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mpileup.c
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/* mpileup.c -- mpileup subcommand. Previously bam_plcmd.c from samtools
Copyright (C) 2008-2021 Genome Research Ltd.
Portions copyright (C) 2009-2012 Broad Institute.
Author: Heng Li <lh3@sanger.ac.uk>
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 <math.h>
#include <stdio.h>
#include <stdlib.h>
#include <unistd.h>
#include <ctype.h>
#include <string.h>
#include <strings.h>
#include <limits.h>
#include <inttypes.h>
#include <errno.h>
#include <sys/stat.h>
#include <getopt.h>
#include <htslib/sam.h>
#include <htslib/faidx.h>
#include <htslib/kstring.h>
#include <htslib/khash_str2int.h>
#include <htslib/hts_os.h>
#include <assert.h>
#include "regidx.h"
#include "bcftools.h"
#include "bam2bcf.h"
#include "bam_sample.h"
#include "gvcf.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_POS (1<<9)
#define MPLP_PRINT_MAPQ (1<<10)
#define MPLP_PER_SAMPLE (1<<11)
#define MPLP_SMART_OVERLAPS (1<<12)
#define MPLP_REALN_PARTIAL (1<<13)
typedef struct _mplp_aux_t mplp_aux_t;
typedef struct _mplp_pileup_t mplp_pileup_t;
// Data shared by all bam files
typedef struct {
int min_mq, flag, min_baseQ, max_baseQ, delta_baseQ, capQ_thres, max_depth,
max_indel_depth, max_read_len, fmt_flag, ambig_reads;
int rflag_require, rflag_filter, output_type;
int openQ, extQ, tandemQ, min_support; // for indels
double min_frac; // for indels
double indel_bias;
char *reg_fname, *pl_list, *fai_fname, *output_fname;
int reg_is_file, record_cmd_line, n_threads;
faidx_t *fai;
regidx_t *bed, *reg; // bed: skipping regions, reg: index-jump to regions
regitr_t *bed_itr, *reg_itr;
int bed_logic; // 1: include region, 0: exclude region
gvcf_t *gvcf;
// auxiliary structures for calling
bcf_callaux_t *bca;
bcf_callret1_t *bcr;
bcf_call_t bc;
bam_mplp_t iter;
mplp_aux_t **mplp_data;
int nfiles;
char **files;
mplp_pileup_t *gplp;
int *n_plp;
const bam_pileup1_t **plp;
bam_smpl_t *bsmpl;
kstring_t buf;
bcf1_t *bcf_rec;
htsFile *bcf_fp;
bcf_hdr_t *bcf_hdr;
int argc;
char **argv;
} mplp_conf_t;
typedef struct {
char *ref[2];
int ref_id[2];
int ref_len[2];
} mplp_ref_t;
#define MPLP_REF_INIT {{NULL,NULL},{-1,-1},{0,0}}
// Data specific to each bam file
struct _mplp_aux_t {
samFile *fp;
hts_itr_t *iter;
bam_hdr_t *h;
mplp_ref_t *ref;
const mplp_conf_t *conf;
int bam_id;
hts_idx_t *idx; // maintained only with more than one -r regions
};
// Data passed to htslib/mpileup
struct _mplp_pileup_t {
int n;
int *n_plp, *m_plp;
bam_pileup1_t **plp;
};
static int mplp_get_ref(mplp_aux_t *ma, int tid, char **ref, int *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;
tmp = r->ref_id[0]; r->ref_id[0] = r->ref_id[1]; r->ref_id[1] = tmp;
tmp = r->ref_len[0]; r->ref_len[0] = r->ref_len[1]; r->ref_len[1] = tmp;
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_seq(ma->conf->fai,
ma->h->target_name[r->ref_id[0]],
0,
INT_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 int mplp_func(void *data, bam1_t *b)
{
char *ref;
mplp_aux_t *ma = (mplp_aux_t*)data;
int ret, ref_len;
while (1)
{
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)) continue; // exclude unmapped reads
if (ma->conf->rflag_require && !(ma->conf->rflag_require&b->core.flag)) continue;
if (ma->conf->rflag_filter && ma->conf->rflag_filter&b->core.flag) continue;
if (ma->conf->bed)
{
// test overlap
regitr_t *itr = ma->conf->bed_itr;
int beg = b->core.pos, end = bam_endpos(b)-1;
int overlap = regidx_overlap(ma->conf->bed, ma->h->target_name[b->core.tid],beg,end, itr);
if ( !ma->conf->bed_logic && !overlap )
{
// exclude only reads which are fully contained in the region
while ( regitr_overlap(itr) )
{
if ( beg < itr->beg ) { overlap = 1; break; }
if ( end > itr->end ) { overlap = 1; break; }
}
}
if ( !overlap ) continue;
}
if ( bam_smpl_get_sample_id(ma->conf->bsmpl,ma->bam_id,b)<0 ) 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 %"PRId64" is outside of %d [ref:%d]\n",
__func__, (int64_t) b->core.pos, ref_len, b->core.tid);
continue;
}
} else {
has_ref = 0;
}
// Allow sufficient room for bam_aux_append of ZQ tag without
// a realloc and consequent breakage of pileup's cached pointers.
if (has_ref && (ma->conf->flag &MPLP_REALN) && !bam_aux_get(b, "ZQ")) {
// Doing sam_prob_realn later is problematic as it adds to
// the tag list (ZQ or BQ), which causes a realloc of b->data.
// This happens after pileup has built a hash table on the
// read name. It's a deficiency in pileup IMO.
// We could implement a new sam_prob_realn that returns ZQ
// somewhere else and cache it ourselves (pileup clientdata),
// but for now we simply use a workaround.
//
// We create a fake tag of the correct length, which we remove
// just prior calling sam_prob_realn so we can guarantee there is
// room. (We can't just make room now as bam_copy1 removes it
// again).
if (b->core.l_qseq > 500) {
uint8_t *ZQ = malloc((uint32_t)b->core.l_qseq+1);
memset(ZQ, '@', b->core.l_qseq);
ZQ[b->core.l_qseq] = 0;
bam_aux_append(b, "_Q", 'Z', b->core.l_qseq+1, ZQ);
free(ZQ);
} else {
static uint8_t ZQ[501] =
"@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@"
"@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@"
"@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@"
"@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@"
"@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@"
"@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@"
"@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@"
"@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@"
"@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@"
"@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@@";
ZQ[b->core.l_qseq] = 0;
bam_aux_append(b, "_Q", 'Z', b->core.l_qseq+1, ZQ);
ZQ[b->core.l_qseq] = '@';
}
}
if (has_ref && ma->conf->capQ_thres > 10) {
int q = sam_cap_mapq(b, ref, ref_len, ma->conf->capQ_thres);
if (q < 0) continue; // skip
else if (b->core.qual > q) b->core.qual = q;
}
if (b->core.qual < ma->conf->min_mq) continue;
else if ((ma->conf->flag&MPLP_NO_ORPHAN) && (b->core.flag&BAM_FPAIRED) && !(b->core.flag&BAM_FPROPER_PAIR)) continue;
return ret;
};
return ret;
}
// Called once per new bam added to the pileup.
// We cache sample information here so we don't have to keep recomputing this
// on each and every pileup column. If FMT/SCR annotation is requested, a flag
// is set to indicate the presence of a soft clip.
//
// Cd is an arbitrary block of data we can write into, which ends up in
// the pileup structures. We stash the sample ID there:
// has_soft_clip .. cd->i & 1
// sample_id .. cd->i >> 1
static int pileup_constructor(void *data, const bam1_t *b, bam_pileup_cd *cd)
{
mplp_aux_t *ma = (mplp_aux_t *)data;
int n = bam_smpl_get_sample_id(ma->conf->bsmpl, ma->bam_id, (bam1_t *)b);
cd->i = 0;
PLP_SET_SAMPLE_ID(cd->i, n);
// Whether read has a soft-clip is used in mplp_realn's heuristics.
// TODO: consider whether clip length is beneficial to use?
int i;
for (i=0; i<b->core.n_cigar; i++) {
int cig = bam_get_cigar(b)[i] & BAM_CIGAR_MASK;
if (cig == BAM_CSOFT_CLIP) {
PLP_SET_SOFT_CLIP(cd->i);
break;
}
}
if (ma->conf->flag & MPLP_REALN) {
int i, tot_ins = 0;
uint32_t *cigar = bam_get_cigar(b);
int p = 0;
for (i=0; i<b->core.n_cigar; i++) {
int cig = cigar[i] & BAM_CIGAR_MASK;
if (bam_cigar_type(cig) & 2)
p += cigar[i] >> BAM_CIGAR_SHIFT;
if (cig == BAM_CINS || cig == BAM_CDEL || cig == BAM_CREF_SKIP) {
tot_ins += cigar[i] >> BAM_CIGAR_SHIFT;
// Possible further optimsation, check tot_ins==1 later
// (and remove break) so we can detect single bp indels.
// We may want to focus BAQ on more complex regions only.
PLP_SET_INDEL(cd->i);
break;
}
// TODO: proper p->cd struct and have cd->i as a size rather
// than a flag.
// Then aggregate together the sizes and if just 1 size for all
// reads or 2 sizes for approx 50/50 split in all reads, then
// treat this as a well-aligned variant and don't run BAQ.
}
}
return 0;
}
static void group_smpl(mplp_pileup_t *m, bam_smpl_t *bsmpl, int n, int *n_plp, const bam_pileup1_t **plp)
{
int i, j;
memset(m->n_plp, 0, m->n * sizeof(int));
for (i = 0; i < n; ++i) // iterate over all bams
{
for (j = 0; j < n_plp[i]; ++j) // iterate over all reads available at this position
{
const bam_pileup1_t *p = plp[i] + j;
int id = PLP_SAMPLE_ID(p->cd.i);
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] = (bam_pileup1_t*) realloc(m->plp[id], sizeof(bam_pileup1_t) * m->m_plp[id]);
}
m->plp[id][m->n_plp[id]++] = *p;
}
}
}
static void flush_bcf_records(mplp_conf_t *conf, htsFile *fp, bcf_hdr_t *hdr, bcf1_t *rec)
{
if ( !conf->gvcf )
{
if ( rec && bcf_write1(fp, hdr, rec)!=0 ) error("[%s] Error: failed to write the record to %s\n", __func__,conf->output_fname?conf->output_fname:"standard output");
return;
}
if ( !rec )
{
gvcf_write(conf->gvcf, fp, hdr, NULL, 0);
return;
}
int is_ref = 0;
if ( rec->n_allele==1 ) is_ref = 1;
else if ( rec->n_allele==2 )
{
// second allele is mpileup's X, not a variant
if ( rec->d.allele[1][0]=='<' && rec->d.allele[1][1]=='*' && rec->d.allele[1][2]=='>' ) is_ref = 1;
}
rec = gvcf_write(conf->gvcf, fp, hdr, rec, is_ref);
if ( rec && bcf_write1(fp,hdr,rec)!=0 ) error("[%s] Error: failed to write the record to %s\n", __func__,conf->output_fname?conf->output_fname:"standard output");
}
/*
* Loops for an indel at this position.
*
* Only reads that overlap an indel loci get realigned. This considerably
* reduces the cost of running BAQ while keeping the main benefits.
*
* TODO: also consider only realigning reads that don't span the indel
* by more than a certain amount either-side. Ie focus BAQ only on reads
* ending adjacent to the indel, where the alignment is most likely to
* be wrong. (2nd TODO: do this based on sequence context; STRs bad, unique
* data good.)
*
* NB: this may sadly realign after we've already used the data. Hmm...
*/
static void mplp_realn(int n, int *n_plp, const bam_pileup1_t **plp,
int flag, int max_read_len,
char *ref, int ref_len, int pos) {
int i, j, has_indel = 0, has_clip = 0, nt = 0;
int min_indel = INT_MAX, max_indel = INT_MIN;
// Is an indel present.
// NB: don't bother even checking if very long as almost guaranteed
// to have indel (and likely soft-clips too).
for (i = 0; i < n; i++) { // iterate over bams
nt += n_plp[i];
for (j = 0; j < n_plp[i]; j++) { // iterate over reads
bam_pileup1_t *p = (bam_pileup1_t *)plp[i] + j;
has_indel += (PLP_HAS_INDEL(p->cd.i) || p->indel) ? 1 : 0;
// Has_clip is almost always true for very long reads
// (eg PacBio CCS), but these rarely matter as the clip
// is likely a long way from this indel.
has_clip += (PLP_HAS_SOFT_CLIP(p->cd.i)) ? 1 : 0;
if (max_indel < p->indel)
max_indel = p->indel;
if (min_indel > p->indel)
min_indel = p->indel;
}
}
if (flag & MPLP_REALN_PARTIAL) {
if (has_indel == 0 ||
(has_clip < 0.2*nt && max_indel == min_indel &&
(has_indel < 0.1*nt /*|| has_indel > 0.9*nt*/ || has_indel == 1)))
return;
}
// Realign
for (i = 0; i < n; i++) { // iterate over bams
for (j = 0; j < n_plp[i]; j++) { // iterate over reads
const bam_pileup1_t *p = plp[i] + j;
bam1_t *b = p->b;
// Avoid doing multiple times.
//
// Note we cannot modify p->cd.i here with a PLP_SET macro
// because the cd item is held by mpileup in an lbnode_t
// struct and copied over to the pileup struct for each
// iteration, essentially making p->cd.i read only.
//
// We could use our own structure (p->cd.p), allocated during
// the constructor, but for simplicity we play dirty and
// abuse an unused flag bit instead.
if (b->core.flag & 32768)
continue;
b->core.flag |= 32768;
if (b->core.l_qseq > max_read_len)
continue;
// Check p->cigar_ind and see what cigar elements are before
// and after. How close is this location to the end of the
// read? Only realign if we don't span by more than X bases.
//
// Again, best only done on deeper data as BAQ helps
// disproportionately more on shallow data sets.
//
// This rescues some of the false negatives that are caused by
// systematic reduction in quality due to sample vs ref alignment.
// At deep coverage we skip realigning more reads as we have sufficient depth.
// This rescues for false negatives. At shallow depth we pay for this with
// more FP so are more stringent on spanning size.
#define REALN_DIST (40+10*(nt<40)+10*(nt<20))
uint32_t *cig = bam_get_cigar(b);
int ncig = b->core.n_cigar;
// Don't realign reads where indel is in middle?
// On long read data we don't care about soft-clips at the ends.
// For short read data, we always calc BAQ on these as they're
// a common source of false positives.
if ((flag & MPLP_REALN_PARTIAL) && nt > 15 && ncig > 1) {
// Left & right cigar op match.
int lr = b->core.l_qseq > 500;
int lm = 0, rm = 0, k;
for (k = 0; k < ncig; k++) {
int cop = bam_cigar_op(cig[k]);
if (lr && (cop == BAM_CHARD_CLIP || cop == BAM_CSOFT_CLIP))
continue;
if (cop == BAM_CMATCH || cop == BAM_CDIFF ||
cop == BAM_CEQUAL)
lm += bam_cigar_oplen(cig[k]);
else
break;
}
for (k = ncig-1; k >= 0; k--) {
int cop = bam_cigar_op(cig[k]);
if (lr && (cop == BAM_CHARD_CLIP || cop == BAM_CSOFT_CLIP))
continue;
if (cop == BAM_CMATCH || cop == BAM_CDIFF ||
cop == BAM_CEQUAL)
rm += bam_cigar_oplen(cig[k]);
else
break;
}
if (lm >= REALN_DIST*4 && rm >= REALN_DIST*4)
continue;
if (lm >= REALN_DIST && rm >= REALN_DIST &&
has_clip < (0.15+0.05*(nt>20))*nt)
continue;
}
if (b->core.l_qseq > 500) {
// don't do BAQ on long-read data if it's going to
// cause us to have a large band-with and costly in CPU
int rl = bam_cigar2rlen(b->core.n_cigar, bam_get_cigar(b));
if (abs(rl - b->core.l_qseq) * b->core.l_qseq >= 500000)
continue;
}
// Fudge: make room for ZQ tag.
uint8_t *_Q = bam_aux_get(b, "_Q");
if (_Q) bam_aux_del(b, _Q);
sam_prob_realn(b, ref, ref_len, (flag & MPLP_REDO_BAQ) ? 7 : 3);
}
}
return;
}
static int mpileup_reg(mplp_conf_t *conf, uint32_t beg, uint32_t end)
{
bam_hdr_t *hdr = conf->mplp_data[0]->h; // header of first file in input list
int ret, i, tid, pos, ref_len;
char *ref;
while ( (ret=bam_mplp_auto(conf->iter, &tid, &pos, conf->n_plp, conf->plp)) > 0)
{
if ( pos<beg || pos>end ) continue;
if ( conf->bed && tid >= 0 )
{
int overlap = regidx_overlap(conf->bed, hdr->target_name[tid], pos, pos, NULL);
if ( !conf->bed_logic ) overlap = overlap ? 0 : 1;
if ( !overlap ) continue;
}
int has_ref = mplp_get_ref(conf->mplp_data[0], tid, &ref, &ref_len);
if (has_ref && (conf->flag & MPLP_REALN))
mplp_realn(conf->nfiles, conf->n_plp, conf->plp, conf->flag,
conf->max_read_len, ref, ref_len, pos);
int total_depth, _ref0, ref16;
for (i = total_depth = 0; i < conf->nfiles; ++i) total_depth += conf->n_plp[i];
group_smpl(conf->gplp, conf->bsmpl, conf->nfiles, conf->n_plp, conf->plp);
_ref0 = (ref && pos < ref_len)? ref[pos] : 'N';
ref16 = seq_nt16_table[_ref0];
bcf_callaux_clean(conf->bca, &conf->bc);
for (i = 0; i < conf->gplp->n; ++i)
bcf_call_glfgen(conf->gplp->n_plp[i], conf->gplp->plp[i], ref16, conf->bca, conf->bcr + i);
conf->bc.tid = tid; conf->bc.pos = pos;
bcf_call_combine(conf->gplp->n, conf->bcr, conf->bca, ref16, &conf->bc);
bcf_clear1(conf->bcf_rec);
bcf_call2bcf(&conf->bc, conf->bcf_rec, conf->bcr, conf->fmt_flag,
conf->bca, 0);
flush_bcf_records(conf, conf->bcf_fp, conf->bcf_hdr, conf->bcf_rec);
// call indels; todo: subsampling with total_depth>max_indel_depth instead of ignoring?
// check me: rghash in bcf_call_gap_prep() should have no effect, reads mplp_func already excludes them
if (!(conf->flag&MPLP_NO_INDEL) && total_depth < conf->max_indel_depth
&& (bcf_callaux_clean(conf->bca, &conf->bc),
bcf_call_gap_prep(conf->gplp->n, conf->gplp->n_plp, conf->gplp->plp, pos, conf->bca, ref) >= 0))
{
for (i = 0; i < conf->gplp->n; ++i)
bcf_call_glfgen(conf->gplp->n_plp[i], conf->gplp->plp[i], -1, conf->bca, conf->bcr + i);
if (bcf_call_combine(conf->gplp->n, conf->bcr, conf->bca, -1, &conf->bc) >= 0)
{
bcf_clear1(conf->bcf_rec);
bcf_call2bcf(&conf->bc, conf->bcf_rec, conf->bcr, conf->fmt_flag, conf->bca, ref);
flush_bcf_records(conf, conf->bcf_fp, conf->bcf_hdr, conf->bcf_rec);
}
}
}
return 0;
}
static int mpileup(mplp_conf_t *conf)
{
if (conf->nfiles == 0) {
fprintf(stderr,"[%s] no input file/data given\n", __func__);
exit(EXIT_FAILURE);
}
mplp_ref_t mp_ref = MPLP_REF_INIT;
conf->gplp = (mplp_pileup_t *) calloc(1,sizeof(mplp_pileup_t));
conf->mplp_data = (mplp_aux_t**) calloc(conf->nfiles, sizeof(mplp_aux_t*));
conf->plp = (const bam_pileup1_t**) calloc(conf->nfiles, sizeof(bam_pileup1_t*));
conf->n_plp = (int*) calloc(conf->nfiles, sizeof(int));
// Allow to run mpileup on multiple regions in one go. This comes at cost: the bai index
// must be kept in the memory for the whole time which can be a problem with many bams.
// Therefore if none or only one region is requested, we initialize the bam iterator as
// before and free the index. Only when multiple regions are queried, we keep the index.
int nregs = 0;
if ( conf->reg_fname )
{
if ( conf->reg_is_file )
{
conf->reg = regidx_init(conf->reg_fname,NULL,NULL,0,NULL);
if ( !conf->reg ) {
fprintf(stderr,"Could not parse the regions: %s\n", conf->reg_fname);
exit(EXIT_FAILURE);
}
}
else
{
conf->reg = regidx_init(NULL,regidx_parse_reg,NULL,sizeof(char*),NULL);
if ( regidx_insert_list(conf->reg,conf->reg_fname,',') !=0 ) {
fprintf(stderr,"Could not parse the regions: %s\n", conf->reg_fname);
exit(EXIT_FAILURE);
}
}
nregs = regidx_nregs(conf->reg);
conf->reg_itr = regitr_init(conf->reg);
regitr_loop(conf->reg_itr); // region iterator now positioned at the first region
}
// read the header of each file in the list and initialize data
// beware: mpileup has always assumed that tid's are consistent in the headers, add sanity check at least!
bam_hdr_t *hdr = NULL; // header of first file in input list
int i;
for (i = 0; i < conf->nfiles; ++i) {
bam_hdr_t *h_tmp;
conf->mplp_data[i] = (mplp_aux_t*) calloc(1, sizeof(mplp_aux_t));
conf->mplp_data[i]->fp = sam_open(conf->files[i], "rb");
if ( !conf->mplp_data[i]->fp )
{
fprintf(stderr, "[%s] failed to open %s: %s\n", __func__, conf->files[i], strerror(errno));
exit(EXIT_FAILURE);
}
if (hts_set_opt(conf->mplp_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(conf->mplp_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);
}
conf->mplp_data[i]->conf = conf;
conf->mplp_data[i]->ref = &mp_ref;
h_tmp = sam_hdr_read(conf->mplp_data[i]->fp);
if ( !h_tmp ) {
fprintf(stderr,"[%s] fail to read the header of %s\n", __func__, conf->files[i]);
exit(EXIT_FAILURE);
}
conf->mplp_data[i]->h = i ? hdr : h_tmp; // for j==0, "h" has not been set yet
conf->mplp_data[i]->bam_id = bam_smpl_add_bam(conf->bsmpl,h_tmp->text,conf->files[i]);
if ( conf->mplp_data[i]->bam_id<0 )
{
// no usable readgroups in this bam, it can be skipped
sam_close(conf->mplp_data[i]->fp);
free(conf->mplp_data[i]);
bam_hdr_destroy(h_tmp);
free(conf->files[i]);
if ( i+1<conf->nfiles ) memmove(&conf->files[i],&conf->files[i+1],sizeof(*conf->files)*(conf->nfiles-i-1));
conf->nfiles--;
i--;
continue;
}
if (conf->reg) {
hts_idx_t *idx = sam_index_load(conf->mplp_data[i]->fp, conf->files[i]);
if (idx == NULL) {
fprintf(stderr, "[%s] fail to load index for %s\n", __func__, conf->files[i]);
exit(EXIT_FAILURE);
}
conf->buf.l = 0;
ksprintf(&conf->buf,"%s:%u-%u",conf->reg_itr->seq,conf->reg_itr->beg+1,conf->reg_itr->end+1);
conf->mplp_data[i]->iter = sam_itr_querys(idx, conf->mplp_data[i]->h, conf->buf.s);
if ( !conf->mplp_data[i]->iter )
{
conf->mplp_data[i]->iter = sam_itr_querys(idx, conf->mplp_data[i]->h, conf->reg_itr->seq);
if ( conf->mplp_data[i]->iter ) {
fprintf(stderr,"[E::%s] fail to parse region '%s'\n", __func__, conf->buf.s);
exit(EXIT_FAILURE);
}
fprintf(stderr,"[E::%s] the sequence \"%s\" not found: %s\n",__func__,conf->reg_itr->seq,conf->files[i]);
exit(EXIT_FAILURE);
}
if ( nregs==1 ) // no need to keep the index in memory
hts_idx_destroy(idx);
else
conf->mplp_data[i]->idx = idx;
}
if ( !hdr ) hdr = h_tmp; /* save the header of first file in list */
else {
// FIXME: check consistency between h and h_tmp
bam_hdr_destroy(h_tmp);
// we store only the first file's header; it's (alleged to be)
// compatible with the i-th file's target_name lookup needs
conf->mplp_data[i]->h = hdr;
}
}
if ( !hdr ) {
fprintf(stderr, "[%s] failed to find a file header with usable read groups\n", __func__);
exit(EXIT_FAILURE);
}
// allocate data storage proportionate to number of samples being studied sm->n
bam_smpl_get_samples(conf->bsmpl, &conf->gplp->n);
conf->gplp->n_plp = (int*) calloc(conf->gplp->n, sizeof(int));
conf->gplp->m_plp = (int*) calloc(conf->gplp->n, sizeof(int));
conf->gplp->plp = (bam_pileup1_t**) calloc(conf->gplp->n, sizeof(bam_pileup1_t*));
fprintf(stderr, "[%s] %d samples in %d input files\n", __func__, conf->gplp->n, conf->nfiles);
// write the VCF header
conf->bcf_fp = hts_open(conf->output_fname?conf->output_fname:"-", hts_bcf_wmode2(conf->output_type,conf->output_fname));
if (conf->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);
}
if ( conf->n_threads ) hts_set_threads(conf->bcf_fp, conf->n_threads);
// BCF header creation
conf->bcf_hdr = bcf_hdr_init("w");
conf->buf.l = 0;
if (conf->record_cmd_line)
{
ksprintf(&conf->buf, "##bcftoolsVersion=%s+htslib-%s\n",bcftools_version(),hts_version());
bcf_hdr_append(conf->bcf_hdr, conf->buf.s);
conf->buf.l = 0;
ksprintf(&conf->buf, "##bcftoolsCommand=mpileup");
for (i=1; i<conf->argc; i++) ksprintf(&conf->buf, " %s", conf->argv[i]);
kputc('\n', &conf->buf);
bcf_hdr_append(conf->bcf_hdr, conf->buf.s);
}
if (conf->fai_fname)
{
conf->buf.l = 0;
ksprintf(&conf->buf, "##reference=file://%s\n", conf->fai_fname);
bcf_hdr_append(conf->bcf_hdr, conf->buf.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<hdr->n_targets; i++)
{
conf->buf.l = 0;
ksprintf(&conf->buf, "##contig=<ID=%s,length=%d>", hdr->target_name[i], hdr->target_len[i]);
bcf_hdr_append(conf->bcf_hdr, conf->buf.s);
}
conf->buf.l = 0;
bcf_hdr_append(conf->bcf_hdr,"##ALT=<ID=*,Description=\"Represents allele(s) other than observed.\">");
bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=INDEL,Number=0,Type=Flag,Description=\"Indicates that the variant is an INDEL.\">");
bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=IDV,Number=1,Type=Integer,Description=\"Maximum number of raw reads supporting an indel\">");
bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=IMF,Number=1,Type=Float,Description=\"Maximum fraction of raw reads supporting an indel\">");
bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=DP,Number=1,Type=Integer,Description=\"Raw read depth\">");
if ( conf->fmt_flag&B2B_INFO_VDB )
bcf_hdr_append(conf->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\">");
if (conf->fmt_flag & B2B_INFO_ZSCORE) {
if ( conf->fmt_flag&B2B_INFO_RPB )
bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=RPBZ,Number=1,Type=Float,Description=\"Mann-Whitney U-z test of Read Position Bias (closer to 0 is better)\">");
bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=MQBZ,Number=1,Type=Float,Description=\"Mann-Whitney U-z test of Mapping Quality Bias (closer to 0 is better)\">");
bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=BQBZ,Number=1,Type=Float,Description=\"Mann-Whitney U-z test of Base Quality Bias (closer to 0 is better)\">");
bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=MQSBZ,Number=1,Type=Float,Description=\"Mann-Whitney U-z test of Mapping Quality vs Strand Bias (closer to 0 is better)\">");
if ( conf->fmt_flag&B2B_INFO_SCB )
bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=SCBZ,Number=1,Type=Float,Description=\"Mann-Whitney U-z test of Soft-Clip Length Bias (closer to 0 is better)\">");
} else {
if ( conf->fmt_flag&B2B_INFO_RPB )
bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=RPB,Number=1,Type=Float,Description=\"Mann-Whitney U test of Read Position Bias (bigger is better)\">");
bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=MQB,Number=1,Type=Float,Description=\"Mann-Whitney U test of Mapping Quality Bias (bigger is better)\">");
bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=BQB,Number=1,Type=Float,Description=\"Mann-Whitney U test of Base Quality Bias (bigger is better)\">");
bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=MQSB,Number=1,Type=Float,Description=\"Mann-Whitney U test of Mapping Quality vs Strand Bias (bigger is better)\">");
}
bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=FS,Number=1,Type=Float,Description=\"Phred-scaled p-value using Fisher's exact test to detect strand bias\">");
#if CDF_MWU_TESTS
bcf_hdr_append(conf->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(conf->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(conf->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(conf->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(conf->bcf_hdr,"##INFO=<ID=SGB,Number=1,Type=Float,Description=\"Segregation based metric.\">");
bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=MQ0F,Number=1,Type=Float,Description=\"Fraction of MQ0 reads (smaller is better)\">");
bcf_hdr_append(conf->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(conf->bcf_hdr,"##INFO=<ID=QS,Number=R,Type=Float,Description=\"Auxiliary tag used for calling\">");
bcf_hdr_append(conf->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(conf->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(conf->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(conf->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(conf->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(conf->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(conf->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(conf->bcf_hdr,"##FORMAT=<ID=AD,Number=R,Type=Integer,Description=\"Allelic depths (high-quality bases)\">");
if ( conf->fmt_flag&B2B_FMT_ADF )
bcf_hdr_append(conf->bcf_hdr,"##FORMAT=<ID=ADF,Number=R,Type=Integer,Description=\"Allelic depths on the forward strand (high-quality bases)\">");
if ( conf->fmt_flag&B2B_FMT_ADR )
bcf_hdr_append(conf->bcf_hdr,"##FORMAT=<ID=ADR,Number=R,Type=Integer,Description=\"Allelic depths on the reverse strand (high-quality bases)\">");
if ( conf->fmt_flag&B2B_FMT_QS )
bcf_hdr_append(conf->bcf_hdr,"##FORMAT=<ID=QS,Number=R,Type=Integer,Description=\"Phred-score allele quality sum used by `call -mG` and `+trio-dnm`\">");
if ( conf->fmt_flag&B2B_INFO_AD )
bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=AD,Number=R,Type=Integer,Description=\"Total allelic depths (high-quality bases)\">");
if ( conf->fmt_flag&B2B_INFO_ADF )
bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=ADF,Number=R,Type=Integer,Description=\"Total allelic depths on the forward strand (high-quality bases)\">");
if ( conf->fmt_flag&B2B_INFO_SCR )
bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=SCR,Number=1,Type=Integer,Description=\"Number of soft-clipped reads (at high-quality bases)\">");
if ( conf->fmt_flag&B2B_FMT_SCR )
bcf_hdr_append(conf->bcf_hdr,"##FORMAT=<ID=SCR,Number=1,Type=Integer,Description=\"Per-sample number of soft-clipped reads (at high-quality bases)\">");
if ( conf->fmt_flag&B2B_INFO_ADR )
bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=ADR,Number=R,Type=Integer,Description=\"Total allelic depths on the reverse strand (high-quality bases)\">");
if ( conf->gvcf )
gvcf_update_header(conf->gvcf, conf->bcf_hdr);
int nsmpl;
const char **smpl = bam_smpl_get_samples(conf->bsmpl, &nsmpl);
for (i=0; i<nsmpl; i++)
bcf_hdr_add_sample(conf->bcf_hdr, smpl[i]);
if ( bcf_hdr_write(conf->bcf_fp, conf->bcf_hdr)!=0 ) error("[%s] Error: failed to write the header to %s\n",__func__,conf->output_fname?conf->output_fname:"standard output");
conf->bca = bcf_call_init(-1., conf->min_baseQ, conf->max_baseQ,
conf->delta_baseQ);
conf->bcr = (bcf_callret1_t*) calloc(nsmpl, sizeof(bcf_callret1_t));
conf->bca->openQ = conf->openQ, conf->bca->extQ = conf->extQ, conf->bca->tandemQ = conf->tandemQ;
conf->bca->indel_bias = conf->indel_bias;
conf->bca->min_frac = conf->min_frac;
conf->bca->min_support = conf->min_support;
conf->bca->per_sample_flt = conf->flag & MPLP_PER_SAMPLE;
conf->bca->fmt_flag = conf->fmt_flag;
conf->bca->ambig_reads = conf->ambig_reads;
conf->bc.bcf_hdr = conf->bcf_hdr;
conf->bc.n = nsmpl;
conf->bc.PL = (int32_t*) malloc(15 * nsmpl * sizeof(*conf->bc.PL));
conf->bc.QS = (int32_t*) malloc(nsmpl*sizeof(*conf->bc.QS)*B2B_MAX_ALLELES);
for (i=0; i<nsmpl; i++)
conf->bcr[i].QS = conf->bc.QS + i*B2B_MAX_ALLELES;
if (conf->fmt_flag)
{
assert( sizeof(float)==sizeof(int32_t) );
conf->bc.DP4 = (int32_t*) malloc(nsmpl * sizeof(int32_t) * 4);
conf->bc.fmt_arr = (uint8_t*) malloc(nsmpl * sizeof(float)); // all fmt_flag fields, float and int32
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
conf->bc.ADR = (int32_t*) malloc((nsmpl+1)*B2B_MAX_ALLELES*sizeof(int32_t));
conf->bc.ADF = (int32_t*) malloc((nsmpl+1)*B2B_MAX_ALLELES*sizeof(int32_t));
for (i=0; i<nsmpl; i++)
{
conf->bcr[i].ADR = conf->bc.ADR + (i+1)*B2B_MAX_ALLELES;
conf->bcr[i].ADF = conf->bc.ADF + (i+1)*B2B_MAX_ALLELES;
}
}
if ( conf->fmt_flag&(B2B_INFO_SCR|B2B_FMT_SCR) )
conf->bc.SCR = (int32_t*) malloc((nsmpl+1)*sizeof(*conf->bc.SCR));
}
// init mpileup
conf->iter = bam_mplp_init(conf->nfiles, mplp_func, (void**)conf->mplp_data);
if ( conf->flag & MPLP_SMART_OVERLAPS ) bam_mplp_init_overlaps(conf->iter);
fprintf(stderr, "[%s] maximum number of reads per input file set to -d %d\n", __func__, conf->max_depth);
if ( (double)conf->max_depth * conf->nfiles > 1<<20)
fprintf(stderr, "Warning: Potential memory hog, up to %.0fM reads in the pileup!\n", (double)conf->max_depth*conf->nfiles);
if ( (double)conf->max_depth * conf->nfiles / nsmpl < 250 )
fprintf(stderr, "Note: The maximum per-sample depth with -d %d is %.1fx\n", conf->max_depth,(double)conf->max_depth * conf->nfiles / nsmpl);
bam_mplp_set_maxcnt(conf->iter, conf->max_depth);
conf->max_indel_depth = conf->max_indel_depth * nsmpl;
conf->bcf_rec = bcf_init1();
bam_mplp_constructor(conf->iter, pileup_constructor);
// Run mpileup for multiple regions
if ( nregs )
{
int ireg = 0;
do
{
// first region is already positioned
if ( ireg++ > 0 )
{
conf->buf.l = 0;
ksprintf(&conf->buf,"%s:%u-%u",conf->reg_itr->seq,conf->reg_itr->beg+1,conf->reg_itr->end+1);
for (i=0; i<conf->nfiles; i++)
{
hts_itr_destroy(conf->mplp_data[i]->iter);
conf->mplp_data[i]->iter = sam_itr_querys(conf->mplp_data[i]->idx, conf->mplp_data[i]->h, conf->buf.s);
if ( !conf->mplp_data[i]->iter )
{
conf->mplp_data[i]->iter = sam_itr_querys(conf->mplp_data[i]->idx, conf->mplp_data[i]->h, conf->reg_itr->seq);
if ( conf->mplp_data[i]->iter ) {
fprintf(stderr,"[E::%s] fail to parse region '%s'\n", __func__, conf->buf.s);
exit(EXIT_FAILURE);
}
fprintf(stderr,"[E::%s] the sequence \"%s\" not found: %s\n",__func__,conf->reg_itr->seq,conf->files[i]);
exit(EXIT_FAILURE);
}
bam_mplp_reset(conf->iter);
}
}
mpileup_reg(conf,conf->reg_itr->beg,conf->reg_itr->end);
}
while ( regitr_loop(conf->reg_itr) );
}
else
mpileup_reg(conf,0,UINT32_MAX);
flush_bcf_records(conf, conf->bcf_fp, conf->bcf_hdr, NULL);
// clean up
free(conf->bc.tmp.s);
bcf_destroy1(conf->bcf_rec);
if (conf->bcf_fp)
{
if ( hts_close(conf->bcf_fp)!=0 ) error("[%s] Error: close failed .. %s\n", __func__,conf->output_fname);
bcf_hdr_destroy(conf->bcf_hdr);
bcf_call_destroy(conf->bca);
free(conf->bc.PL);
free(conf->bc.DP4);
free(conf->bc.ADR);
free(conf->bc.ADF);
free(conf->bc.SCR);
free(conf->bc.QS);
free(conf->bc.fmt_arr);
free(conf->bcr);
}
if ( conf->gvcf ) gvcf_destroy(conf->gvcf);
free(conf->buf.s);
for (i = 0; i < conf->gplp->n; ++i) free(conf->gplp->plp[i]);
free(conf->gplp->plp); free(conf->gplp->n_plp); free(conf->gplp->m_plp); free(conf->gplp);
bam_mplp_destroy(conf->iter);
bam_hdr_destroy(hdr);
for (i = 0; i < conf->nfiles; ++i) {
if ( nregs>1 ) hts_idx_destroy(conf->mplp_data[i]->idx);
sam_close(conf->mplp_data[i]->fp);
if ( conf->mplp_data[i]->iter) hts_itr_destroy(conf->mplp_data[i]->iter);
free(conf->mplp_data[i]);
}
if ( conf->reg_itr ) regitr_destroy(conf->reg_itr);
free(conf->mplp_data); free(conf->plp); free(conf->n_plp);
free(mp_ref.ref[0]);
free(mp_ref.ref[1]);
return 0;
}
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 = (char**) 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++)