#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)
{
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);
- ks_resize(s, b->core.l_qseq);
+ // 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]);
j += ol;
} else if (op == BAM_CHARD_CLIP) {
/* do nothing */
- } else if (op == BAM_CDEL || op == BAM_CPAD) {
+ } 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 bam_pad2unpad(bamFile in, bamFile out)
+int load_unpadded_ref(faidx_t *fai, char *ref_name, int ref_len, kstring_t *seq)
{
- bam_header_t *h;
- bam1_t *b;
+ 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 n2 = 0, m2 = 0, *posmap = 0;
+ int ret = 0, n2 = 0, m2 = 0, *posmap = 0;
- h = bam_header_read(in);
- /* TODO - The reference sequence lengths in the BAM + SAM headers should be updated */
- bam_header_write(out, h);
b = bam_init1();
r.l = r.m = q.l = q.m = 0; r.s = q.s = 0;
- while (bam_read1(in, b) >= 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) {
- int i, k;
- /*
- fprintf(stderr, "[depad] Found embedded reference %s\n", bam1_qname(b));
- */
+ // 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 %i in BAM header, but %ld in embedded reference\n", bam1_qname(b), 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 = realloc(posmap, r.m * sizeof(int));
- for (i = k = 0; i < r.l; ++i) {
- posmap[i] = k;
- if (r.s[i]) ++k;
- }
+ 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));
+ fprintf(stderr, "[depad] ERROR: Read '%s' has CIGAR but no RNAME\n", bam1_qname(b));
return -1;
- } else if (b->core.tid != r_tid) {
- fprintf(stderr, "[depad] ERROR: Missing %s embedded reference sequence\n", h->target_name[b->core.tid]);
+ } else if (b->core.tid == r_tid) {
+ ; // good case, reference available
+ } 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]);
- if (bam_cigar_op(cigar[0]) == BAM_CHARD_CLIP) {
+ 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]);
}
}
- /* Include any pads if starts with an insert */
- 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));
/* 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]) {
} 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]);
- if (bam_cigar_op(cigar[b->core.n_cigar-1]) == BAM_CHARD_CLIP) {
+ 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 operators, e.g. 5M2P10M -> 15M */
+ /* 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]) == BAM_CMATCH && bam_cigar_op(cigar2[i-1]) == BAM_CPAD && bam_cigar_op(cigar2[i-2]) == BAM_CMATCH)
- cigar2[i] += cigar2[i-2], cigar2[i-2] = cigar2[i-1] = 0;
+ 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];
}
- bam_write1(out, b);
+ 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);
- bam_header_destroy(h);
- return 0;
+ 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 = calloc(strlen(header->text) + 1, 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[])
{
- bamFile in, out;
- int result=0;
- if (argc == 1) {
- fprintf(stderr, "Usage: samtools depad <in.bam>\n");
- return 1;
- }
- in = strcmp(argv[1], "-")? bam_open(argv[1], "r") : bam_dopen(fileno(stdin), "r");
- out = bam_dopen(fileno(stdout), "w");
- result = bam_pad2unpad(in, out);
- bam_close(in); bam_close(out);
- return result;
+ 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
+ free(fn_list); free(fn_out);
+ if (h != in->header) bam_header_destroy(h);
+ samclose(in);
+ samclose(out);
+ fai_destroy(fai);
+ 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;
}
projects / samtools.git / commitdiff