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bam_plcmd.c
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/* bam_plcmd.c -- mpileup subcommand.
Copyright (C) 2008-2015, 2019-2021 Genome Research Ltd.
Portions copyright (C) 2009-2012 Broad Institute.
Author: Heng Li <[email protected]>
Permission is hereby granted, free of charge, to any person obtaining a copy
of this software and associated documentation files (the "Software"), to deal
in the Software without restriction, including without limitation the rights
to use, copy, modify, merge, publish, distribute, sublicense, and/or sell
copies of the Software, and to permit persons to whom the Software is
furnished to do so, subject to the following conditions:
The above copyright notice and this permission notice shall be included in
all copies or substantial portions of the Software.
THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR
IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY,
FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL
THE AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER
LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING
FROM, OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER
DEALINGS IN THE SOFTWARE. */
#include <config.h>
#include <math.h>
#include <stdio.h>
#include <stdlib.h>
#include <unistd.h>
#include <ctype.h>
#include <string.h>
#include <strings.h>
#include <limits.h>
#include <errno.h>
#include <sys/stat.h>
#include <getopt.h>
#include <inttypes.h>
#include <htslib/sam.h>
#include <htslib/faidx.h>
#include <htslib/kstring.h>
#include <htslib/klist.h>
#include <htslib/khash_str2int.h>
#include <htslib/cram.h>
#include "samtools.h"
#include "bedidx.h"
#include "sam_opts.h"
#include "bam_plbuf.h"
#define dummy_free(p)
KLIST_INIT(auxlist, char *, dummy_free)
static inline int printw(int c, FILE *fp)
{
char buf[16];
int l, x;
if (c == 0) return fputc('0', fp);
for (l = 0, x = c < 0? -c : c; x > 0; x /= 10) buf[l++] = x%10 + '0';
if (c < 0) buf[l++] = '-';
buf[l] = 0;
for (x = 0; x < l/2; ++x) {
int y = buf[x]; buf[x] = buf[l-1-x]; buf[l-1-x] = y;
}
fputs(buf, fp);
return 0;
}
int pileup_seq(FILE *fp, const bam_pileup1_t *p, hts_pos_t pos,
hts_pos_t ref_len, const char *ref, kstring_t *ks,
int rev_del, int no_ins, int no_ins_mods,
int no_del, int no_ends)
{
no_ins_mods |= no_ins;
int j;
hts_base_mod_state *m = p->cd.p;
if (!no_ends && p->is_head) {
putc('^', fp);
putc(p->b->core.qual > 93? 126 : p->b->core.qual + 33, fp);
}
if (!p->is_del) {
int c = p->qpos < p->b->core.l_qseq
? seq_nt16_str[bam_seqi(bam_get_seq(p->b), p->qpos)]
: 'N';
if (ref) {
int rb = pos < ref_len? ref[pos] : 'N';
if (c == '=' || seq_nt16_table[c] == seq_nt16_table[rb]) c = bam_is_rev(p->b)? ',' : '.';
else c = bam_is_rev(p->b)? tolower(c) : toupper(c);
} else {
if (c == '=') c = bam_is_rev(p->b)? ',' : '.';
else c = bam_is_rev(p->b)? tolower(c) : toupper(c);
}
putc(c, fp);
if (m) {
int nm;
hts_base_mod mod[256];
if ((nm = bam_mods_at_qpos(p->b, p->qpos, m, mod, 256)) > 0) {
putc('[', fp);
int j;
for (j = 0; j < nm && j < 256; j++) {
char qual[20];
if (mod[j].qual >= 0)
sprintf(qual, "%d", mod[j].qual);
else
*qual = 0;
if (mod[j].modified_base < 0)
// ChEBI
fprintf(fp, "%c(%d)%s", "+-"[mod[j].strand],
-mod[j].modified_base, qual);
else
fprintf(fp, "%c%c%s", "+-"[mod[j].strand],
mod[j].modified_base, qual);
}
putc(']', fp);
}
}
} else putc(p->is_refskip? (bam_is_rev(p->b)? '<' : '>') : ((bam_is_rev(p->b) && rev_del) ? '#' : '*'), fp);
int del_len = -p->indel;
if (p->indel > 0) {
int len = bam_plp_insertion_mod(p, m && !no_ins_mods ? m : NULL,
ks, &del_len);
if (len < 0) {
print_error("mpileup", "bam_plp_insertion() failed");
return -1;
}
if (no_ins < 2) {
putc('+', fp);
printw(len, fp);
}
if (!no_ins) {
if (bam_is_rev(p->b)) {
char pad = rev_del ? '#' : '*';
int in_mod = 0;
for (j = 0; j < ks->l; j++) {
if (ks->s[j] == '[') in_mod = 1;
else if (ks->s[j] == ']') in_mod = 0;
putc(ks->s[j] != '*'
? (in_mod ? ks->s[j] : tolower(ks->s[j]))
: pad, fp);
}
} else {
int in_mod = 0;
for (j = 0; j < ks->l; j++) {
if (ks->s[j] == '[') in_mod = 1;
if (ks->s[j] == ']') in_mod = 0;
putc(in_mod ? ks->s[j] : toupper(ks->s[j]), fp);
}
}
}
}
if (del_len > 0) {
if (no_del < 2)
printw(-del_len, fp);
if (!no_del) {
for (j = 1; j <= del_len; ++j) {
int c = (ref && (int)pos+j < ref_len)? ref[pos+j] : 'N';
putc(bam_is_rev(p->b)? tolower(c) : toupper(c), fp);
}
}
}
if (!no_ends && p->is_tail) putc('$', fp);
return 0;
}
#include "sample.h"
#define MPLP_NO_COMP (1<<2)
#define MPLP_NO_ORPHAN (1<<3)
#define MPLP_REALN (1<<4)
#define MPLP_NO_INDEL (1<<5)
#define MPLP_REDO_BAQ (1<<6)
#define MPLP_ILLUMINA13 (1<<7)
#define MPLP_IGNORE_RG (1<<8)
#define MPLP_SMART_OVERLAPS (1<<10)
#define MPLP_PRINT_MAPQ_CHAR (1<<11)
#define MPLP_PRINT_QPOS (1<<12)
#define MPLP_PRINT_QNAME (1<<13)
#define MPLP_PRINT_FLAG (1<<14)
#define MPLP_PRINT_RNAME (1<<15)
#define MPLP_PRINT_POS (1<<16)
#define MPLP_PRINT_MAPQ (1<<17)
#define MPLP_PRINT_CIGAR (1<<18)
#define MPLP_PRINT_RNEXT (1<<19)
#define MPLP_PRINT_PNEXT (1<<20)
#define MPLP_PRINT_TLEN (1<<21)
#define MPLP_PRINT_SEQ (1<<22)
#define MPLP_PRINT_QUAL (1<<23)
#define MPLP_PRINT_MODS (1<<24)
#define MPLP_PRINT_QPOS5 (1<<25)
#define MPLP_PRINT_LAST (1<<26) // terminator for loop
#define MPLP_MAX_DEPTH 8000
#define MPLP_MAX_INDEL_DEPTH 250
typedef struct {
int min_mq, flag, min_baseQ, capQ_thres, max_depth, max_indel_depth, all, rev_del;
int rflag_require, rflag_filter;
char *reg, *pl_list, *fai_fname, *output_fname;
faidx_t *fai;
void *bed, *rghash, *auxlist;
int argc;
char **argv;
char sep, empty, no_ins, no_ins_mods, no_del, no_ends;
sam_global_args ga;
} mplp_conf_t;
typedef struct {
char *ref[2];
int ref_id[2];
hts_pos_t ref_len[2];
} mplp_ref_t;
#define MPLP_REF_INIT {{NULL,NULL},{-1,-1},{0,0}}
typedef struct {
samFile *fp;
hts_itr_t *iter;
sam_hdr_t *h;
mplp_ref_t *ref;
const mplp_conf_t *conf;
} mplp_aux_t;
typedef struct {
int n;
int *n_plp, *m_plp;
bam_pileup1_t **plp;
} mplp_pileup_t;
static int build_auxlist(mplp_conf_t *conf, char *optstring) {
if (!optstring)
return 0;
void *colhash = khash_str2int_init();
if (!colhash)
return 1;
struct active_cols {
char *name;
int supported;
};
const struct active_cols colnames[11] = {
{"QNAME", 1}, {"FLAG", 1}, {"RNAME", 1}, {"POS", 1}, {"MAPQ", 1}, {"CIGAR", 0}, {"RNEXT", 1}, {"PNEXT", 1}, {"TLEN", 0}, {"SEQ", 0}, {"QUAL", 0}
};
int i, f = MPLP_PRINT_QNAME, colno = 11;
for (i = 0; i < colno; i++, f <<= 1)
if (colnames[i].supported)
khash_str2int_set(colhash, colnames[i].name, f);
conf->auxlist = kl_init(auxlist);
if (!conf->auxlist)
return 1;
char *save_p;
char *tag = strtok_r(optstring, ",", &save_p);
while (tag) {
if (khash_str2int_get(colhash, tag, &f) == 0) {
conf->flag |= f;
} else {
if (strlen(tag) != 2) {
fprintf(stderr, "[%s] tag '%s' has more than two characters or not supported\n", __func__, tag);
} else {
char **tag_p = kl_pushp(auxlist, conf->auxlist);
*tag_p = tag;
}
}
tag = strtok_r(NULL, ",", &save_p);
}
khash_str2int_destroy(colhash);
return 0;
}
static int mplp_get_ref(mplp_aux_t *ma, int tid, char **ref, hts_pos_t *ref_len) {
mplp_ref_t *r = ma->ref;
//printf("get ref %d {%d/%p, %d/%p}\n", tid, r->ref_id[0], r->ref[0], r->ref_id[1], r->ref[1]);
if (!r || !ma->conf->fai) {
*ref = NULL;
return 0;
}
// Do we need to reference count this so multiple mplp_aux_t can
// track which references are in use?
// For now we just cache the last two. Sufficient?
if (tid == r->ref_id[0]) {
*ref = r->ref[0];
*ref_len = r->ref_len[0];
return 1;
}
if (tid == r->ref_id[1]) {
// Last, swap over
int tmp_id;
hts_pos_t tmp_len;
tmp_id = r->ref_id[0]; r->ref_id[0] = r->ref_id[1]; r->ref_id[1] = tmp_id;
tmp_len = r->ref_len[0]; r->ref_len[0] = r->ref_len[1]; r->ref_len[1] = tmp_len;
char *tc;
tc = r->ref[0]; r->ref[0] = r->ref[1]; r->ref[1] = tc;
*ref = r->ref[0];
*ref_len = r->ref_len[0];
return 1;
}
// New, so migrate to old and load new
free(r->ref[1]);
r->ref[1] = r->ref[0];
r->ref_id[1] = r->ref_id[0];
r->ref_len[1] = r->ref_len[0];
r->ref_id[0] = tid;
r->ref[0] = faidx_fetch_seq64(ma->conf->fai,
sam_hdr_tid2name(ma->h, r->ref_id[0]),
0,
HTS_POS_MAX,
&r->ref_len[0]);
if (!r->ref[0]) {
r->ref[0] = NULL;
r->ref_id[0] = -1;
r->ref_len[0] = 0;
*ref = NULL;
return 0;
}
*ref = r->ref[0];
*ref_len = r->ref_len[0];
return 1;
}
// Initialise and destroy the base modifier state data. This is called
// as each new read is added or removed from the pileups.
static
int pileup_cd_create(void *data, const bam1_t *b, bam_pileup_cd *cd) {
int ret;
hts_base_mod_state *m = hts_base_mod_state_alloc();
ret = bam_parse_basemod(b, m);
cd->p = m;
return ret;
}
static
int pileup_cd_destroy(void *data, const bam1_t *b, bam_pileup_cd *cd) {
hts_base_mod_state_free(cd->p);
return 0;
}
static void
print_empty_pileup(FILE *fp, const mplp_conf_t *conf, const char *tname,
hts_pos_t pos, int n, const char *ref, hts_pos_t ref_len)
{
int i;
fprintf(fp, "%s\t%"PRIhts_pos"\t%c", tname, pos+1, (ref && pos < ref_len)? ref[pos] : 'N');
for (i = 0; i < n; ++i) {
fputs("\t0\t*\t*", fp);
int flag_value = MPLP_PRINT_MAPQ_CHAR;
while(flag_value < MPLP_PRINT_LAST) {
if (flag_value != MPLP_PRINT_MODS && (conf->flag & flag_value))
fputs("\t*", fp);
flag_value <<= 1;
}
if (conf->auxlist) {
int t = 0;
while(t++ < ((klist_t(auxlist) *)conf->auxlist)->size)
fputs("\t*", fp);
}
}
putc('\n', fp);
}
static int mplp_func(void *data, bam1_t *b)
{
char *ref;
mplp_aux_t *ma = (mplp_aux_t*)data;
int ret, skip = 0;
hts_pos_t ref_len;
do {
int has_ref;
ret = ma->iter? sam_itr_next(ma->fp, ma->iter, b) : sam_read1(ma->fp, ma->h, b);
if (ret < 0) break;
// The 'B' cigar operation is not part of the specification, considering as obsolete.
// bam_remove_B(b);
if (b->core.tid < 0 || (b->core.flag&BAM_FUNMAP)) { // exclude unmapped reads
skip = 1;
continue;
}
if (ma->conf->rflag_require && !(ma->conf->rflag_require&b->core.flag)) { skip = 1; continue; }
if (ma->conf->rflag_filter && ma->conf->rflag_filter&b->core.flag) { skip = 1; continue; }
if (ma->conf->bed && ma->conf->all == 0) { // test overlap
skip = !bed_overlap(ma->conf->bed, sam_hdr_tid2name(ma->h, b->core.tid), b->core.pos, bam_endpos(b));
if (skip) continue;
}
if (ma->conf->rghash) { // exclude read groups
uint8_t *rg = bam_aux_get(b, "RG");
skip = (rg && khash_str2int_get(ma->conf->rghash, (const char*)(rg+1), NULL)==0);
if (skip) continue;
}
if (ma->conf->flag & MPLP_ILLUMINA13) {
int i;
uint8_t *qual = bam_get_qual(b);
for (i = 0; i < b->core.l_qseq; ++i)
qual[i] = qual[i] > 31? qual[i] - 31 : 0;
}
if (ma->conf->fai && b->core.tid >= 0) {
has_ref = mplp_get_ref(ma, b->core.tid, &ref, &ref_len);
if (has_ref && ref_len <= b->core.pos) { // exclude reads outside of the reference sequence
fprintf(stderr,"[%s] Skipping because %"PRIhts_pos" is outside of %"PRIhts_pos" [ref:%d]\n",
__func__, (int64_t) b->core.pos, ref_len, b->core.tid);
skip = 1;
continue;
}
} else {
has_ref = 0;
}
skip = 0;
if (has_ref && (ma->conf->flag&MPLP_REALN)) sam_prob_realn(b, ref, ref_len, (ma->conf->flag & MPLP_REDO_BAQ)? 7 : 3);
if (has_ref && ma->conf->capQ_thres > 10) {
int q = sam_cap_mapq(b, ref, ref_len, ma->conf->capQ_thres);
if (q < 0) skip = 1;
else if (b->core.qual > q) b->core.qual = q;
}
if (b->core.qual < ma->conf->min_mq) skip = 1;
else if ((ma->conf->flag&MPLP_NO_ORPHAN) && (b->core.flag&BAM_FPAIRED) && !(b->core.flag&BAM_FPROPER_PAIR)) skip = 1;
} while (skip);
return ret;
}
/*
* Performs pileup
* @param conf configuration for this pileup
* @param n number of files specified in fn
* @param fn filenames
* @param fn_idx index filenames
*/
static int mpileup(mplp_conf_t *conf, int n, char **fn, char **fn_idx)
{
mplp_aux_t **data;
int i, tid, *n_plp, tid0 = 0, max_depth;
hts_pos_t pos, beg0 = 0, end0 = HTS_POS_MAX, ref_len;
const bam_pileup1_t **plp;
mplp_ref_t mp_ref = MPLP_REF_INIT;
bam_mplp_t iter;
sam_hdr_t *h = NULL; /* header of first file in input list */
char *ref;
FILE *pileup_fp = 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));
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(stderr,"[%s] no input file/data given\n", __func__);
exit(EXIT_FAILURE);
}
// read the header of each file in the list and initialize data
refs_t *refs = NULL;
for (i = 0; i < n; ++i) {
sam_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(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(stderr, "Failed to set CRAM_OPT_DECODE_MD value\n");
exit(EXIT_FAILURE);
}
if (!refs && conf->fai_fname) {
if (hts_set_fai_filename(data[i]->fp, conf->fai_fname) != 0) {
fprintf(stderr, "[%s] failed to process %s: %s\n",
__func__, conf->fai_fname, strerror(errno));
exit(EXIT_FAILURE);
}
refs = cram_get_refs(data[i]->fp);
} else if (conf->fai_fname) {
if (hts_set_opt(data[i]->fp, CRAM_OPT_SHARED_REF, refs) != 0) {
fprintf(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(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 : sam_hdr_str(h_tmp));
if (conf->reg) {
hts_idx_t *idx = NULL;
// If index filename has not been specfied, look in BAM folder
if (fn_idx != NULL) {
idx = sam_index_load2(data[i]->fp, fn[i], fn_idx[i]);
} else {
idx = sam_index_load(data[i]->fp, fn[i]);
}
if (idx == NULL) {
fprintf(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(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, tid0 = data[i]->iter->tid;
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
sam_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;
}
}
fprintf(stderr, "[%s] %d samples in %d input files\n", __func__, sm->n, n);
pileup_fp = conf->output_fname? fopen(conf->output_fname, "w") : stdout;
if (pileup_fp == NULL) {
fprintf(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_PRINT_MODS) {
bam_mplp_constructor(iter, pileup_cd_create);
bam_mplp_destructor(iter, pileup_cd_destroy);
}
if ( conf->flag & MPLP_SMART_OVERLAPS ) bam_mplp_init_overlaps(iter);
if ( !conf->max_depth ) {
max_depth = INT_MAX;
fprintf(stderr, "[%s] Max depth set to maximum value (%d)\n", __func__, INT_MAX);
} else {
max_depth = conf->max_depth;
if ( max_depth * n > 1<<20 )
fprintf(stderr, "[%s] Combined max depth is above 1M. Potential memory hog!\n", __func__);
}
bam_mplp_set_maxcnt(iter, max_depth);
int ret;
int last_tid = -1;
hts_pos_t last_pos = -1;
// begin pileup
while ( (ret=bam_mplp64_auto(iter, &tid, &pos, n_plp, plp)) > 0) {
if (conf->reg && (pos < beg0 || pos >= end0)) continue; // out of the region requested
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->all) {
// Deal with missing portions of previous tids
while (tid > last_tid) {
if (last_tid >= 0 && !conf->reg) {
while (++last_pos < sam_hdr_tid2len(h, last_tid)) {
if (conf->bed && bed_overlap(conf->bed, sam_hdr_tid2name(h, last_tid), last_pos, last_pos + 1) == 0)
continue;
print_empty_pileup(pileup_fp, conf, sam_hdr_tid2name(h, last_tid), last_pos, n, ref, ref_len);
}
}
last_tid++;
last_pos = -1;
if (conf->all < 2)
break;
}
}
if (conf->all) {
// Deal with missing portion of current tid
while (++last_pos < pos) {
if (conf->reg && last_pos < beg0) continue; // out of range; skip
if (conf->bed && bed_overlap(conf->bed, sam_hdr_tid2name(h, tid), last_pos, last_pos + 1) == 0)
continue;
print_empty_pileup(pileup_fp, conf, sam_hdr_tid2name(h, tid), last_pos, n, ref, ref_len);
}
last_tid = tid;
last_pos = pos;
}
if (conf->bed && tid >= 0 && !bed_overlap(conf->bed, sam_hdr_tid2name(h, tid), pos, pos+1)) continue;
fprintf(pileup_fp, "%s\t%"PRIhts_pos"\t%c", sam_hdr_tid2name(h, 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);
int flag_value = MPLP_PRINT_MAPQ_CHAR;
while(flag_value < MPLP_PRINT_LAST) {
if (flag_value != MPLP_PRINT_MODS
&& (conf->flag & flag_value))
fputs("\t*", pileup_fp);
flag_value <<= 1;
}
if (conf->auxlist) {
int t = 0;
while(t++ < ((klist_t(auxlist) *)conf->auxlist)->size)
fputs("\t*", pileup_fp);
}
} else {
int n = 0;
kstring_t ks = KS_INITIALIZE;
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) {
n++;
if (pileup_seq(pileup_fp, plp[i] + j, pos, ref_len,
ref, &ks, conf->rev_del,
conf->no_ins, conf->no_ins_mods,
conf->no_del, conf->no_ends) < 0) {
ret = 1;
goto fail;
}
}
}
if (!n) putc('*', pileup_fp);
/* Print base qualities */
n = 0;
ks_free(&ks);
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);
n++;
}
}
if (!n) putc('*', pileup_fp);
/* Print selected columns */
int flag_value = MPLP_PRINT_MAPQ_CHAR;
while(flag_value < MPLP_PRINT_LAST) {
if (flag_value != MPLP_PRINT_MODS
&& (conf->flag & flag_value)) {
n = 0;
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 ) continue;
if (n > 0 && flag_value != MPLP_PRINT_MAPQ_CHAR) putc(',', pileup_fp);
n++;
switch (flag_value) {
case MPLP_PRINT_MAPQ_CHAR:
c = p->b->core.qual + 33;
if (c > 126) c = 126;
putc(c, pileup_fp);
break;
case MPLP_PRINT_QPOS:
// query position in current orientation
fprintf(pileup_fp, "%d", p->qpos + 1);
break;
case MPLP_PRINT_QPOS5: {
// query position in 5' to 3' orientation
int pos5 = bam_is_rev(p->b)
? p->b->core.l_qseq-p->qpos + p->is_del
: p->qpos + 1;
fprintf(pileup_fp, "%d", pos5);
break;
}
case MPLP_PRINT_QNAME:
fputs(bam_get_qname(p->b), pileup_fp);
break;
case MPLP_PRINT_FLAG:
fprintf(pileup_fp, "%d", p->b->core.flag);
break;
case MPLP_PRINT_RNAME:
if (p->b->core.tid >= 0)
fputs(sam_hdr_tid2name(h, p->b->core.tid), pileup_fp);
else
putc('*', pileup_fp);
break;
case MPLP_PRINT_POS:
fprintf(pileup_fp, "%"PRId64, (int64_t) p->b->core.pos + 1);
break;
case MPLP_PRINT_MAPQ:
fprintf(pileup_fp, "%d", p->b->core.qual);
break;
case MPLP_PRINT_RNEXT:
if (p->b->core.mtid >= 0)
fputs(sam_hdr_tid2name(h, p->b->core.mtid), pileup_fp);
else
putc('*', pileup_fp);
break;
case MPLP_PRINT_PNEXT:
fprintf(pileup_fp, "%"PRId64, (int64_t) p->b->core.mpos + 1);
break;
}
}
if (!n) putc('*', pileup_fp);
}
flag_value <<= 1;
}
/* Print selected tags */
klist_t(auxlist) *auxlist_p = ((klist_t(auxlist) *)conf->auxlist);
if (auxlist_p && auxlist_p->size) {
kliter_t(auxlist) *aux;
for (aux = kl_begin(auxlist_p); aux != kl_end(auxlist_p); aux = kl_next(aux)) {
n = 0;
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 ) continue;
if (n > 0) putc(conf->sep, pileup_fp);
n++;
uint8_t* tag_u = bam_aux_get(p->b, kl_val(aux));
if (!tag_u) {
putc(conf->empty , pileup_fp);
continue;
}
int tag_supported = 0;
/* Tag value is string */
if (*tag_u == 'Z' || *tag_u == 'H') {
char *tag_s = bam_aux2Z(tag_u);
if (!tag_s) continue;
fputs(tag_s, pileup_fp);
tag_supported = 1;
}
/* Tag value is integer */
if (*tag_u == 'I' || *tag_u == 'i' || *tag_u == 'C' || *tag_u == 'c' || *tag_u == 'S' || *tag_u == 's') {
int64_t tag_i = bam_aux2i(tag_u);
fprintf(pileup_fp, "%" PRId64 "", tag_i);
tag_supported = 1;
}
/* Tag value is float */
if (*tag_u == 'd' || *tag_u == 'f') {
double tag_f = bam_aux2f(tag_u);
fprintf(pileup_fp, "%lf", tag_f);
tag_supported = 1;
}
/* Tag value is character */
if (*tag_u == 'A') {
char tag_c = bam_aux2A(tag_u);
putc(tag_c, pileup_fp);
tag_supported = 1;
}
if (!tag_supported) putc('*', pileup_fp);
}
if (!n) putc('*', pileup_fp);
}
}
}
}
putc('\n', pileup_fp);
}
if (ret < 0) {
print_error("mpileup", "error reading from input file");
ret = EXIT_FAILURE;
goto fail;
}
if (conf->all) {
// Handle terminating region
if (last_tid < 0 && conf->reg && conf->all > 1) {
last_tid = tid0;
last_pos = beg0-1;
mplp_get_ref(data[0], tid0, &ref, &ref_len);
}
while (last_tid >= 0 && last_tid < sam_hdr_nref(h)) {
while (++last_pos < sam_hdr_tid2len(h, last_tid)) {
if (last_pos >= end0) break;
if (conf->bed && bed_overlap(conf->bed, sam_hdr_tid2name(h, last_tid), last_pos, last_pos + 1) == 0)
continue;
print_empty_pileup(pileup_fp, conf, sam_hdr_tid2name(h, last_tid), last_pos, n, ref, ref_len);
}
last_tid++;
last_pos = -1;
if (conf->all < 2 || conf->reg)
break;
}
}
fail:
// clean up
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);
bam_mplp_destroy(iter);
sam_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;
}
static int is_url(const char *s)
{
static const char uri_scheme_chars[] =
"ABCDEFGHIJKLMNOPQRSTUVWXYZabcdefghijklmnopqrstuvwxyz0123456789+.-";
return s[strspn(s, uri_scheme_chars)] == ':';
}
#define MAX_PATH_LEN 1024
int read_file_list(const char *file_list,int *n,char **argv[])
{
char buf[MAX_PATH_LEN];
int len, nfiles = 0;
char **files = NULL;
struct stat sb;
*n = 0;
*argv = NULL;
FILE *fh = fopen(file_list,"r");
if ( !fh )
{
fprintf(stderr,"%s: %s\n", file_list,strerror(errno));
return 1;
}
files = calloc(nfiles,sizeof(char*));
nfiles = 0;
while ( fgets(buf,MAX_PATH_LEN,fh) )
{
// allow empty lines and trailing spaces
len = strlen(buf);
while ( len>0 && isspace(buf[len-1]) ) len--;
if ( !len ) continue;
// check sanity of the file list
buf[len] = 0;
if (! (is_url(buf) || stat(buf, &sb) == 0))
{
// no such file, check if it is safe to print its name
int i, safe_to_print = 1;
for (i=0; i<len; i++)
if (!isprint(buf[i])) { safe_to_print = 0; break; }
if ( safe_to_print )
fprintf(stderr,"The file list \"%s\" appears broken, could not locate: %s\n", file_list,buf);
else
fprintf(stderr,"Does the file \"%s\" really contain a list of files and do all exist?\n", file_list);
return 1;
}
nfiles++;
files = realloc(files,nfiles*sizeof(char*));
files[nfiles-1] = strdup(buf);
}
fclose(fh);
if ( !nfiles )
{
fprintf(stderr,"No files read from %s\n", file_list);
return 1;
}
*argv = files;
*n = nfiles;
return 0;
}
#undef MAX_PATH_LEN
static void print_usage(FILE *fp, const mplp_conf_t *mplp)
{
char *tmp_require = bam_flag2str(mplp->rflag_require);
char *tmp_filter = bam_flag2str(mplp->rflag_filter);
// Display usage information, formatted for the standard 80 columns.
// (The unusual string formatting here aids the readability of this
// source code in 80 columns, to the extent that's possible.)
fprintf(fp,
"\n"
"Usage: samtools mpileup [options] in1.bam [in2.bam [...]]\n"
"\n"
"Input options:\n"
" -6, --illumina1.3+ quality is in the Illumina-1.3+ encoding\n"
" -A, --count-orphans do not discard anomalous read pairs\n"
" -b, --bam-list FILE list of input BAM filenames, one per line\n"
" -B, --no-BAQ disable BAQ (per-Base Alignment Quality)\n"
" -C, --adjust-MQ INT adjust mapping quality; recommended:50, disable:0 [0]\n"
" -d, --max-depth INT max per-file depth; avoids excessive memory usage [%d]\n", mplp->max_depth);
fprintf(fp,
" -E, --redo-BAQ recalculate BAQ on the fly, ignore existing BQs\n"
" -f, --fasta-ref FILE faidx indexed reference sequence file\n"
" -G, --exclude-RG FILE exclude read groups listed in FILE\n"
" -l, --positions FILE skip unlisted positions (chr pos) or regions (BED)\n"
" -q, --min-MQ INT skip alignments with mapQ smaller than INT [%d]\n", mplp->min_mq);
fprintf(fp,
" -Q, --min-BQ INT skip bases with baseQ/BAQ smaller than INT [%d]\n", mplp->min_baseQ);
fprintf(fp,
" -r, --region REG region in which pileup is generated\n"
" -R, --ignore-RG ignore RG tags (one BAM = one sample)\n"
" --rf, --incl-flags STR|INT required flags: include reads with any of the mask bits set [%s]\n", tmp_require);
fprintf(fp,
" --ff, --excl-flags STR|INT filter flags: skip reads with any of the mask bits set\n"
" [%s]\n", tmp_filter);
fprintf(fp,
" -x, --ignore-overlaps-removal, --disable-overlap-removal\n"
" disable read-pair overlap detection and removal\n"
" -X, --customized-index use customized index files\n" // -X flag for index filename
"\n"
"Output options:\n"
" -o, --output FILE write output to FILE [standard output]\n"
" -O, --output-BP output base positions on reads, current orientation\n"
" --output-BP-5 output base positions on reads, 5' to 3' orientation\n"
" -M, --output-mods output base modifications\n"
" -s, --output-MQ output mapping quality\n"
" --output-QNAME output read names\n"
" --output-extra STR output extra read fields and read tag values\n"
" --output-sep CHAR set the separator character for tag lists [,]\n"
" --output-empty CHAR set the no value character for tag lists [*]\n"
" --no-output-ins skip insertion sequence after +NUM\n"
" Use twice for complete insertion removal\n"
" --no-output-ins-mods don't display base modifications within insertions\n"
" --no-output-del skip deletion sequence after -NUM\n"
" Use twice for complete deletion removal\n"
" --no-output-ends remove ^MQUAL and $ markup in sequence column\n"
" --reverse-del use '#' character for deletions on the reverse strand\n"
" -a output all positions (including zero depth)\n"
" -a -a (or -aa) output absolutely all positions, including unused ref. sequences\n"
"\n"
"Generic options:\n");
sam_global_opt_help(fp, "-.--.--.");
fprintf(fp, "\n"
"Note that using \"samtools mpileup\" to generate BCF or VCF files has been\n"
"removed. To output these formats, please use \"bcftools mpileup\" instead.\n");
free(tmp_require);
free(tmp_filter);
}
int bam_mpileup(int argc, char *argv[])
{
int c;
const char *file_list = NULL;
char **fn = NULL;
int nfiles = 0, use_orphan = 0, has_index_file = 0;
mplp_conf_t mplp;
memset(&mplp, 0, sizeof(mplp_conf_t));
mplp.min_baseQ = 13;
mplp.capQ_thres = 0;
mplp.max_depth = MPLP_MAX_DEPTH;
mplp.flag = MPLP_NO_ORPHAN | MPLP_REALN | MPLP_SMART_OVERLAPS;
mplp.argc = argc; mplp.argv = argv;
mplp.rflag_filter = BAM_FUNMAP | BAM_FSECONDARY | BAM_FQCFAIL | BAM_FDUP;
mplp.output_fname = NULL;
mplp.all = 0;
mplp.rev_del = 0;