#include <string.h>
#include <assert.h>
#include <unistd.h>
#include "kstring.h"
#include "sam_header.h"
#include "sam.h"
#include "bam.h"
#include "faidx.h"
bam_header_t *bam_header_dup(const bam_header_t *h0); /*in sam.c*/
static void replace_cigar(bam1_t *b, int n, uint32_t *cigar)
{
if (n != b->core.n_cigar) {
int o = b->core.l_qname + b->core.n_cigar * 4;
if (b->data_len + (n - b->core.n_cigar) * 4 > b->m_data) {
b->m_data = b->data_len + (n - b->core.n_cigar) * 4;
kroundup32(b->m_data);
b->data = (uint8_t*)realloc(b->data, b->m_data);
}
memmove(b->data + b->core.l_qname + n * 4, b->data + o, b->data_len - o);
memcpy(b->data + b->core.l_qname, cigar, n * 4);
b->data_len += (n - b->core.n_cigar) * 4;
b->core.n_cigar = n;
} else memcpy(b->data + b->core.l_qname, cigar, n * 4);
}
#define write_cigar(_c, _n, _m, _v) do { \
if (_n == _m) { \
_m = _m? _m<<1 : 4; \
_c = (uint32_t*)realloc(_c, _m * 4); \
} \
_c[_n++] = (_v); \
} while (0)
static void unpad_seq(bam1_t *b, kstring_t *s)
{
int k, j, i;
int length;
uint32_t *cigar = bam1_cigar(b);
uint8_t *seq = bam1_seq(b);
// b->core.l_qseq gives length of the SEQ entry (including soft clips, S)
// We need the padded length after alignment from the CIGAR (excluding
// soft clips S, but including pads from CIGAR D operations)
length = 0;
for (k = 0; k < b->core.n_cigar; ++k) {
int op, ol;
op= bam_cigar_op(cigar[k]);
ol = bam_cigar_oplen(cigar[k]);
if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF || op == BAM_CDEL)
length += ol;
}
ks_resize(s, length);
for (k = 0, s->l = 0, j = 0; k < b->core.n_cigar; ++k) {
int op, ol;
op = bam_cigar_op(cigar[k]);
ol = bam_cigar_oplen(cigar[k]);
if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) {
for (i = 0; i < ol; ++i, ++j) s->s[s->l++] = bam1_seqi(seq, j);
} else if (op == BAM_CSOFT_CLIP) {
j += ol;
} else if (op == BAM_CHARD_CLIP) {
/* do nothing */
} else if (op == BAM_CDEL) {
for (i = 0; i < ol; ++i) s->s[s->l++] = 0;
} else {
fprintf(stderr, "[depad] ERROR: Didn't expect CIGAR op %c in read %s\n", BAM_CIGAR_STR[op], bam1_qname(b));
assert(-1);
}
}
assert(length == s->l);
}
int load_unpadded_ref(faidx_t *fai, char *ref_name, int ref_len, kstring_t *seq)
{
char base;
char *fai_ref = 0;
int fai_ref_len = 0, k;
fai_ref = fai_fetch(fai, ref_name, &fai_ref_len);
if (fai_ref_len != ref_len) {
fprintf(stderr, "[depad] ERROR: FASTA sequence %s length %i, expected %i\n", ref_name, fai_ref_len, ref_len);
free(fai_ref);
return -1;
}
ks_resize(seq, ref_len);
seq->l = 0;
for (k = 0; k < ref_len; ++k) {
base = fai_ref[k];
if (base == '-' || base == '*') {
// Map gaps to null to match unpad_seq function
seq->s[seq->l++] = 0;
} else {
int i = bam_nt16_table[(int)base];
if (i == 0 || i==16) { // Equals maps to 0, anything unexpected to 16
fprintf(stderr, "[depad] ERROR: Invalid character %c (ASCII %i) in FASTA sequence %s\n", base, (int)base, ref_name);
free(fai_ref);
return -1;
}
seq->s[seq->l++] = i;
}
}
assert(ref_len == seq->l);
free(fai_ref);
return 0;
}
int get_unpadded_len(faidx_t *fai, char *ref_name, int padded_len)
{
char base;
char *fai_ref = 0;
int fai_ref_len = 0, k;
int bases=0, gaps=0;
fai_ref = fai_fetch(fai, ref_name, &fai_ref_len);
if (fai_ref_len != padded_len) {
fprintf(stderr, "[depad] ERROR: FASTA sequence '%s' length %i, expected %i\n", ref_name, fai_ref_len, padded_len);
free(fai_ref);
return -1;
}
for (k = 0; k < padded_len; ++k) {
//fprintf(stderr, "[depad] checking base %i of %i or %i\n", k+1, ref_len, strlen(fai_ref));
base = fai_ref[k];
if (base == '-' || base == '*') {
gaps += 1;
} else {
int i = bam_nt16_table[(int)base];
if (i == 0 || i==16) { // Equals maps to 0, anything unexpected to 16
fprintf(stderr, "[depad] ERROR: Invalid character %c (ASCII %i) in FASTA sequence '%s'\n", base, (int)base, ref_name);
free(fai_ref);
return -1;
}
bases += 1;
}
}
free(fai_ref);
assert (padded_len == bases + gaps);
return bases;
}
inline int * update_posmap(int *posmap, kstring_t ref)
{
int i, k;
posmap = realloc(posmap, ref.m * sizeof(int));
for (i = k = 0; i < ref.l; ++i) {
posmap[i] = k;
if (ref.s[i]) ++k;
}
return posmap;
}
int bam_pad2unpad(samfile_t *in, samfile_t *out, faidx_t *fai)
{
bam_header_t *h = 0;
bam1_t *b = 0;
kstring_t r, q;
int r_tid = -1;
uint32_t *cigar2 = 0;
int ret = 0, n2 = 0, m2 = 0, *posmap = 0;
b = bam_init1();
r.l = r.m = q.l = q.m = 0; r.s = q.s = 0;
int read_ret;
h = in->header;
while ((read_ret = samread(in, b)) >= 0) { // read one alignment from `in'
uint32_t *cigar = bam1_cigar(b);
n2 = 0;
if (b->core.pos == 0 && b->core.tid >= 0 && strcmp(bam1_qname(b), h->target_name[b->core.tid]) == 0) {
// fprintf(stderr, "[depad] Found embedded reference '%s'\n", bam1_qname(b));
r_tid = b->core.tid;
unpad_seq(b, &r);
if (h->target_len[r_tid] != r.l) {
fprintf(stderr, "[depad] ERROR: (Padded) length of '%s' is %d in BAM header, but %ld in embedded reference\n", bam1_qname(b), h->target_len[r_tid], r.l);
return -1;
}
if (fai) {
// Check the embedded reference matches the FASTA file
if (load_unpadded_ref(fai, h->target_name[b->core.tid], h->target_len[b->core.tid], &q)) {
fprintf(stderr, "[depad] ERROR: Failed to load embedded reference '%s' from FASTA\n", h->target_name[b->core.tid]);
return -1;
}
assert(r.l == q.l);
int i;
for (i = 0; i < r.l; ++i) {
if (r.s[i] != q.s[i]) {
// Show gaps as ASCII 45
fprintf(stderr, "[depad] ERROR: Embedded sequence and reference FASTA don't match for %s base %i, '%c' vs '%c'\n",
h->target_name[b->core.tid], i+1,
r.s[i] ? bam_nt16_rev_table[(int)r.s[i]] : 45,
q.s[i] ? bam_nt16_rev_table[(int)q.s[i]] : 45);
return -1;
}
}
}
write_cigar(cigar2, n2, m2, bam_cigar_gen(b->core.l_qseq, BAM_CMATCH));
replace_cigar(b, n2, cigar2);
posmap = update_posmap(posmap, r);
} else if (b->core.n_cigar > 0) {
int i, k, op;
if (b->core.tid < 0) {
fprintf(stderr, "[depad] ERROR: Read '%s' has CIGAR but no RNAME\n", bam1_qname(b));
return -1;
} else if (b->core.tid == r_tid) {
; // good case, reference available
//fprintf(stderr, "[depad] Have ref '%s' for read '%s'\n", h->target_name[b->core.tid], bam1_qname(b));
} else if (fai) {
if (load_unpadded_ref(fai, h->target_name[b->core.tid], h->target_len[b->core.tid], &r)) {
fprintf(stderr, "[depad] ERROR: Failed to load '%s' from reference FASTA\n", h->target_name[b->core.tid]);
return -1;
}
posmap = update_posmap(posmap, r);
r_tid = b->core.tid;
// fprintf(stderr, "[depad] Loaded %s from FASTA file\n", h->target_name[b->core.tid]);
} else {
fprintf(stderr, "[depad] ERROR: Missing %s embedded reference sequence (and no FASTA file)\n", h->target_name[b->core.tid]);
return -1;
}
unpad_seq(b, &q);
if (bam_cigar_op(cigar[0]) == BAM_CSOFT_CLIP) {
write_cigar(cigar2, n2, m2, cigar[0]);
} else if (bam_cigar_op(cigar[0]) == BAM_CHARD_CLIP) {
write_cigar(cigar2, n2, m2, cigar[0]);
if (b->core.n_cigar > 2 && bam_cigar_op(cigar[1]) == BAM_CSOFT_CLIP) {
write_cigar(cigar2, n2, m2, cigar[1]);
}
}
/* Determine CIGAR operator for each base in the aligned read */
for (i = 0, k = b->core.pos; i < q.l; ++i, ++k)
q.s[i] = q.s[i]? (r.s[k]? BAM_CMATCH : BAM_CINS) : (r.s[k]? BAM_CDEL : BAM_CPAD);
/* Include any pads if starts with an insert */
if (q.s[0] == BAM_CINS) {
for (k = 0; k+1 < b->core.pos && !r.s[b->core.pos - k - 1]; ++k);
if (k) write_cigar(cigar2, n2, m2, bam_cigar_gen(k, BAM_CPAD));
}
/* Count consecutive CIGAR operators to turn into a CIGAR string */
for (i = k = 1, op = q.s[0]; i < q.l; ++i) {
if (op != q.s[i]) {
write_cigar(cigar2, n2, m2, bam_cigar_gen(k, op));
op = q.s[i]; k = 1;
} else ++k;
}
write_cigar(cigar2, n2, m2, bam_cigar_gen(k, op));
if (bam_cigar_op(cigar[b->core.n_cigar-1]) == BAM_CSOFT_CLIP) {
write_cigar(cigar2, n2, m2, cigar[b->core.n_cigar-1]);
} else if (bam_cigar_op(cigar[b->core.n_cigar-1]) == BAM_CHARD_CLIP) {
if (b->core.n_cigar > 2 && bam_cigar_op(cigar[b->core.n_cigar-2]) == BAM_CSOFT_CLIP) {
write_cigar(cigar2, n2, m2, cigar[b->core.n_cigar-2]);
}
write_cigar(cigar2, n2, m2, cigar[b->core.n_cigar-1]);
}
/* Remove redundant P operators between M/X/=/D operators, e.g. 5M2P10M -> 15M */
int pre_op, post_op;
for (i = 2; i < n2; ++i)
if (bam_cigar_op(cigar2[i-1]) == BAM_CPAD) {
pre_op = bam_cigar_op(cigar2[i-2]);
post_op = bam_cigar_op(cigar2[i]);
/* Note don't need to check for X/= as code above will use M only */
if ((pre_op == BAM_CMATCH || pre_op == BAM_CDEL) && (post_op == BAM_CMATCH || post_op == BAM_CDEL)) {
/* This is a redundant P operator */
cigar2[i-1] = 0; // i.e. 0M
/* If had same operator either side, combine them in post_op */
if (pre_op == post_op) {
/* If CIGAR M, could treat as simple integers since BAM_CMATCH is zero*/
cigar2[i] = bam_cigar_gen(bam_cigar_oplen(cigar2[i-2]) + bam_cigar_oplen(cigar2[i]), post_op);
cigar2[i-2] = 0; // i.e. 0M
}
}
}
/* Remove the zero'd operators (0M) */
for (i = k = 0; i < n2; ++i)
if (cigar2[i]) cigar2[k++] = cigar2[i];
n2 = k;
replace_cigar(b, n2, cigar2);
b->core.pos = posmap[b->core.pos];
if (b->core.mtid < 0 || b->core.mpos < 0) {
/* Nice case, no mate to worry about*/
// fprintf(stderr, "[depad] Read '%s' mate not mapped\n", bam1_qname(b));
/* TODO - Warning if FLAG says mate should be mapped? */
/* Clean up funny input where mate position is given but mate reference is missing: */
b->core.mtid = -1;
b->core.mpos = -1;
} else if (b->core.mtid == b->core.tid) {
/* Nice case, same reference */
// fprintf(stderr, "[depad] Read '%s' mate mapped to same ref\n", bam1_qname(b));
b->core.mpos = posmap[b->core.mpos];
} else {
/* Nasty case, Must load alternative posmap */
// fprintf(stderr, "[depad] Loading reference '%s' temporarily\n", h->target_name[b->core.mtid]);
if (!fai) {
fprintf(stderr, "[depad] ERROR: Needed reference %s sequence for mate (and no FASTA file)\n", h->target_name[b->core.mtid]);
return -1;
}
/* Temporarily load the other reference sequence */
if (load_unpadded_ref(fai, h->target_name[b->core.mtid], h->target_len[b->core.mtid], &r)) {
fprintf(stderr, "[depad] ERROR: Failed to load '%s' from reference FASTA\n", h->target_name[b->core.mtid]);
return -1;
}
posmap = update_posmap(posmap, r);
b->core.mpos = posmap[b->core.mpos];
/* Restore the reference and posmap*/
if (load_unpadded_ref(fai, h->target_name[b->core.tid], h->target_len[b->core.tid], &r)) {
fprintf(stderr, "[depad] ERROR: Failed to load '%s' from reference FASTA\n", h->target_name[b->core.tid]);
return -1;
}
posmap = update_posmap(posmap, r);
}
}
samwrite(out, b);
}
if (read_ret < -1) {
fprintf(stderr, "[depad] truncated file.\n");
ret = 1;
}
free(r.s); free(q.s); free(posmap);
bam_destroy1(b);
return ret;
}
bam_header_t * fix_header(bam_header_t *old, faidx_t *fai)
{
int i = 0, unpadded_len = 0;
bam_header_t *header = 0 ;
header = bam_header_dup(old);
for (i = 0; i < old->n_targets; ++i) {
unpadded_len = get_unpadded_len(fai, old->target_name[i], old->target_len[i]);
if (unpadded_len < 0) {
fprintf(stderr, "[depad] ERROR getting unpadded length of '%s', padded length %i\n", old->target_name[i], old->target_len[i]);
} else {
header->target_len[i] = unpadded_len;
//fprintf(stderr, "[depad] Recalculating '%s' length %i -> %i\n", old->target_name[i], old->target_len[i], header->target_len[i]);
}
}
/* Duplicating the header allocated new buffer for header string */
/* After modifying the @SQ lines it will only get smaller, since */
/* the LN entries will be the same or shorter, and we'll remove */
/* any MD entries (MD5 checksums). */
assert(strlen(old->text) == strlen(header->text));
assert (0==strcmp(old->text, header->text));
const char *text;
text = old->text;
header->text[0] = '\0'; /* Resuse the allocated buffer */
char * newtext = header->text;
char * end=NULL;
while (text[0]=='@') {
end = strchr(text, '\n');
assert(end != 0);
if (text[1]=='S' && text[2]=='Q' && text[3]=='\t') {
/* TODO - edit the @SQ line here to remove MD and fix LN. */
/* For now just remove the @SQ line, and samtools will */
/* automatically generate a minimal replacement with LN. */
/* However, that discards any other tags like AS, SP, UR. */
//fprintf(stderr, "[depad] Removing @SQ line\n");
} else {
/* Copy this line to the new header */
strncat(newtext, text, end - text + 1);
}
text = end + 1;
}
assert (text[0]=='\0');
/* Check we didn't overflow the buffer */
assert (strlen(header->text) <= strlen(old->text));
if (strlen(header->text) < header->l_text) {
//fprintf(stderr, "[depad] Reallocating header buffer\n");
assert (newtext == header->text);
newtext = malloc(strlen(header->text) + 1);
strcpy(newtext, header->text);
free(header->text);
header->text = newtext;
header->l_text = strlen(newtext);
}
//fprintf(stderr, "[depad] Here is the new header (pending @SQ lines),\n\n%s\n(end)\n", header->text);
return header;
}
static int usage(int is_long_help);
int main_pad2unpad(int argc, char *argv[])
{
samfile_t *in = 0, *out = 0;
bam_header_t *h = 0;
faidx_t *fai = 0;
int c, is_bamin = 1, compress_level = -1, is_bamout = 1, is_long_help = 0;
char in_mode[5], out_mode[5], *fn_out = 0, *fn_list = 0, *fn_ref = 0;
int ret=0;
/* parse command-line options */
strcpy(in_mode, "r"); strcpy(out_mode, "w");
while ((c = getopt(argc, argv, "Sso:u1T:?")) >= 0) {
switch (c) {
case 'S': is_bamin = 0; break;
case 's': assert(compress_level == -1); is_bamout = 0; break;
case 'o': fn_out = strdup(optarg); break;
case 'u': assert(is_bamout == 1); compress_level = 0; break;
case '1': assert(is_bamout == 1); compress_level = 1; break;
case 'T': fn_ref = strdup(optarg); break;
case '?': is_long_help = 1; break;
default: return usage(is_long_help);
}
}
if (argc == optind) return usage(is_long_help);
if (is_bamin) strcat(in_mode, "b");
if (is_bamout) strcat(out_mode, "b");
strcat(out_mode, "h");
if (compress_level >= 0) {
char tmp[2];
tmp[0] = compress_level + '0'; tmp[1] = '\0';
strcat(out_mode, tmp);
}
// Load FASTA reference (also needed for SAM -> BAM if missing header)
if (fn_ref) {
fn_list = samfaipath(fn_ref);
fai = fai_load(fn_ref);
}
// open file handlers
if ((in = samopen(argv[optind], in_mode, fn_list)) == 0) {
fprintf(stderr, "[depad] failed to open \"%s\" for reading.\n", argv[optind]);
ret = 1;
goto depad_end;
}
if (in->header == 0) {
fprintf(stderr, "[depad] failed to read the header from \"%s\".\n", argv[optind]);
ret = 1;
goto depad_end;
}
if (in->header->text == 0 || in->header->l_text == 0) {
fprintf(stderr, "[depad] Warning - failed to read any header text from \"%s\".\n", argv[optind]);
assert (0 == in->header->l_text);
assert (0 == in->header->text);
}
if (fn_ref) {
h = fix_header(in->header, fai);
} else {
fprintf(stderr, "[depad] Warning - reference lengths will not be corrected without FASTA reference\n");
h = in->header;
}
if ((out = samopen(fn_out? fn_out : "-", out_mode, h)) == 0) {
fprintf(stderr, "[depad] failed to open \"%s\" for writing.\n", fn_out? fn_out : "standard output");
ret = 1;
goto depad_end;
}
// Do the depad
ret = bam_pad2unpad(in, out, fai);
depad_end:
// close files, free and return
if (fai) fai_destroy(fai);
if (h != in->header) bam_header_destroy(h);
samclose(in);
samclose(out);
free(fn_list); free(fn_out);
return ret;
}
static int usage(int is_long_help)
{
fprintf(stderr, "\n");
fprintf(stderr, "Usage: samtools depad <in.bam>\n\n");
fprintf(stderr, "Options: -s output is SAM (default is BAM)\n");
fprintf(stderr, " -S input is SAM (default is BAM)\n");
fprintf(stderr, " -u uncompressed BAM output (can't use with -s)\n");
fprintf(stderr, " -1 fast compression BAM output (can't use with -s)\n");
fprintf(stderr, " -T FILE reference sequence file [null]\n");
fprintf(stderr, " -o FILE output file name [stdout]\n");
fprintf(stderr, " -? longer help\n");
fprintf(stderr, "\n");
if (is_long_help)
fprintf(stderr, "Notes:\n\
\n\
1. Requires embedded reference sequences (before the reads for that reference),\n\
with the future aim to also support a FASTA padded reference sequence file.\n\
\n\
2. The input padded alignment read's CIGAR strings must not use P or I operators.\n\
\n");
return 1;
}
