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1 #include <string.h>
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2 #include <assert.h>
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3 #include <unistd.h>
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4 #include "kstring.h"
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5 #include "sam_header.h"
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6 #include "sam.h"
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7 #include "bam.h"
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8 #include "faidx.h"
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9
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10 bam_header_t *bam_header_dup(const bam_header_t *h0); /*in sam.c*/
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11
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12 static void replace_cigar(bam1_t *b, int n, uint32_t *cigar)
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13 {
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14 if (n != b->core.n_cigar) {
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15 int o = b->core.l_qname + b->core.n_cigar * 4;
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16 if (b->data_len + (n - b->core.n_cigar) * 4 > b->m_data) {
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17 b->m_data = b->data_len + (n - b->core.n_cigar) * 4;
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18 kroundup32(b->m_data);
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19 b->data = (uint8_t*)realloc(b->data, b->m_data);
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20 }
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21 memmove(b->data + b->core.l_qname + n * 4, b->data + o, b->data_len - o);
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22 memcpy(b->data + b->core.l_qname, cigar, n * 4);
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23 b->data_len += (n - b->core.n_cigar) * 4;
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24 b->core.n_cigar = n;
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25 } else memcpy(b->data + b->core.l_qname, cigar, n * 4);
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26 }
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27
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28 #define write_cigar(_c, _n, _m, _v) do { \
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29 if (_n == _m) { \
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30 _m = _m? _m<<1 : 4; \
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31 _c = (uint32_t*)realloc(_c, _m * 4); \
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32 } \
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33 _c[_n++] = (_v); \
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34 } while (0)
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35
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36 static void unpad_seq(bam1_t *b, kstring_t *s)
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37 {
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38 int k, j, i;
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39 int length;
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40 uint32_t *cigar = bam1_cigar(b);
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41 uint8_t *seq = bam1_seq(b);
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42 // b->core.l_qseq gives length of the SEQ entry (including soft clips, S)
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43 // We need the padded length after alignment from the CIGAR (excluding
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44 // soft clips S, but including pads from CIGAR D operations)
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45 length = 0;
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46 for (k = 0; k < b->core.n_cigar; ++k) {
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47 int op, ol;
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48 op= bam_cigar_op(cigar[k]);
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49 ol = bam_cigar_oplen(cigar[k]);
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50 if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF || op == BAM_CDEL)
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51 length += ol;
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52 }
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53 ks_resize(s, length);
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54 for (k = 0, s->l = 0, j = 0; k < b->core.n_cigar; ++k) {
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55 int op, ol;
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56 op = bam_cigar_op(cigar[k]);
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57 ol = bam_cigar_oplen(cigar[k]);
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58 if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) {
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59 for (i = 0; i < ol; ++i, ++j) s->s[s->l++] = bam1_seqi(seq, j);
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60 } else if (op == BAM_CSOFT_CLIP) {
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61 j += ol;
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62 } else if (op == BAM_CHARD_CLIP) {
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63 /* do nothing */
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64 } else if (op == BAM_CDEL) {
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65 for (i = 0; i < ol; ++i) s->s[s->l++] = 0;
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66 } else {
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67 fprintf(stderr, "[depad] ERROR: Didn't expect CIGAR op %c in read %s\n", BAM_CIGAR_STR[op], bam1_qname(b));
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68 assert(-1);
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69 }
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70 }
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71 assert(length == s->l);
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72 }
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73
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74 int load_unpadded_ref(faidx_t *fai, char *ref_name, int ref_len, kstring_t *seq)
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75 {
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76 char base;
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77 char *fai_ref = 0;
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78 int fai_ref_len = 0, k;
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79
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80 fai_ref = fai_fetch(fai, ref_name, &fai_ref_len);
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81 if (fai_ref_len != ref_len) {
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82 fprintf(stderr, "[depad] ERROR: FASTA sequence %s length %i, expected %i\n", ref_name, fai_ref_len, ref_len);
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83 free(fai_ref);
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84 return -1;
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85 }
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86 ks_resize(seq, ref_len);
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87 seq->l = 0;
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88 for (k = 0; k < ref_len; ++k) {
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89 base = fai_ref[k];
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90 if (base == '-' || base == '*') {
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91 // Map gaps to null to match unpad_seq function
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92 seq->s[seq->l++] = 0;
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93 } else {
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94 int i = bam_nt16_table[(int)base];
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95 if (i == 0 || i==16) { // Equals maps to 0, anything unexpected to 16
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96 fprintf(stderr, "[depad] ERROR: Invalid character %c (ASCII %i) in FASTA sequence %s\n", base, (int)base, ref_name);
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97 free(fai_ref);
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98 return -1;
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99 }
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100 seq->s[seq->l++] = i;
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101 }
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102 }
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103 assert(ref_len == seq->l);
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104 free(fai_ref);
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105 return 0;
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106 }
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107
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108 int get_unpadded_len(faidx_t *fai, char *ref_name, int padded_len)
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109 {
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110 char base;
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111 char *fai_ref = 0;
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112 int fai_ref_len = 0, k;
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113 int bases=0, gaps=0;
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114
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115 fai_ref = fai_fetch(fai, ref_name, &fai_ref_len);
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116 if (fai_ref_len != padded_len) {
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117 fprintf(stderr, "[depad] ERROR: FASTA sequence '%s' length %i, expected %i\n", ref_name, fai_ref_len, padded_len);
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118 free(fai_ref);
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119 return -1;
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120 }
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121 for (k = 0; k < padded_len; ++k) {
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122 //fprintf(stderr, "[depad] checking base %i of %i or %i\n", k+1, ref_len, strlen(fai_ref));
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123 base = fai_ref[k];
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124 if (base == '-' || base == '*') {
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125 gaps += 1;
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126 } else {
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127 int i = bam_nt16_table[(int)base];
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128 if (i == 0 || i==16) { // Equals maps to 0, anything unexpected to 16
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129 fprintf(stderr, "[depad] ERROR: Invalid character %c (ASCII %i) in FASTA sequence '%s'\n", base, (int)base, ref_name);
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130 free(fai_ref);
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131 return -1;
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132 }
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133 bases += 1;
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134 }
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135 }
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136 free(fai_ref);
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137 assert (padded_len == bases + gaps);
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138 return bases;
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139 }
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140
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141 inline int * update_posmap(int *posmap, kstring_t ref)
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142 {
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143 int i, k;
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144 posmap = realloc(posmap, ref.m * sizeof(int));
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145 for (i = k = 0; i < ref.l; ++i) {
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146 posmap[i] = k;
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147 if (ref.s[i]) ++k;
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148 }
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149 return posmap;
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150 }
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151
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152 int bam_pad2unpad(samfile_t *in, samfile_t *out, faidx_t *fai)
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153 {
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154 bam_header_t *h = 0;
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155 bam1_t *b = 0;
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156 kstring_t r, q;
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157 int r_tid = -1;
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158 uint32_t *cigar2 = 0;
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159 int ret = 0, n2 = 0, m2 = 0, *posmap = 0;
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160
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161 b = bam_init1();
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162 r.l = r.m = q.l = q.m = 0; r.s = q.s = 0;
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163 int read_ret;
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164 h = in->header;
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165 while ((read_ret = samread(in, b)) >= 0) { // read one alignment from `in'
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166 uint32_t *cigar = bam1_cigar(b);
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167 n2 = 0;
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168 if (b->core.pos == 0 && b->core.tid >= 0 && strcmp(bam1_qname(b), h->target_name[b->core.tid]) == 0) {
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169 // fprintf(stderr, "[depad] Found embedded reference '%s'\n", bam1_qname(b));
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170 r_tid = b->core.tid;
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171 unpad_seq(b, &r);
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172 if (h->target_len[r_tid] != r.l) {
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173 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);
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174 return -1;
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175 }
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176 if (fai) {
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177 // Check the embedded reference matches the FASTA file
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178 if (load_unpadded_ref(fai, h->target_name[b->core.tid], h->target_len[b->core.tid], &q)) {
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179 fprintf(stderr, "[depad] ERROR: Failed to load embedded reference '%s' from FASTA\n", h->target_name[b->core.tid]);
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180 return -1;
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181 }
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182 assert(r.l == q.l);
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183 int i;
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184 for (i = 0; i < r.l; ++i) {
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185 if (r.s[i] != q.s[i]) {
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186 // Show gaps as ASCII 45
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187 fprintf(stderr, "[depad] ERROR: Embedded sequence and reference FASTA don't match for %s base %i, '%c' vs '%c'\n",
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188 h->target_name[b->core.tid], i+1,
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189 r.s[i] ? bam_nt16_rev_table[(int)r.s[i]] : 45,
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190 q.s[i] ? bam_nt16_rev_table[(int)q.s[i]] : 45);
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191 return -1;
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192 }
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193 }
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194 }
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195 write_cigar(cigar2, n2, m2, bam_cigar_gen(b->core.l_qseq, BAM_CMATCH));
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196 replace_cigar(b, n2, cigar2);
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197 posmap = update_posmap(posmap, r);
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198 } else if (b->core.n_cigar > 0) {
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199 int i, k, op;
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200 if (b->core.tid < 0) {
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201 fprintf(stderr, "[depad] ERROR: Read '%s' has CIGAR but no RNAME\n", bam1_qname(b));
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202 return -1;
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203 } else if (b->core.tid == r_tid) {
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204 ; // good case, reference available
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205 //fprintf(stderr, "[depad] Have ref '%s' for read '%s'\n", h->target_name[b->core.tid], bam1_qname(b));
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206 } else if (fai) {
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207 if (load_unpadded_ref(fai, h->target_name[b->core.tid], h->target_len[b->core.tid], &r)) {
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208 fprintf(stderr, "[depad] ERROR: Failed to load '%s' from reference FASTA\n", h->target_name[b->core.tid]);
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209 return -1;
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210 }
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211 posmap = update_posmap(posmap, r);
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212 r_tid = b->core.tid;
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213 // fprintf(stderr, "[depad] Loaded %s from FASTA file\n", h->target_name[b->core.tid]);
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214 } else {
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215 fprintf(stderr, "[depad] ERROR: Missing %s embedded reference sequence (and no FASTA file)\n", h->target_name[b->core.tid]);
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216 return -1;
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217 }
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218 unpad_seq(b, &q);
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219 if (bam_cigar_op(cigar[0]) == BAM_CSOFT_CLIP) {
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220 write_cigar(cigar2, n2, m2, cigar[0]);
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221 } else if (bam_cigar_op(cigar[0]) == BAM_CHARD_CLIP) {
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222 write_cigar(cigar2, n2, m2, cigar[0]);
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223 if (b->core.n_cigar > 2 && bam_cigar_op(cigar[1]) == BAM_CSOFT_CLIP) {
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224 write_cigar(cigar2, n2, m2, cigar[1]);
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225 }
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226 }
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227 /* Determine CIGAR operator for each base in the aligned read */
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228 for (i = 0, k = b->core.pos; i < q.l; ++i, ++k)
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229 q.s[i] = q.s[i]? (r.s[k]? BAM_CMATCH : BAM_CINS) : (r.s[k]? BAM_CDEL : BAM_CPAD);
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230 /* Include any pads if starts with an insert */
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231 if (q.s[0] == BAM_CINS) {
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232 for (k = 0; k+1 < b->core.pos && !r.s[b->core.pos - k - 1]; ++k);
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233 if (k) write_cigar(cigar2, n2, m2, bam_cigar_gen(k, BAM_CPAD));
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234 }
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235 /* Count consecutive CIGAR operators to turn into a CIGAR string */
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236 for (i = k = 1, op = q.s[0]; i < q.l; ++i) {
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237 if (op != q.s[i]) {
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238 write_cigar(cigar2, n2, m2, bam_cigar_gen(k, op));
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239 op = q.s[i]; k = 1;
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240 } else ++k;
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241 }
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242 write_cigar(cigar2, n2, m2, bam_cigar_gen(k, op));
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243 if (bam_cigar_op(cigar[b->core.n_cigar-1]) == BAM_CSOFT_CLIP) {
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244 write_cigar(cigar2, n2, m2, cigar[b->core.n_cigar-1]);
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245 } else if (bam_cigar_op(cigar[b->core.n_cigar-1]) == BAM_CHARD_CLIP) {
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246 if (b->core.n_cigar > 2 && bam_cigar_op(cigar[b->core.n_cigar-2]) == BAM_CSOFT_CLIP) {
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247 write_cigar(cigar2, n2, m2, cigar[b->core.n_cigar-2]);
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248 }
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249 write_cigar(cigar2, n2, m2, cigar[b->core.n_cigar-1]);
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250 }
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251 /* Remove redundant P operators between M/X/=/D operators, e.g. 5M2P10M -> 15M */
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252 int pre_op, post_op;
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253 for (i = 2; i < n2; ++i)
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254 if (bam_cigar_op(cigar2[i-1]) == BAM_CPAD) {
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255 pre_op = bam_cigar_op(cigar2[i-2]);
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256 post_op = bam_cigar_op(cigar2[i]);
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257 /* Note don't need to check for X/= as code above will use M only */
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258 if ((pre_op == BAM_CMATCH || pre_op == BAM_CDEL) && (post_op == BAM_CMATCH || post_op == BAM_CDEL)) {
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259 /* This is a redundant P operator */
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260 cigar2[i-1] = 0; // i.e. 0M
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261 /* If had same operator either side, combine them in post_op */
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262 if (pre_op == post_op) {
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263 /* If CIGAR M, could treat as simple integers since BAM_CMATCH is zero*/
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264 cigar2[i] = bam_cigar_gen(bam_cigar_oplen(cigar2[i-2]) + bam_cigar_oplen(cigar2[i]), post_op);
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265 cigar2[i-2] = 0; // i.e. 0M
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266 }
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267 }
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268 }
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269 /* Remove the zero'd operators (0M) */
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270 for (i = k = 0; i < n2; ++i)
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271 if (cigar2[i]) cigar2[k++] = cigar2[i];
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272 n2 = k;
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273 replace_cigar(b, n2, cigar2);
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274 b->core.pos = posmap[b->core.pos];
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275 if (b->core.mtid < 0 || b->core.mpos < 0) {
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276 /* Nice case, no mate to worry about*/
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277 // fprintf(stderr, "[depad] Read '%s' mate not mapped\n", bam1_qname(b));
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278 /* TODO - Warning if FLAG says mate should be mapped? */
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279 /* Clean up funny input where mate position is given but mate reference is missing: */
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280 b->core.mtid = -1;
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281 b->core.mpos = -1;
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282 } else if (b->core.mtid == b->core.tid) {
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283 /* Nice case, same reference */
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284 // fprintf(stderr, "[depad] Read '%s' mate mapped to same ref\n", bam1_qname(b));
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285 b->core.mpos = posmap[b->core.mpos];
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286 } else {
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287 /* Nasty case, Must load alternative posmap */
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288 // fprintf(stderr, "[depad] Loading reference '%s' temporarily\n", h->target_name[b->core.mtid]);
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289 if (!fai) {
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290 fprintf(stderr, "[depad] ERROR: Needed reference %s sequence for mate (and no FASTA file)\n", h->target_name[b->core.mtid]);
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291 return -1;
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292 }
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293 /* Temporarily load the other reference sequence */
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294 if (load_unpadded_ref(fai, h->target_name[b->core.mtid], h->target_len[b->core.mtid], &r)) {
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295 fprintf(stderr, "[depad] ERROR: Failed to load '%s' from reference FASTA\n", h->target_name[b->core.mtid]);
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296 return -1;
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297 }
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298 posmap = update_posmap(posmap, r);
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299 b->core.mpos = posmap[b->core.mpos];
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300 /* Restore the reference and posmap*/
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301 if (load_unpadded_ref(fai, h->target_name[b->core.tid], h->target_len[b->core.tid], &r)) {
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302 fprintf(stderr, "[depad] ERROR: Failed to load '%s' from reference FASTA\n", h->target_name[b->core.tid]);
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303 return -1;
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304 }
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305 posmap = update_posmap(posmap, r);
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306 }
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307 }
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308 samwrite(out, b);
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309 }
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310 if (read_ret < -1) {
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311 fprintf(stderr, "[depad] truncated file.\n");
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312 ret = 1;
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313 }
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314 free(r.s); free(q.s); free(posmap);
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315 bam_destroy1(b);
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316 return ret;
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317 }
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318
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319 bam_header_t * fix_header(bam_header_t *old, faidx_t *fai)
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320 {
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321 int i = 0, unpadded_len = 0;
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322 bam_header_t *header = 0 ;
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323
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324 header = bam_header_dup(old);
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325 for (i = 0; i < old->n_targets; ++i) {
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326 unpadded_len = get_unpadded_len(fai, old->target_name[i], old->target_len[i]);
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327 if (unpadded_len < 0) {
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328 fprintf(stderr, "[depad] ERROR getting unpadded length of '%s', padded length %i\n", old->target_name[i], old->target_len[i]);
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329 } else {
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330 header->target_len[i] = unpadded_len;
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331 //fprintf(stderr, "[depad] Recalculating '%s' length %i -> %i\n", old->target_name[i], old->target_len[i], header->target_len[i]);
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332 }
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333 }
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334 /* Duplicating the header allocated new buffer for header string */
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335 /* After modifying the @SQ lines it will only get smaller, since */
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336 /* the LN entries will be the same or shorter, and we'll remove */
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337 /* any MD entries (MD5 checksums). */
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338 assert(strlen(old->text) == strlen(header->text));
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339 assert (0==strcmp(old->text, header->text));
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340 const char *text;
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341 text = old->text;
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342 header->text[0] = '\0'; /* Resuse the allocated buffer */
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343 char * newtext = header->text;
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344 char * end=NULL;
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345 while (text[0]=='@') {
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346 end = strchr(text, '\n');
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347 assert(end != 0);
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348 if (text[1]=='S' && text[2]=='Q' && text[3]=='\t') {
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349 /* TODO - edit the @SQ line here to remove MD and fix LN. */
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350 /* For now just remove the @SQ line, and samtools will */
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351 /* automatically generate a minimal replacement with LN. */
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352 /* However, that discards any other tags like AS, SP, UR. */
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353 //fprintf(stderr, "[depad] Removing @SQ line\n");
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354 } else {
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355 /* Copy this line to the new header */
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356 strncat(newtext, text, end - text + 1);
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357 }
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358 text = end + 1;
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359 }
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360 assert (text[0]=='\0');
|
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361 /* Check we didn't overflow the buffer */
|
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362 assert (strlen(header->text) <= strlen(old->text));
|
|
363 if (strlen(header->text) < header->l_text) {
|
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364 //fprintf(stderr, "[depad] Reallocating header buffer\n");
|
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365 assert (newtext == header->text);
|
|
366 newtext = malloc(strlen(header->text) + 1);
|
|
367 strcpy(newtext, header->text);
|
|
368 free(header->text);
|
|
369 header->text = newtext;
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|
370 header->l_text = strlen(newtext);
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|
371 }
|
|
372 //fprintf(stderr, "[depad] Here is the new header (pending @SQ lines),\n\n%s\n(end)\n", header->text);
|
|
373 return header;
|
|
374 }
|
|
375
|
|
376 static int usage(int is_long_help);
|
|
377
|
|
378 int main_pad2unpad(int argc, char *argv[])
|
|
379 {
|
|
380 samfile_t *in = 0, *out = 0;
|
|
381 bam_header_t *h = 0;
|
|
382 faidx_t *fai = 0;
|
|
383 int c, is_bamin = 1, compress_level = -1, is_bamout = 1, is_long_help = 0;
|
|
384 char in_mode[5], out_mode[5], *fn_out = 0, *fn_list = 0, *fn_ref = 0;
|
|
385 int ret=0;
|
|
386
|
|
387 /* parse command-line options */
|
|
388 strcpy(in_mode, "r"); strcpy(out_mode, "w");
|
|
389 while ((c = getopt(argc, argv, "Sso:u1T:?")) >= 0) {
|
|
390 switch (c) {
|
|
391 case 'S': is_bamin = 0; break;
|
|
392 case 's': assert(compress_level == -1); is_bamout = 0; break;
|
|
393 case 'o': fn_out = strdup(optarg); break;
|
|
394 case 'u': assert(is_bamout == 1); compress_level = 0; break;
|
|
395 case '1': assert(is_bamout == 1); compress_level = 1; break;
|
|
396 case 'T': fn_ref = strdup(optarg); break;
|
|
397 case '?': is_long_help = 1; break;
|
|
398 default: return usage(is_long_help);
|
|
399 }
|
|
400 }
|
|
401 if (argc == optind) return usage(is_long_help);
|
|
402
|
|
403 if (is_bamin) strcat(in_mode, "b");
|
|
404 if (is_bamout) strcat(out_mode, "b");
|
|
405 strcat(out_mode, "h");
|
|
406 if (compress_level >= 0) {
|
|
407 char tmp[2];
|
|
408 tmp[0] = compress_level + '0'; tmp[1] = '\0';
|
|
409 strcat(out_mode, tmp);
|
|
410 }
|
|
411
|
|
412 // Load FASTA reference (also needed for SAM -> BAM if missing header)
|
|
413 if (fn_ref) {
|
|
414 fn_list = samfaipath(fn_ref);
|
|
415 fai = fai_load(fn_ref);
|
|
416 }
|
|
417 // open file handlers
|
|
418 if ((in = samopen(argv[optind], in_mode, fn_list)) == 0) {
|
|
419 fprintf(stderr, "[depad] failed to open \"%s\" for reading.\n", argv[optind]);
|
|
420 ret = 1;
|
|
421 goto depad_end;
|
|
422 }
|
|
423 if (in->header == 0) {
|
|
424 fprintf(stderr, "[depad] failed to read the header from \"%s\".\n", argv[optind]);
|
|
425 ret = 1;
|
|
426 goto depad_end;
|
|
427 }
|
|
428 if (in->header->text == 0 || in->header->l_text == 0) {
|
|
429 fprintf(stderr, "[depad] Warning - failed to read any header text from \"%s\".\n", argv[optind]);
|
|
430 assert (0 == in->header->l_text);
|
|
431 assert (0 == in->header->text);
|
|
432 }
|
|
433 if (fn_ref) {
|
|
434 h = fix_header(in->header, fai);
|
|
435 } else {
|
|
436 fprintf(stderr, "[depad] Warning - reference lengths will not be corrected without FASTA reference\n");
|
|
437 h = in->header;
|
|
438 }
|
|
439 if ((out = samopen(fn_out? fn_out : "-", out_mode, h)) == 0) {
|
|
440 fprintf(stderr, "[depad] failed to open \"%s\" for writing.\n", fn_out? fn_out : "standard output");
|
|
441 ret = 1;
|
|
442 goto depad_end;
|
|
443 }
|
|
444
|
|
445 // Do the depad
|
|
446 ret = bam_pad2unpad(in, out, fai);
|
|
447
|
|
448 depad_end:
|
|
449 // close files, free and return
|
|
450 if (fai) fai_destroy(fai);
|
|
451 if (h != in->header) bam_header_destroy(h);
|
|
452 samclose(in);
|
|
453 samclose(out);
|
|
454 free(fn_list); free(fn_out);
|
|
455 return ret;
|
|
456 }
|
|
457
|
|
458 static int usage(int is_long_help)
|
|
459 {
|
|
460 fprintf(stderr, "\n");
|
|
461 fprintf(stderr, "Usage: samtools depad <in.bam>\n\n");
|
|
462 fprintf(stderr, "Options: -s output is SAM (default is BAM)\n");
|
|
463 fprintf(stderr, " -S input is SAM (default is BAM)\n");
|
|
464 fprintf(stderr, " -u uncompressed BAM output (can't use with -s)\n");
|
|
465 fprintf(stderr, " -1 fast compression BAM output (can't use with -s)\n");
|
|
466 fprintf(stderr, " -T FILE reference sequence file [null]\n");
|
|
467 fprintf(stderr, " -o FILE output file name [stdout]\n");
|
|
468 fprintf(stderr, " -? longer help\n");
|
|
469 fprintf(stderr, "\n");
|
|
470 if (is_long_help)
|
|
471 fprintf(stderr, "Notes:\n\
|
|
472 \n\
|
|
473 1. Requires embedded reference sequences (before the reads for that reference),\n\
|
|
474 with the future aim to also support a FASTA padded reference sequence file.\n\
|
|
475 \n\
|
|
476 2. The input padded alignment read's CIGAR strings must not use P or I operators.\n\
|
|
477 \n");
|
|
478 return 1;
|
|
479 }
|