10 void bam_fillmd1_core(bam1_t *b, char *ref, int is_equal, int max_nm)
12 uint8_t *seq = bam1_seq(b);
13 uint32_t *cigar = bam1_cigar(b);
14 bam1_core_t *c = &b->core;
17 uint8_t *old_md, *old_nm;
18 int32_t old_nm_i = -1, nm = 0;
20 str = (kstring_t*)calloc(1, sizeof(kstring_t));
21 for (i = y = 0, x = c->pos; i < c->n_cigar; ++i) {
22 int j, l = cigar[i]>>4, op = cigar[i]&0xf;
23 if (op == BAM_CMATCH) {
24 for (j = 0; j < l; ++j) {
26 int c1 = bam1_seqi(seq, z), c2 = bam_nt16_table[(int)ref[x+j]];
27 if (ref[x+j] == 0) break; // out of boundary
28 if ((c1 == c2 && c1 != 15 && c2 != 15) || c1 == 0) { // a match
29 if (is_equal) seq[z/2] &= (z&1)? 0xf0 : 0x0f;
32 ksprintf(str, "%d", u);
39 } else if (op == BAM_CDEL) {
40 ksprintf(str, "%d", u);
42 for (j = 0; j < l; ++j) {
43 if (ref[x+j] == 0) break;
49 } else if (op == BAM_CINS || op == BAM_CSOFT_CLIP) {
51 if (op == BAM_CINS) nm += l;
52 } else if (op == BAM_CREF_SKIP) {
56 ksprintf(str, "%d", u);
58 if (max_nm > 0 && nm >= max_nm) {
59 for (i = y = 0, x = c->pos; i < c->n_cigar; ++i) {
60 int j, l = cigar[i]>>4, op = cigar[i]&0xf;
61 if (op == BAM_CMATCH) {
62 for (j = 0; j < l; ++j) {
64 int c1 = bam1_seqi(seq, z), c2 = bam_nt16_table[(int)ref[x+j]];
65 if (ref[x+j] == 0) break; // out of boundary
66 if ((c1 == c2 && c1 != 15 && c2 != 15) || c1 == 0) { // a match
67 seq[z/2] |= (z&1)? 0x0f : 0xf0;
73 } else if (op == BAM_CDEL || op == BAM_CREF_SKIP) x += l;
74 else if (op == BAM_CINS || op == BAM_CSOFT_CLIP) y += l;
78 old_nm = bam_aux_get(b, "NM");
79 if (c->flag & BAM_FUNMAP) return;
80 if (old_nm) old_nm_i = bam_aux2i(old_nm);
81 if (!old_nm) bam_aux_append(b, "NM", 'i', 4, (uint8_t*)&nm);
82 else if (nm != old_nm_i) {
83 fprintf(stderr, "[bam_fillmd1] different NM for read '%s': %d -> %d\n", bam1_qname(b), old_nm_i, nm);
84 bam_aux_del(b, old_nm);
85 bam_aux_append(b, "NM", 'i', 4, (uint8_t*)&nm);
88 old_md = bam_aux_get(b, "MD");
89 if (!old_md) bam_aux_append(b, "MD", 'Z', str->l + 1, (uint8_t*)str->s);
92 if (strlen((char*)old_md+1) == str->l) {
93 for (i = 0; i < str->l; ++i)
94 if (toupper(old_md[i+1]) != toupper(str->s[i]))
96 if (i < str->l) is_diff = 1;
99 fprintf(stderr, "[bam_fillmd1] different MD for read '%s': '%s' -> '%s'\n", bam1_qname(b), old_md+1, str->s);
100 bam_aux_del(b, old_md);
101 bam_aux_append(b, "MD", 'Z', str->l + 1, (uint8_t*)str->s);
104 free(str->s); free(str);
107 void bam_fillmd1(bam1_t *b, char *ref, int is_equal)
109 bam_fillmd1_core(b, ref, is_equal, 0);
114 #define MIN_REF_LEN 10
116 int bam_realn(bam1_t *b, const char *ref)
118 int k, l_ref, score, n_cigar;
119 uint32_t *cigar = bam1_cigar(b);
120 uint8_t *s_ref = 0, *s_read = 0, *seq;
122 bam1_core_t *c = &b->core;
123 // set S/W parameters
124 par = ka_param_blast; par.gap_open = 4; par.gap_ext = 1; par.gap_end_open = par.gap_end_ext = 0;
125 // calculate the length of the reference in the alignment
126 for (k = l_ref = 0; k < c->n_cigar; ++k) {
127 if ((cigar[k]&0xf) == BAM_CREF_SKIP) break; // do not do realignment if there is an `N' operation
128 if ((cigar[k]&0xf) == BAM_CMATCH || (cigar[k]&0xf) == BAM_CDEL)
129 l_ref += cigar[k]>>4;
131 if (k != c->n_cigar || l_ref < MIN_REF_LEN) return -1;
132 for (k = 0; k < l_ref; ++k)
133 if (ref[c->pos + k] == 0) return -1; // the read stands out of the reference
135 s_ref = calloc(l_ref, 1);
136 s_read = calloc(c->l_qseq, 1);
137 for (k = 0, seq = bam1_seq(b); k < c->l_qseq; ++k)
138 s_read[k] = bam_nt16_nt4_table[bam1_seqi(seq, k)];
139 for (k = 0; k < l_ref; ++k)
140 s_ref[k] = bam_nt16_nt4_table[(int)bam_nt16_table[(int)ref[c->pos + k]]];
142 cigar = ka_global_core(s_ref, l_ref, s_read, c->l_qseq, &par, &score, &n_cigar);
143 if (score <= 0) { // realignment failed
144 free(cigar); free(s_ref); free(s_read);
147 // copy over the alignment
148 if (4 * (n_cigar - (int)c->n_cigar) + b->data_len > b->m_data) { // enlarge b->data
149 b->m_data = 4 * (n_cigar - (int)c->n_cigar) + b->data_len;
150 kroundup32(b->m_data);
151 b->data = realloc(b->data, b->m_data);
153 if (n_cigar != (int)c->n_cigar) { // move data
154 memmove(b->data + c->l_qname + 4 * n_cigar, bam1_seq(b), b->data_len - c->l_qname - 4 * c->n_cigar);
155 b->data_len += 4 * (n_cigar - (int)c->n_cigar);
157 memcpy(bam1_cigar(b), cigar, n_cigar * 4);
158 c->n_cigar = n_cigar;
159 free(s_ref); free(s_read); free(cigar);
163 int bam_fillmd(int argc, char *argv[])
165 int c, is_equal = 0, tid = -2, ret, len, is_bam_out, is_sam_in, is_uncompressed, max_nm = 0, is_realn;
166 samfile_t *fp, *fpout = 0;
168 char *ref = 0, mode_w[8], mode_r[8];
171 is_bam_out = is_sam_in = is_uncompressed = is_realn = 0;
172 mode_w[0] = mode_r[0] = 0;
173 strcpy(mode_r, "r"); strcpy(mode_w, "w");
174 while ((c = getopt(argc, argv, "reubSn:")) >= 0) {
176 case 'r': is_realn = 1; break;
177 case 'e': is_equal = 1; break;
178 case 'b': is_bam_out = 1; break;
179 case 'u': is_uncompressed = is_bam_out = 1; break;
180 case 'S': is_sam_in = 1; break;
181 case 'n': max_nm = atoi(optarg); break;
182 default: fprintf(stderr, "[bam_fillmd] unrecognized option '-%c'\n", c); return 1;
185 if (!is_sam_in) strcat(mode_r, "b");
186 if (is_bam_out) strcat(mode_w, "b");
187 else strcat(mode_w, "h");
188 if (is_uncompressed) strcat(mode_w, "u");
189 if (optind + 1 >= argc) {
190 fprintf(stderr, "\n");
191 fprintf(stderr, "Usage: samtools fillmd [-eubrS] <aln.bam> <ref.fasta>\n\n");
192 fprintf(stderr, "Options: -e change identical bases to '='\n");
193 fprintf(stderr, " -u uncompressed BAM output (for piping)\n");
194 fprintf(stderr, " -b compressed BAM output\n");
195 fprintf(stderr, " -S the input is SAM with header\n");
196 fprintf(stderr, " -r read-independent local realignment\n\n");
199 fp = samopen(argv[optind], mode_r, 0);
200 if (fp == 0) return 1;
201 if (is_sam_in && (fp->header == 0 || fp->header->n_targets == 0)) {
202 fprintf(stderr, "[bam_fillmd] input SAM does not have header. Abort!\n");
205 fpout = samopen("-", mode_w, fp->header);
206 fai = fai_load(argv[optind+1]);
209 while ((ret = samread(fp, b)) >= 0) {
210 if (b->core.tid >= 0) {
211 if (tid != b->core.tid) {
213 ref = fai_fetch(fai, fp->header->target_name[b->core.tid], &len);
216 fprintf(stderr, "[bam_fillmd] fail to find sequence '%s' in the reference.\n",
217 fp->header->target_name[tid]);
219 if (is_realn) bam_realn(b, ref);
220 if (ref) bam_fillmd1_core(b, ref, is_equal, max_nm);
228 samclose(fp); samclose(fpout);