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bam_plcmd.c
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/* bam_plcmd.c -- mpileup subcommand.
Copyright (C) 2008-2015, 2019 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 "samtools.h"
#include "bedidx.h"
#include "sam_opts.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;
}
static inline 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 j;
if (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);
} 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(p, ks, &del_len);
if (len < 0)
return -1;
putc('+', fp); printw(len, fp);
if (bam_is_rev(p->b)) {
char pad = rev_del ? '#' : '*';
for (j = 0; j < len; j++)
putc(ks->s[j] != '*' ? tolower(ks->s[j]) : pad, fp);
} else {
for (j = 0; j < len; j++)
putc(toupper(ks->s[j]), fp);
}
}
if (del_len > 0) {
printw(-del_len, fp);
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 (p->is_tail) putc('$', fp);
return 0;
}
#include <assert.h>
#include "bam2bcf.h"
#include "sample.h"
#define MPLP_BCF 1
#define MPLP_VCF (1<<1)
#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_PRINT_QPOS (1<<9)
#define MPLP_PER_SAMPLE (1<<11)
#define MPLP_SMART_OVERLAPS (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_MAX_DEPTH 8000
#define MPLP_MAX_INDEL_DEPTH 250
typedef struct {
int min_mq, flag, min_baseQ, capQ_thres, max_depth, max_indel_depth, fmt_flag, all, rev_del;
int rflag_require, rflag_filter;
int openQ, extQ, tandemQ, min_support; // for indels
double min_frac; // for indels
char *reg, *pl_list, *fai_fname, *output_fname;
faidx_t *fai;
void *bed, *rghash, *auxlist;
int argc;
char **argv;
char sep, empty;
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;
}
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);
if (conf->flag & MPLP_PRINT_QPOS)
fputs("\t*", fp);
int flag_value = MPLP_PRINT_QNAME;
while(flag_value < MPLP_PRINT_QUAL + 1) {
if (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;
}
static void group_smpl(mplp_pileup_t *m, bam_sample_t *sm, kstring_t *buf,
int n, char *const*fn, int *n_plp, const bam_pileup1_t **plp, int ignore_rg)
{
int i, j;
memset(m->n_plp, 0, m->n * sizeof(int));
for (i = 0; i < n; ++i) {
for (j = 0; j < n_plp[i]; ++j) {
const bam_pileup1_t *p = plp[i] + j;
uint8_t *q;
int id = -1;
q = ignore_rg? NULL : bam_aux_get(p->b, "RG");
if (q) id = bam_smpl_rg2smid(sm, fn[i], (char*)q+1, buf);
if (id < 0) id = bam_smpl_rg2smid(sm, fn[i], 0, buf);
if (id < 0 || id >= m->n) {
assert(q); // otherwise a bug
fprintf(stderr, "[%s] Read group %s used in file %s but absent from the header or an alignment missing read group.\n", __func__, (char*)q+1, fn[i]);
exit(EXIT_FAILURE);
}
if (m->n_plp[id] == m->m_plp[id]) {
m->m_plp[id] = m->m_plp[id]? m->m_plp[id]<<1 : 8;
m->plp[id] = realloc(m->plp[id], sizeof(bam_pileup1_t) * m->m_plp[id]);
}
m->plp[id][m->n_plp[id]++] = *p;
}
}
}
/*
* 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)
{
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, *n_plp, tid0 = 0, max_depth, max_indel_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;
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(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) {
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 (conf->fai_fname && 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);
}
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->flag & MPLP_BCF) {
// Collect read group IDs with PL (platform) listed in pl_list (note: fragile, strstr search)
rghash = bcf_call_add_rg(rghash, sam_hdr_str(h_tmp), conf->pl_list);
}
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);
if (conf->flag & MPLP_BCF)
{
const char *mode;
// 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*));
// write the VCF header
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(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 < sam_hdr_nref(h); i++)
{
str.l = 0;
ksprintf(&str, "##contig=<ID=%s,length=%"PRId64">", sam_hdr_tid2name(h, i), (int64_t) sam_hdr_tid2len(h, 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);
if (bcf_hdr_write(bcf_fp, bcf_hdr) != 0) {
print_error_errno("mpileup", "Failed to write VCF/BCF header to \"%s\"",
conf->output_fname? conf->output_fname : "standard output");
exit(EXIT_FAILURE);
}
// 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") : 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_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__);
}
// Only used when writing BCF
max_indel_depth = conf->max_indel_depth * sm->n;
bam_mplp_set_maxcnt(iter, max_depth);
bcf1_t *bcf_rec = bcf_init1();
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->flag & MPLP_BCF) {
int total_depth, _ref0, ref16;
if (conf->bed && tid >= 0 && !bed_overlap(conf->bed, sam_hdr_tid2name(h, tid), pos, pos+1)) continue;
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);
if (bcf_write1(bcf_fp, bcf_hdr, bcf_rec) != 0) {
print_error_errno("mpileup", "Failed to write VCF/BCF record to \"%s\"",
conf->output_fname?conf->output_fname:"standard output");
exit(EXIT_FAILURE);
}
// 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);
if (bcf_write1(bcf_fp, bcf_hdr, bcf_rec) != 0) {
print_error_errno("mpileup", "Failed to write VCF/BCF record to \"%s\"",
conf->output_fname?conf->output_fname:"standard output");
exit(EXIT_FAILURE);
}
}
}
} else {
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);
if (conf->flag & MPLP_PRINT_QPOS)
fputs("\t*", pileup_fp);
int flag_value = MPLP_PRINT_QNAME;
while(flag_value < MPLP_PRINT_QUAL + 1) {
if (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) < 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 mpileup positions */
if (conf->flag & MPLP_PRINT_QPOS) {
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(',', pileup_fp);
n++;
fprintf(pileup_fp, "%d", p->qpos + 1);
}
if (!n) putc('*', pileup_fp);
}
/* Print selected columns */
int flag_value = MPLP_PRINT_QNAME;
while(flag_value < MPLP_PRINT_QUAL + 1) {
if (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) putc(',', pileup_fp);
n++;
switch (flag_value) {
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:
c = p->b->core.qual + 33;
if (c > 126) c = 126;
putc(c, pileup_fp);
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;
}
/* 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 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 value is float */
if (*tag_u == 'd' || *tag_u == 'f') {
double tag_f = bam_aux2f(tag_u);
fprintf(pileup_fp, "%lf", tag_f);
}
/* Tag value is character */
if (*tag_u == 'A') {
char tag_c = bam_aux2A(tag_u);
putc(tag_c, pileup_fp);
}
}
if (!n) putc('*', pileup_fp);
}
}
}
}
putc('\n', pileup_fp);
}
}
if (conf->all && !(conf->flag & MPLP_BCF)) {
// 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
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);
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;