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1 /* written by Jonas Behr, Regina Bohnert and Gunnar Raetsch, FML Tuebingen, Germany, 2010 */
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2
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3 #include <stdio.h>
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4 #include <assert.h>
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5 #include "sam.h"
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6 #include "get_reads_direct.h"
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7
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8 #include <vector>
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9 using std::vector;
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10 #include <string>
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11 using std::string;
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12
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13 #ifdef __cplusplus
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14 extern "C" {
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15 #endif
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16
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17 typedef struct {
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18 int beg, end;
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19 samfile_t *in;
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20 } tmpstruct_t;
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21
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22 typedef struct {
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23 uint64_t u, v;
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24 } pair64_t;
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25
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26 static inline int is_overlap(uint32_t beg, uint32_t end, const bam1_t *b)
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27 {
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28 uint32_t rbeg = b->core.pos;
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29 uint32_t rend = b->core.n_cigar? bam_calend(&b->core, bam1_cigar(b)) : b->core.pos + 1;
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30 return (rend > beg && rbeg < end);
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31 }
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32
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33 pair64_t * get_chunk_coordinates(const bam_index_t *idx, int tid, int beg, int end, int* cnt_off);
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34
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35 int bam_fetch_reads(bamFile fp, const bam_index_t *idx, int tid, int beg, int end, void *data, bam_header_t* header, vector<CRead*>* reads, char strand);
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36
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37 // callback for bam_plbuf_init()
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38 static int pileup_func(uint32_t tid, uint32_t pos, int n, const bam_pileup1_t *pl, void *data)
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39 {
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40 //tmpstruct_t *tmp = (tmpstruct_t*)data;
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41 //if ((int)pos >= tmp->beg && (int)pos < tmp->end)
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42 // printf("%s\t%d\t%d\n", tmp->in->header->target_name[tid], pos + 1, n);
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43 return 0;
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44 }
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45 #ifdef __cplusplus
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46 }
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47 #endif
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48 int parse_sam_line(char* line, CRead* read);
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49 int set_strand(char c);
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50 //void parse_cigar(bam1_t* b, CRead* read);
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51 void parse_cigar(bam1_t* b, CRead* read, bam_header_t* header);
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52
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53
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54 int get_reads_from_bam(char* filename, char* region, vector<CRead*>* reads, char strand, int lsubsample)
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55 {
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56 subsample = lsubsample;
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57 set_strand(strand);
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58
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59 srand (time(NULL));
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60 //srand (1234);
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61 tmpstruct_t tmp;
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62 tmp.in = samopen(filename, "rb", 0);
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63 if (tmp.in == 0) {
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64 fprintf(stderr, "Fail to open BAM file %s\n", filename);
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65 return 1;
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66 }
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67 int ref;
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68 bam_index_t *idx;
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69 bam_plbuf_t *buf;
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70 idx = bam_index_load(filename); // load BAM index
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71 if (idx == 0) {
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72 fprintf(stderr, "BAM indexing file is not available.\n");
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73 return 1;
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74 }
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75 bam_parse_region(tmp.in->header, region, &ref,
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76 &tmp.beg, &tmp.end); // parse the region
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77 if (ref < 0) {
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78 fprintf(stderr, "Invalid region %s\n", region);
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79 return 1;
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80 }
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81
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82 buf = bam_plbuf_init(pileup_func, &tmp); // initialize pileup
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83
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84 bam_fetch_reads(tmp.in->x.bam, idx, ref, tmp.beg, tmp.end, buf, tmp.in->header, reads, strand);
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85 //fprintf(stdout, "intron_list: %d \n", intron_list->size());
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86
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87 bam_plbuf_push(0, buf); // finalize pileup
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88 bam_index_destroy(idx);
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89 bam_plbuf_destroy(buf);
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90 samclose(tmp.in);
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91 return 0;
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92 }
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93
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94
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95 int bam_fetch_reads(bamFile fp, const bam_index_t *idx, int tid, int beg, int end, void *data, bam_header_t* header, vector<CRead*>* reads, char strand)
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96 {
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97 int n_off;
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98 pair64_t *off = get_chunk_coordinates(idx, tid, beg, end, &n_off);
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99 if (off == 0) return 0;
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100 {
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101 // retrive alignments
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102 uint64_t curr_off;
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103 int i, ret, n_seeks;
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104 n_seeks = 0; i = -1; curr_off = 0;
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105 bam1_t *b = (bam1_t*)calloc(1, sizeof(bam1_t));
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106 for (;;) {
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107 if (curr_off == 0 || curr_off >= off[i].v) { // then jump to the next chunk
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108 if (i == n_off - 1) break; // no more chunks
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109 if (i >= 0) assert(curr_off == off[i].v); // otherwise bug
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110 if (i < 0 || off[i].v != off[i+1].u) { // not adjacent chunks; then seek
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111 bam_seek(fp, off[i+1].u, SEEK_SET);
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112 curr_off = bam_tell(fp);
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113 ++n_seeks;
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114 }
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115 ++i;
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116 }
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117 if ((ret = bam_read1(fp, b)) > 0) {
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118 curr_off = bam_tell(fp);
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119 if (b->core.tid != tid || b->core.pos >= end) break; // no need to proceed
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120 else if (is_overlap(beg, end, b))
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121 {
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122 int rr = rand();
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123 if ((rr%1000 < subsample))
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124 {
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125 CRead* read = new CRead();
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126 parse_cigar(b, read, header);
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127
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128 if (strand == '0' || strand==read->strand[0])
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129 {
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130 reads->push_back(read);
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131 }
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132 else if (read->strand[0]=='0')
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133 {
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134 reads->push_back(read);
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135 }
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136 //else if (read->strand[0]=='0'&&((b->core.flag & g_flag_off) >0))
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137 //{
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138 // //fprintf(stdout, "(-)-strand; read->strand[0]==0, num_exons: %i \n", read->block_starts.size());
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139 // // this flag means that the read has been reversed for alignment
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140 // // flag bit set and (-)-strand requested
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141 // reads->push_back(read);
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142 //}
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143 //else if (read->strand[0]=='0'&&(g_flag_on>0&&(b->core.flag & g_flag_on)==0))
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144 //{
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145 // //fprintf(stdout, "(+)-strand; read->strand[0]==0, num_exons: %i \n", read->block_starts.size());
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146 // // (+)-strand requested and flag bit not set
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147 // reads->push_back(read);
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148 //}
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149 }
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150 }
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151 } else break; // end of file
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152 }
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153 // fprintf(stderr, "[bam_fetch] # seek calls: %d\n", n_seeks);
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154 bam_destroy1(b);
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155 }
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156 free(off);
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157 return 0;
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158 }
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159
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160 void parse_cigar(bam1_t* b, CRead* read, bam_header_t* header)
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161 {
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162 read->start_pos = b->core.pos+1;
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163 read->set_strand('0');
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164
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165 for (int k = 0; k < b->core.n_cigar; ++k)
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166 {
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167 int op = bam1_cigar(b)[k] & BAM_CIGAR_MASK; // operation
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168 int l = bam1_cigar(b)[k] >> BAM_CIGAR_SHIFT; // length
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169 //fprintf(stdout, "op:%d l:%d\n", op, l);
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170 if (op == BAM_CMATCH)
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171 {
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172 if (k==0)
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173 {
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174 read->block_lengths.push_back(l);
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175 read->block_starts.push_back(0);
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176 }
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177 else
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178 {
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179 int op_prev = bam1_cigar(b)[k-1] & BAM_CIGAR_MASK;
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180 int l_prev = bam1_cigar(b)[k-1] >> BAM_CIGAR_SHIFT;
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181 if (op_prev==BAM_CREF_SKIP)// intron before
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182 {
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183 if (read->block_lengths.size()>=1)
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184 {
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185 int last_block_start = (*(read->block_starts.end()-1));
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186 int intron_start = last_block_start+(*(read->block_lengths.end()-1));
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187 read->block_lengths.push_back(l);
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188 read->block_starts.push_back(intron_start+l_prev);
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189 }
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190 else
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191 {
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192 // start of first block was not a match
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193 read->block_lengths.push_back(l);
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194 read->block_starts.push_back(0);
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195 }
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196 }
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197 else
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198 {
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199 if (read->block_lengths.size()>=1 && op == BAM_CDEL)// if it is an insertion then the matching block is not inreased
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200 (*(read->block_lengths.end()-1))+=l;
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201 else
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202 {
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203 //char *samline = bam_format1(header, b);
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204 //printf("header: %s \n", samline);
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205 }
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206 }
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207 }
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208 }
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209 else if (op == BAM_CDEL)
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210 {
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211 if (k>0 && read->block_lengths.size()>=1)
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212 (*(read->block_lengths.end()-1))+=l;
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213 }
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214 else if (op == BAM_CREF_SKIP)//intron
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215 {}
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216 else if (op == BAM_CINS || op == BAM_CSOFT_CLIP)
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217 {}
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218 }
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219 // parse auxiliary data
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220 uint8_t* s = bam1_aux(b);
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221 uint8_t* end = b->data + b->data_len;
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222 while (s < end)
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223 {
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224 uint8_t type, key[2];
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225 key[0] = s[0]; key[1] = s[1];
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226 s += 2; type = *s; ++s;
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227 //fprintf(stdout, "\n%c%c:%c\n", key[0], key[1], type);
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228 if (type == 'A')
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229 {
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230 if ( key[0] =='X' && key[1] == 'S')
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231 {
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232 read->set_strand((char) *s);
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233 }
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234 ++s;
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235 }
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236 else if (type == 'C')
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237 {
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238 if ( key[0] =='H' && key[1] == '0')
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239 {
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240 uint8_t matches = *s;
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241 read->matches = (int) matches;
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242 }
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243 if ( key[0] =='N' && key[1] == 'M')
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244 {
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245 uint8_t mismatches = *s;
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246 read->mismatches = (int) mismatches;
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247 }
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248 if ( key[0] =='H' && key[1] == 'I')
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249 {
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250 uint8_t mai = *s;
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251 read->multiple_alignment_index = (int) mai;
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252 }
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253
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254 ++s;
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255 }
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256 else if (type == 'c') { ++s; }
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257 else if (type == 'S') { s += 2; }
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258 else if (type == 's') { s += 2; }
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259 else if (type == 'I') { s += 4; }
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260 else if (type == 'i') { s += 4; }
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261 else if (type == 'f') { s += 4; }
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262 else if (type == 'd') { s += 8; }
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263 else if (type == 'Z') { ++s; }
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264 else if (type == 'H') { ++s; }
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265 }
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266 }
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267
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268 int set_strand(char c)
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269 {
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270 if (c=='+')
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271 {
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272 char* fl = (char*) "0x0010";
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273 g_flag_on = strtol(fl, 0, 0);
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274 g_flag_off = 0;
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275 }
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276 else if (c=='-')
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277 {
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278 char* fl = (char*) "0x0010";
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279 g_flag_off = strtol(fl, 0, 0);
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280 g_flag_on = 0;
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281 }
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282 }
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283
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