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1 #include <unistd.h>
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2 #include <assert.h>
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3 #include <string.h>
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4 #include <ctype.h>
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5 #include <math.h>
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6 #include "faidx.h"
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7 #include "sam.h"
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8 #include "kstring.h"
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9 #include "kaln.h"
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10 #include "kprobaln.h"
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11
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12 #define USE_EQUAL 1
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13 #define DROP_TAG 2
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14 #define BIN_QUAL 4
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15 #define UPDATE_NM 8
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16 #define UPDATE_MD 16
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17 #define HASH_QNM 32
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18
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19 char bam_nt16_nt4_table[] = { 4, 0, 1, 4, 2, 4, 4, 4, 3, 4, 4, 4, 4, 4, 4, 4 };
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20
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21 int bam_aux_drop_other(bam1_t *b, uint8_t *s);
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22
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23 void bam_fillmd1_core(bam1_t *b, char *ref, int flag, int max_nm)
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24 {
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25 uint8_t *seq = bam1_seq(b);
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26 uint32_t *cigar = bam1_cigar(b);
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27 bam1_core_t *c = &b->core;
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28 int i, x, y, u = 0;
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29 kstring_t *str;
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30 int32_t old_nm_i = -1, nm = 0;
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31
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32 str = (kstring_t*)calloc(1, sizeof(kstring_t));
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33 for (i = y = 0, x = c->pos; i < c->n_cigar; ++i) {
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34 int j, l = cigar[i]>>4, op = cigar[i]&0xf;
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35 if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) {
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36 for (j = 0; j < l; ++j) {
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37 int z = y + j;
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38 int c1 = bam1_seqi(seq, z), c2 = bam_nt16_table[(int)ref[x+j]];
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39 if (ref[x+j] == 0) break; // out of boundary
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40 if ((c1 == c2 && c1 != 15 && c2 != 15) || c1 == 0) { // a match
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41 if (flag&USE_EQUAL) seq[z/2] &= (z&1)? 0xf0 : 0x0f;
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42 ++u;
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43 } else {
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44 kputw(u, str); kputc(ref[x+j], str);
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45 u = 0; ++nm;
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46 }
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47 }
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48 if (j < l) break;
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49 x += l; y += l;
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50 } else if (op == BAM_CDEL) {
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51 kputw(u, str); kputc('^', str);
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52 for (j = 0; j < l; ++j) {
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53 if (ref[x+j] == 0) break;
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54 kputc(ref[x+j], str);
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55 }
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56 u = 0;
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57 if (j < l) break;
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58 x += l; nm += l;
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59 } else if (op == BAM_CINS || op == BAM_CSOFT_CLIP) {
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60 y += l;
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61 if (op == BAM_CINS) nm += l;
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62 } else if (op == BAM_CREF_SKIP) {
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63 x += l;
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64 }
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65 }
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66 kputw(u, str);
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67 // apply max_nm
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68 if (max_nm > 0 && nm >= max_nm) {
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69 for (i = y = 0, x = c->pos; i < c->n_cigar; ++i) {
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70 int j, l = cigar[i]>>4, op = cigar[i]&0xf;
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71 if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) {
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72 for (j = 0; j < l; ++j) {
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73 int z = y + j;
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74 int c1 = bam1_seqi(seq, z), c2 = bam_nt16_table[(int)ref[x+j]];
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75 if (ref[x+j] == 0) break; // out of boundary
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76 if ((c1 == c2 && c1 != 15 && c2 != 15) || c1 == 0) { // a match
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77 seq[z/2] |= (z&1)? 0x0f : 0xf0;
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78 bam1_qual(b)[z] = 0;
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79 }
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80 }
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81 if (j < l) break;
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82 x += l; y += l;
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83 } else if (op == BAM_CDEL || op == BAM_CREF_SKIP) x += l;
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84 else if (op == BAM_CINS || op == BAM_CSOFT_CLIP) y += l;
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85 }
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86 }
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87 // update NM
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88 if (flag & UPDATE_NM) {
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89 uint8_t *old_nm = bam_aux_get(b, "NM");
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90 if (c->flag & BAM_FUNMAP) return;
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91 if (old_nm) old_nm_i = bam_aux2i(old_nm);
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92 if (!old_nm) bam_aux_append(b, "NM", 'i', 4, (uint8_t*)&nm);
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93 else if (nm != old_nm_i) {
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94 fprintf(stderr, "[bam_fillmd1] different NM for read '%s': %d -> %d\n", bam1_qname(b), old_nm_i, nm);
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95 bam_aux_del(b, old_nm);
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96 bam_aux_append(b, "NM", 'i', 4, (uint8_t*)&nm);
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97 }
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98 }
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99 // update MD
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100 if (flag & UPDATE_MD) {
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101 uint8_t *old_md = bam_aux_get(b, "MD");
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102 if (c->flag & BAM_FUNMAP) return;
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103 if (!old_md) bam_aux_append(b, "MD", 'Z', str->l + 1, (uint8_t*)str->s);
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104 else {
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105 int is_diff = 0;
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106 if (strlen((char*)old_md+1) == str->l) {
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107 for (i = 0; i < str->l; ++i)
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108 if (toupper(old_md[i+1]) != toupper(str->s[i]))
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109 break;
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110 if (i < str->l) is_diff = 1;
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111 } else is_diff = 1;
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112 if (is_diff) {
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113 fprintf(stderr, "[bam_fillmd1] different MD for read '%s': '%s' -> '%s'\n", bam1_qname(b), old_md+1, str->s);
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114 bam_aux_del(b, old_md);
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115 bam_aux_append(b, "MD", 'Z', str->l + 1, (uint8_t*)str->s);
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116 }
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117 }
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118 }
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119 // drop all tags but RG
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120 if (flag&DROP_TAG) {
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121 uint8_t *q = bam_aux_get(b, "RG");
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122 bam_aux_drop_other(b, q);
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123 }
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124 // reduce the resolution of base quality
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125 if (flag&BIN_QUAL) {
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126 uint8_t *qual = bam1_qual(b);
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127 for (i = 0; i < b->core.l_qseq; ++i)
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128 if (qual[i] >= 3) qual[i] = qual[i]/10*10 + 7;
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129 }
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130 free(str->s); free(str);
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131 }
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132
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133 void bam_fillmd1(bam1_t *b, char *ref, int flag)
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134 {
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135 bam_fillmd1_core(b, ref, flag, 0);
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136 }
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137
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138 int bam_cap_mapQ(bam1_t *b, char *ref, int thres)
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139 {
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140 uint8_t *seq = bam1_seq(b), *qual = bam1_qual(b);
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141 uint32_t *cigar = bam1_cigar(b);
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142 bam1_core_t *c = &b->core;
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143 int i, x, y, mm, q, len, clip_l, clip_q;
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144 double t;
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145 if (thres < 0) thres = 40; // set the default
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146 mm = q = len = clip_l = clip_q = 0;
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147 for (i = y = 0, x = c->pos; i < c->n_cigar; ++i) {
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148 int j, l = cigar[i]>>4, op = cigar[i]&0xf;
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149 if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) {
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150 for (j = 0; j < l; ++j) {
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151 int z = y + j;
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152 int c1 = bam1_seqi(seq, z), c2 = bam_nt16_table[(int)ref[x+j]];
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153 if (ref[x+j] == 0) break; // out of boundary
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154 if (c2 != 15 && c1 != 15 && qual[z] >= 13) { // not ambiguous
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155 ++len;
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156 if (c1 && c1 != c2 && qual[z] >= 13) { // mismatch
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157 ++mm;
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158 q += qual[z] > 33? 33 : qual[z];
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159 }
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160 }
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161 }
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162 if (j < l) break;
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163 x += l; y += l; len += l;
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164 } else if (op == BAM_CDEL) {
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165 for (j = 0; j < l; ++j)
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166 if (ref[x+j] == 0) break;
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167 if (j < l) break;
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168 x += l;
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169 } else if (op == BAM_CSOFT_CLIP) {
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170 for (j = 0; j < l; ++j) clip_q += qual[y+j];
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171 clip_l += l;
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172 y += l;
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173 } else if (op == BAM_CHARD_CLIP) {
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174 clip_q += 13 * l;
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175 clip_l += l;
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176 } else if (op == BAM_CINS) y += l;
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177 else if (op == BAM_CREF_SKIP) x += l;
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178 }
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179 for (i = 0, t = 1; i < mm; ++i)
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180 t *= (double)len / (i+1);
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181 t = q - 4.343 * log(t) + clip_q / 5.;
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182 if (t > thres) return -1;
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183 if (t < 0) t = 0;
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184 t = sqrt((thres - t) / thres) * thres;
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185 // fprintf(stderr, "%s %lf %d\n", bam1_qname(b), t, q);
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186 return (int)(t + .499);
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187 }
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188
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189 int bam_prob_realn_core(bam1_t *b, const char *ref, int flag)
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190 {
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191 int k, i, bw, x, y, yb, ye, xb, xe, apply_baq = flag&1, extend_baq = flag>>1&1, redo_baq = flag&4;
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192 uint32_t *cigar = bam1_cigar(b);
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193 bam1_core_t *c = &b->core;
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194 kpa_par_t conf = kpa_par_def;
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195 uint8_t *bq = 0, *zq = 0, *qual = bam1_qual(b);
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196 if ((c->flag & BAM_FUNMAP) || b->core.l_qseq == 0) return -1; // do nothing
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197 // test if BQ or ZQ is present
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198 if ((bq = bam_aux_get(b, "BQ")) != 0) ++bq;
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199 if ((zq = bam_aux_get(b, "ZQ")) != 0 && *zq == 'Z') ++zq;
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200 if (bq && redo_baq)
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201 {
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202 bam_aux_del(b, bq-1);
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203 bq = 0;
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204 }
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205 if (bq && zq) { // remove the ZQ tag
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206 bam_aux_del(b, zq-1);
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207 zq = 0;
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208 }
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209 if (bq || zq) {
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210 if ((apply_baq && zq) || (!apply_baq && bq)) return -3; // in both cases, do nothing
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211 if (bq && apply_baq) { // then convert BQ to ZQ
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212 for (i = 0; i < c->l_qseq; ++i)
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213 qual[i] = qual[i] + 64 < bq[i]? 0 : qual[i] - ((int)bq[i] - 64);
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214 *(bq - 3) = 'Z';
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215 } else if (zq && !apply_baq) { // then convert ZQ to BQ
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216 for (i = 0; i < c->l_qseq; ++i)
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217 qual[i] += (int)zq[i] - 64;
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218 *(zq - 3) = 'B';
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219 }
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220 return 0;
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221 }
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222 // find the start and end of the alignment
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223 x = c->pos, y = 0, yb = ye = xb = xe = -1;
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224 for (k = 0; k < c->n_cigar; ++k) {
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225 int op, l;
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226 op = cigar[k]&0xf; l = cigar[k]>>4;
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227 if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) {
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228 if (yb < 0) yb = y;
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229 if (xb < 0) xb = x;
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230 ye = y + l; xe = x + l;
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231 x += l; y += l;
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232 } else if (op == BAM_CSOFT_CLIP || op == BAM_CINS) y += l;
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233 else if (op == BAM_CDEL) x += l;
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234 else if (op == BAM_CREF_SKIP) return -1; // do nothing if there is a reference skip
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235 }
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236 // set bandwidth and the start and the end
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237 bw = 7;
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238 if (abs((xe - xb) - (ye - yb)) > bw)
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239 bw = abs((xe - xb) - (ye - yb)) + 3;
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240 conf.bw = bw;
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241 xb -= yb + bw/2; if (xb < 0) xb = 0;
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242 xe += c->l_qseq - ye + bw/2;
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243 if (xe - xb - c->l_qseq > bw)
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244 xb += (xe - xb - c->l_qseq - bw) / 2, xe -= (xe - xb - c->l_qseq - bw) / 2;
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245 { // glocal
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246 uint8_t *s, *r, *q, *seq = bam1_seq(b), *bq;
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247 int *state;
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248 bq = calloc(c->l_qseq + 1, 1);
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249 memcpy(bq, qual, c->l_qseq);
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250 s = calloc(c->l_qseq, 1);
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251 for (i = 0; i < c->l_qseq; ++i) s[i] = bam_nt16_nt4_table[bam1_seqi(seq, i)];
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252 r = calloc(xe - xb, 1);
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253 for (i = xb; i < xe; ++i) {
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254 if (ref[i] == 0) { xe = i; break; }
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255 r[i-xb] = bam_nt16_nt4_table[bam_nt16_table[(int)ref[i]]];
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256 }
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257 state = calloc(c->l_qseq, sizeof(int));
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258 q = calloc(c->l_qseq, 1);
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259 kpa_glocal(r, xe-xb, s, c->l_qseq, qual, &conf, state, q);
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260 if (!extend_baq) { // in this block, bq[] is capped by base quality qual[]
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261 for (k = 0, x = c->pos, y = 0; k < c->n_cigar; ++k) {
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262 int op = cigar[k]&0xf, l = cigar[k]>>4;
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263 if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) {
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264 for (i = y; i < y + l; ++i) {
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265 if ((state[i]&3) != 0 || state[i]>>2 != x - xb + (i - y)) bq[i] = 0;
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266 else bq[i] = bq[i] < q[i]? bq[i] : q[i];
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267 }
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268 x += l; y += l;
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269 } else if (op == BAM_CSOFT_CLIP || op == BAM_CINS) y += l;
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270 else if (op == BAM_CDEL) x += l;
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271 }
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272 for (i = 0; i < c->l_qseq; ++i) bq[i] = qual[i] - bq[i] + 64; // finalize BQ
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273 } else { // in this block, bq[] is BAQ that can be larger than qual[] (different from the above!)
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274 uint8_t *left, *rght;
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275 left = calloc(c->l_qseq, 1); rght = calloc(c->l_qseq, 1);
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276 for (k = 0, x = c->pos, y = 0; k < c->n_cigar; ++k) {
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277 int op = cigar[k]&0xf, l = cigar[k]>>4;
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278 if (op == BAM_CMATCH || op == BAM_CEQUAL || op == BAM_CDIFF) {
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279 for (i = y; i < y + l; ++i)
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280 bq[i] = ((state[i]&3) != 0 || state[i]>>2 != x - xb + (i - y))? 0 : q[i];
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281 for (left[y] = bq[y], i = y + 1; i < y + l; ++i)
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282 left[i] = bq[i] > left[i-1]? bq[i] : left[i-1];
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283 for (rght[y+l-1] = bq[y+l-1], i = y + l - 2; i >= y; --i)
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284 rght[i] = bq[i] > rght[i+1]? bq[i] : rght[i+1];
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285 for (i = y; i < y + l; ++i)
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286 bq[i] = left[i] < rght[i]? left[i] : rght[i];
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287 x += l; y += l;
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288 } else if (op == BAM_CSOFT_CLIP || op == BAM_CINS) y += l;
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289 else if (op == BAM_CDEL) x += l;
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290 }
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291 for (i = 0; i < c->l_qseq; ++i) bq[i] = 64 + (qual[i] <= bq[i]? 0 : qual[i] - bq[i]); // finalize BQ
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292 free(left); free(rght);
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293 }
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294 if (apply_baq) {
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295 for (i = 0; i < c->l_qseq; ++i) qual[i] -= bq[i] - 64; // modify qual
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296 bam_aux_append(b, "ZQ", 'Z', c->l_qseq + 1, bq);
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297 } else bam_aux_append(b, "BQ", 'Z', c->l_qseq + 1, bq);
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298 free(bq); free(s); free(r); free(q); free(state);
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299 }
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300 return 0;
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301 }
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302
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303 int bam_prob_realn(bam1_t *b, const char *ref)
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304 {
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305 return bam_prob_realn_core(b, ref, 1);
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306 }
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307
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308 int bam_fillmd(int argc, char *argv[])
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309 {
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310 int c, flt_flag, tid = -2, ret, len, is_bam_out, is_sam_in, is_uncompressed, max_nm, is_realn, capQ, baq_flag;
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311 samfile_t *fp, *fpout = 0;
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312 faidx_t *fai;
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313 char *ref = 0, mode_w[8], mode_r[8];
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314 bam1_t *b;
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315
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316 flt_flag = UPDATE_NM | UPDATE_MD;
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317 is_bam_out = is_sam_in = is_uncompressed = is_realn = max_nm = capQ = baq_flag = 0;
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318 mode_w[0] = mode_r[0] = 0;
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319 strcpy(mode_r, "r"); strcpy(mode_w, "w");
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320 while ((c = getopt(argc, argv, "EqreuNhbSC:n:Ad")) >= 0) {
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321 switch (c) {
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322 case 'r': is_realn = 1; break;
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323 case 'e': flt_flag |= USE_EQUAL; break;
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324 case 'd': flt_flag |= DROP_TAG; break;
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325 case 'q': flt_flag |= BIN_QUAL; break;
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326 case 'h': flt_flag |= HASH_QNM; break;
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327 case 'N': flt_flag &= ~(UPDATE_MD|UPDATE_NM); break;
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328 case 'b': is_bam_out = 1; break;
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329 case 'u': is_uncompressed = is_bam_out = 1; break;
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330 case 'S': is_sam_in = 1; break;
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331 case 'n': max_nm = atoi(optarg); break;
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332 case 'C': capQ = atoi(optarg); break;
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333 case 'A': baq_flag |= 1; break;
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334 case 'E': baq_flag |= 2; break;
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335 default: fprintf(stderr, "[bam_fillmd] unrecognized option '-%c'\n", c); return 1;
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336 }
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337 }
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338 if (!is_sam_in) strcat(mode_r, "b");
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339 if (is_bam_out) strcat(mode_w, "b");
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340 else strcat(mode_w, "h");
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341 if (is_uncompressed) strcat(mode_w, "u");
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342 if (optind + 1 >= argc) {
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343 fprintf(stderr, "\n");
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344 fprintf(stderr, "Usage: samtools fillmd [-eubrS] <aln.bam> <ref.fasta>\n\n");
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345 fprintf(stderr, "Options: -e change identical bases to '='\n");
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346 fprintf(stderr, " -u uncompressed BAM output (for piping)\n");
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347 fprintf(stderr, " -b compressed BAM output\n");
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348 fprintf(stderr, " -S the input is SAM with header\n");
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349 fprintf(stderr, " -A modify the quality string\n");
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350 fprintf(stderr, " -r compute the BQ tag (without -A) or cap baseQ by BAQ (with -A)\n");
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351 fprintf(stderr, " -E extended BAQ for better sensitivity but lower specificity\n\n");
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352 return 1;
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353 }
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354 fp = samopen(argv[optind], mode_r, 0);
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355 if (fp == 0) return 1;
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356 if (is_sam_in && (fp->header == 0 || fp->header->n_targets == 0)) {
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357 fprintf(stderr, "[bam_fillmd] input SAM does not have header. Abort!\n");
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358 return 1;
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359 }
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360 fpout = samopen("-", mode_w, fp->header);
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361 fai = fai_load(argv[optind+1]);
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362
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363 b = bam_init1();
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364 while ((ret = samread(fp, b)) >= 0) {
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365 if (b->core.tid >= 0) {
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366 if (tid != b->core.tid) {
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367 free(ref);
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368 ref = fai_fetch(fai, fp->header->target_name[b->core.tid], &len);
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369 tid = b->core.tid;
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370 if (ref == 0)
|
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371 fprintf(stderr, "[bam_fillmd] fail to find sequence '%s' in the reference.\n",
|
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372 fp->header->target_name[tid]);
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373 }
|
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374 if (is_realn) bam_prob_realn_core(b, ref, baq_flag);
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375 if (capQ > 10) {
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376 int q = bam_cap_mapQ(b, ref, capQ);
|
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377 if (b->core.qual > q) b->core.qual = q;
|
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378 }
|
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379 if (ref) bam_fillmd1_core(b, ref, flt_flag, max_nm);
|
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380 }
|
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381 samwrite(fpout, b);
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|
382 }
|
|
383 bam_destroy1(b);
|
|
384
|
|
385 free(ref);
|
|
386 fai_destroy(fai);
|
|
387 samclose(fp); samclose(fpout);
|
|
388 return 0;
|
|
389 }
|