);
bam_header_destroy(h);
for (i = 0; i < n; ++i) {
bam_close(data[i]->fp);
if (data[i]->iter) bam_iter_destroy(data[i]->iter);
free(data[i]);
}
free(data); free(plp); free(ref); free(n_plp);
return 0;
}
static bwa_seq_t *bwa_read_bam(bwa_seqio_t *bs, int n_needed, int *n, int is_comp, int trim_qual)
{
bwa_seq_t *seqs, *p;
int n_seqs, l, i;
long n_trimmed = 0, n_tot = 0;
bam1_t *b;
int res;
b = bam_init1();
n_seqs = 0;
seqs = (bwa_seq_t*)calloc(n_needed, sizeof(bwa_seq_t));
#ifdef USE_HTSLIB
while ((res = sam_read1(bs->fp, bs->h, b)) >= 0) {
#else
while ((res = bam_read1(bs->fp, b)) >= 0) {
#endif
uint8_t *s, *q;
int go = 0;
if ((bs->which & 1) && (b->core.flag & BAM_FREAD1)) go = 1;
if ((bs->which & 2) && (b->core.flag & BAM_FREAD2)) go = 1;
if ((bs->which & 4) && !(b->core.flag& BAM_FREAD1) && !(b->core.flag& BAM_FREAD2))go = 1;
if (go == 0) continue;
l = b->core.l_qseq;
p = &seqs[n_seqs++];
p->tid = -1; // no assigned to a thread
p->qual = 0;
p->full_len = p->clip_len = p->len = l;
n_tot += p->full_len;
#ifdef USE_HTSLIB
s = bam_get_seq(b); q = bam_get_qual(b);
#else
s = bam1_seq(b); q = bam1_qual(b);
#endif
p->seq = (ubyte_t*)calloc(p->len + 1, 1);
p->qual = (ubyte_t*)calloc(p->len + 1, 1);
for (i = 0; i != p->full_len; ++i) {
#ifdef USE_HTSLIB
p->seq[i] = bam_nt16_nt4_table[(int)bam_seqi(s, i)];
#else
p->seq[i] = bam_nt16_nt4_table[(int)bam1_seqi(s, i)];
#endif
p->qual[i] = q[i] + 33 < 126? q[i] + 33 : 126;
}
#ifdef USE_HTSLIB
if (bam_is_rev(b)) { // then reverse
#else
if (bam1_strand(b)) { // then reverse
#endif
seq_reverse(p->len, p->seq, 1);
seq_reverse(p->len, p->qual, 0);
}
if (trim_qual >= 1) n_trimmed += bwa_trim_read(trim_qual, p);
p->rseq = (ubyte_t*)calloc(p->full_len, 1);
memcpy(p->rseq, p->seq, p->len);
seq_reverse(p->len, p->seq, 0); // *IMPORTANT*: will be reversed back in bwa_refine_gapped()
seq_reverse(p->len, p->rseq, is_comp);
#ifdef USE_HTSLIB
p->name = strdup((const char*)bam_get_qname(b));
#else
p->name = strdup((const char*)bam1_qname(b));
#endif
if (n_seqs == n_needed) break;
}
if (res < 0 && res != -1) err_fatal_simple("Error reading bam file");
*n = n_seqs;
if (n_seqs && trim_qual >= 1)
fprintf(stderr, "[bwa_read_seq] %.1f%% bases are trimmed.\n", 100.0f * n_trimmed/n_tot);
if (n_seqs == 0) {
free(seqs);
bam_destroy1(b);
return 0;
}
bam_destroy1(b);
return seqs;
}
#define BARCODE_LOW_QUAL 13
bwa_seq_t *bwa_read_seq(bwa_seqio_t *bs, int n_needed, int *n, int mode, int trim_qual)
{
bwa_seq_t *seqs, *p;
kseq_t *seq = bs->ks;
int n_seqs, l, i, is_comp = mode&BWA_MODE_COMPREAD, is_64 = mode&BWA_MODE_IL13, l_bc = mode>>24;
long n_trimmed = 0, n_tot = 0;
if (l_bc > BWA_MAX_BCLEN) {
fprintf(stderr, "[%s] the maximum barcode length is %d.\n", __func__, BWA_MAX_BCLEN);
return 0;
}
if (bs->is_bam) return bwa_read_bam(bs, n_needed, n, is_comp, trim_qual); // l_bc has no effect for BAM input
n_seqs = 0;
seqs = (bwa_seq_t*)calloc(n_needed, sizeof(bwa_seq_t));
while ((l = kseq_read(seq)) >= 0) {
if ((mode & BWA_MODE_CFY) && (seq->comment.l != 0)) {
// skip reads that are marked to be filtered by Casava
char *s = index(seq->comment.s, ':');
if (s && *(++s) == 'Y') {
continue;
}
}
if (is_64 && seq->qual.l)
for (i = 0; i < seq->qual.l; ++i) seq->qual.s[i] -= 31;
if (seq->seq.l <= l_bc) continue; // sequence length equals or smaller than the barcode length
p = &seqs[n_seqs++];
if (l_bc) { // then trim barcode
for (i = 0; i < l_bc; ++i)
p->bc[i] = (seq->qual.l && seq->qual.s[i]-33 < BARCODE_LOW_QUAL)? tolower(seq->seq.s[i]) : toupper(seq->seq.s[i]);
p->bc[i] = 0;
for (; i < seq->seq.l; ++i)
seq->seq.s[i - l_bc] = seq->seq.s[i];
seq->seq.l -= l_bc; seq->seq.s[seq->seq.l] = 0;
if (seq->qual.l) {
for (i = l_bc; i < seq->qual.l; ++i)
seq->qual.s[i - l_bc] = seq->qual.s[i];
seq->qual.l -= l_bc; seq->qual.s[seq->qual.l] = 0;
}
l = seq->seq.l;
} else p->bc[0] = 0;
p->tid = -1; // no assigned to a thread
p->qual = 0;
p->full_len = p->clip_len = p->len = l;
n_tot += p->full_len;
p->seq = (ubyte_t*)calloc(p->full_len, 1);
for (i = 0; i != p->full_len; ++i)
p->seq[i] = nst_nt4_table[(int)seq->seq.s[i]];
if (seq->qual.l) { // copy quality
p->qual = (ubyte_t*)strdup((char*)seq->qual.s);
if (trim_qual >= 1) n_trimmed += bwa_trim_read(trim_qual, p);
}
p->rseq = (ubyte_t*)calloc(p->full_len, 1);
memcpy(p->rseq, p->seq, p->len);
seq_reverse(p->len, p->seq, 0); // *IMPORTANT*: will be reversed back in bwa_refine_gapped()
seq_reverse(p->len, p->rseq, is_comp);
p->name = strdup((const char*)seq->name.s);
{ // trim /[12]$
int t = strlen(p->name);
if (t > 2 && p->name[t-2] == '/' && (p->name[t-1] == '1' || p->name[t-1] == '2')) p->name[t-2] = '\0';
}
if (n_seqs == n_needed) break;
}
*n = n_seqs;
if (n_seqs && trim_qual >= 1)
fprintf(stderr, "[bwa_read_seq] %.1f%% bases are trimmed.\n", 100.0f * n_trimmed/n_tot);
if (n_seqs == 0) {
free(seqs);
return 0;
}
return seqs;
}
void bwa_free_read_seq(int n_seqs, bwa_seq_t *seqs)
{
int i, j;
for (i = 0; i != n_seqs; ++i) {
bwa_seq_t *p = seqs + i;
for (j = 0; j < p->n_multi; ++j)
if (p->multi[j].cigar) free(p->multi[j].cigar);
free(p->name);
free(p->seq); free(p->rseq); free(p->qual); free(p->aln); free(p->md); free(p->multi);
free(p->cigar);
}
free(seqs);
}