背景

在处理NGS 数据时，尤其是突变的检测。我们需要对bam 文件进行读写。 目前有相对方便的库 pysam 和 htsjdk ，这两个库提供的api 可以方便python 和java 读写，操作BAM。

随着测序深度的提高，尤其是UMI技术的普及，上万层的测序数据下，我们需要提高分析软件的效率，C++是比较好的选择。

C++ 中可以读写BAM 的库有 htslib 、bamtools、Seqan3 等。Seqan3的文档信息比较全，但是我使用文档中的测试案例发现，速度很慢（也许是我打开方式有问题？）；bamtools 和 htslib 的效率挺好的，但是都缺乏详细的文档，用起来真的头大。

本文，粗略的学习了htslib sam.h sam.c 部分的代码，以及参考了陈师傅的gencore 代码，总结了一些htslib的使用方法。（pileup 部分好难，略过~）

C++ 代码

• 获得Cigar

  /*
  @discussion In the CIGAR array, each element is a 32-bit integer. The
   lower 4 bits gives a CIGAR operation and the higher 28 bits keep the
   length of a CIGAR.
  */
  
  string getCigar(const bam1_t *b) {
      uint32_t *data = (uint32_t *)bam_get_cigar(b);
      int cigarNum = b->core.n_cigar;
      stringstream ss;
      for(int i=0; i<cigarNum; i++) {
          uint32_t val = data[i];
          char op = bam_cigar_opchr(val);
          uint32_t len = bam_cigar_oplen(val);
          ss << len << op;
      }
      return ss.str();
  }
  

• 获得序列和质量值

  //Each base is encoded in 4 bits: 1 for A, 2 for C, 4 for G, 8 for T and 15 for N.
  char fourbits2base(uint8_t val) {
      switch(val) {
          case 1:
              return 'A';
          case 2:
              return 'C';
          case 4:
              return 'G';
          case 8:
              return 'T';
          case 15:
              return 'N';
          default:
              cerr << "ERROR: Wrong base with value "<< (int)val << endl ;
              return 'N';
      }
  }
  
  // get seq 序列的信息记录在8bit 的数据结构中，前4bit 是前面的碱基，后4bit 是后面的碱基
  string getSeq(const bam1_t *b) {
      uint8_t *data = bam_get_seq(b);
      int len = b->core.l_qseq;
      string s(len, '\0');
      for(int i=0; i<len; i++) {
          char base;
          if(i%2 == 1)
              base = fourbits2base(data[i/2] & 0xF); 
          else
              base = fourbits2base((data[i/2]>>4) & 0xF);
          s[i] = base;
      }
      return s;
  }
  
  // get seq quality
  string getQual(const bam1_t *b) {
      uint8_t *data = bam_get_qual(b);
      int len = b->core.l_qseq;
      string s(len, '\0');
      for(int i=0; i<len; i++) {
          s[i] = (char)(data[i] + 33); // 转换成打印的ascci
      }
      return s;
  }
  

• 读取完整BAM

  int main(int argc,char *argv[])
  {
      if(argc < 2) {
          cerr << "need bam path";
          return -1;
      }
      samFile *bam_in = sam_open(argv[1],"r"); // open bam file
      bam_hdr_t *bam_header = sam_hdr_read(bam_in); // read header
      bam1_t *aln = NULL;
      aln = bam_init1(); //initialize an alignment
      
  //return >= 0 on successfully reading a new record, -1 on end of stream, < -1 on error
      while (sam_read1(bam_in,bam_header,aln) >= 0) 
      {
          int pos = aln->core.pos ;
          string chr = "*";
          if (aln->core.tid != -1) // 存在无法比对到基因组的reads
              chr = bam_header->target_name[aln->core.tid]; // config name(chromosome)            
          string queryname = bam_get_qname(aln);
          string cigar = getCigar(aln);
          string seq = getSeq(aln);
          string qual = getQual(aln);
  
          cout << "QueryName: " << queryname << '\n' 
              << "Positon: " << chr << '\t' <<  pos <<'\n'
              << "Cigar: " << cigar << '\n'
              << "Seq: " << seq << '\n'
              << "Qual: " << qual << '\n';
          
      }
  
      bam_destroy1(aln); // 回收资源
      bam_hdr_destroy(bam_header);
      sam_close(bam_in);
      cout << "Finshed!\n";
      return 0;
  }
  

• 读取选定区域的BAM

  int main(int argc,char *argv[])
  {
      if(argc < 3) {
          cerr << "need bam path; chrom: start pos-end pos";
          return -1;
      }
      samFile *bam_in = sam_open(argv[1],"r"); // open bam file
      hts_idx_t *bam_index = sam_index_load(bam_in,argv[1]); //load index 
      bam_hdr_t *bam_header = sam_hdr_read(bam_in); // read header
      bam1_t *aln = NULL;
      aln = bam_init1(); //initialize an alignment
  /*
  Regions are parsed by hts_parse_reg(), and take one of the following forms:
  region          | Outputs
  --------------- | -------------
  REF             | All reads with RNAME REF
  REF:            | All reads with RNAME REF
  REF:START       | Reads with RNAME REF overlapping START to end of REF
  REF:-END        | Reads with RNAME REF overlapping start of REF to END
  REF:START-END   | Reads with RNAME REF overlapping START to END
  .               | All reads from the start of the file
  *               | Unmapped reads at the end of the file (RNAME '*' in SAM)
  */
          string regin = argv[2]; 
         
      hts_itr_t *iter = sam_itr_querys(bam_index,bam_header,regin);
      if(!iter) cerr << "invalid regin\n";
     // while (sam_read1(bam_in,bam_header,aln) >= 0)
      while (sam_itr_next(bam_in, iter, aln) >= 0)
      {
          int pos = aln->core.pos ;
          string chr = "*";
          if (aln->core.tid != -1)
              chr = bam_header->target_name[aln->core.tid]; // config name(chromosome)            
          string queryname = bam_get_qname(aln);
          string cigar = getCigar(aln);
          string seq = getSeq(aln);
          string qual = getQual(aln);
          string md = getAux(aln,"MD");
  
          //int64_t newPos = pos - 5;
          cout << "QueryName: " << queryname << '\n' 
              << "Positon: " <<aln->core.tid << '\t' << chr << '\t' <<  pos <<'\n'
              << "Cigar: " << cigar << '\n'
              << md << '\n'
              << "Seq: " << seq << '\n'
              << "Qual: " << qual << '\n';
          
      }
      sam_itr_destroy(iter) ;// free iter
      bam_destroy1(aln);
      bam_hdr_destroy(bam_header);
      sam_close(bam_in);
      cout << "Finshed!\n";
      return 0;
  }
  
  

• 辅助字段的读取

  string getAux(const bam1_t *b,const char tag[2])
  {
      kstring_t res = KS_INITIALIZE;    // 需要初始化
      if(bam_aux_get_str(b,tag,&res) == 1) //kstring的string buffer 没有\0终止
      {
          int len = ks_len(&res);
          char *ks_s = ks_str(&res);
          string s(len, '\0');
          for (int i = 0;i<len;i++ ){
              s[i] = ks_s[i];
          }
          ks_free(&res); // 释放资源
          return s;
      }    
      else 
      {
          cerr << "no tag :" << tag << '\n';
          ks_free(&res);
          return "";
      }
      
  }
  
  int main（）
  {
  。。。
      string md = getAux(aln,"MD");
  。。。
  }