Question: how to use htslib to convert fastq to bam file
gravatar for Luyi Tian
4.4 years ago by
Luyi Tian110
Luyi Tian110 wrote:

I want to write some c code to convert fastq to unmapped bam file. I use htslib to read and write bam file and have some problem. The bam file I generated seems not validated. I guess I missed some part but I dont know what it is. This is my toy code:

// trim_fq_to_bam.c
#include <zlib.h> // for reading compressed .fq file
#include <stdio.h>
#include <string.h>
#include <htslib/sam.h>
#include <htslib/bgzf.h>
#include <htslib/kseq.h>
#include <htslib/hts.h>
KSEQ_INIT(gzFile, gzread)
#define bam1_seq_seti(s, i, c) ( (s)[(i)>>1] = ((s)[(i)>>1] & 0xf<<(((i)&1)<<2)) | (c)<<((~(i)&1)<<2) )

static const char init_header[] = "@HD\tVN:1.4\tSO:unknown\n";

void dump_read(bam1_t* b) {
    printf("->core.tid:(%d)\n", b->core.tid);
    printf("->core.pos:(%d)\n", b->core.pos);
    printf("->core.bin:(%d)\n", b->core.bin);
    printf("->core.qual:(%d)\n", b->core.qual);
    printf("->core.l_qname:(%d)\n", b->core.l_qname);
    printf("->core.flag:(%d)\n", b->core.flag);
    printf("->core.n_cigar:(%d)\n", b->core.n_cigar);
    printf("->core.l_qseq:(%d)\n", b->core.l_qseq);
    printf("->core.mtid:(%d)\n", b->core.mtid);
    printf("->core.mpos:(%d)\n", b->core.mpos);
    printf("->core.isize:(%d)\n", b->core.isize);
    if (b->data) {
        int i;
        for (i = 0; i < b->l_data; ++i) {
            printf("%x ", b->data[i]);
    if (b->core.l_qname) {
        printf("qname: %s\n",bam_get_qname(b));
    if (b->core.l_qseq) {
        int i;
        for (i = 0; i < b->core.l_qseq; ++i) {
            printf("%c", seq_nt16_str[bam_seqi(bam_get_seq(b),i)]);
        for (i = 0; i < b->core.l_qseq; ++i) {


    if (bam_get_l_aux(b)) {
        int i = 0;
        uint8_t* aux = bam_get_aux(b);

        while (i < bam_get_l_aux(b)) {
            i += 2;
            switch (*(aux+i)) {
                case 'Z':
                    while (*(aux+1+i) != '\0') { putc(*(aux+1+i), stdout); ++i; }

int main(int argc, char const *argv[])
    char *fn_in = "test_data/rd1.fq.gz";
    char *fn_out = "test_data/new.bam";
    int l = 0;
    gzFile *f_rd = gzopen(fn_in, "r");
    if (!f_rd){fprintf(stderr, "cant open file: %s\n", fn_in); exit(EXIT_FAILURE);}
    kseq_t *seq;
    seq =  kseq_init(f_rd);
    BGZF *fp = bgzf_open(fn_out,"w");

    // write header
    bam_hdr_t *hdr = bam_hdr_init();
    hdr->l_text = strlen(init_header);
    hdr->text = strdup(init_header);
    hdr->n_targets = 0;
    bam_hdr_write(fp, hdr);

    // main loop, iter through each fastq records
    while ((l = kseq_read(seq)) >= 0){
        printf("%s, %s\n", seq->seq.s, seq->qual.s);
        bam1_t *q = bam_init1();
        //`q->data` structure: qname-cigar-seq-qual-aux
        q->l_data = seq->name.l+1+(int)(1.5*seq->seq.l+(seq->seq.l % 2 != 0)); // +1 includes the tailing '\0'
        if (q->m_data < q->l_data) {
            q->m_data = q->l_data;
            q->data = (uint8_t*)realloc(q->data, q->m_data);
        q->core.flag = BAM_FMUNMAP;
        q->core.l_qname = seq->name.l+1; // +1 includes the tailing '\0'
        q->core.l_qseq = seq->seq.l;
        q->core.n_cigar = 0; // we have no cigar sequence
        memcpy(q->data, seq->name.s, q->core.l_qname); // first set qname
        uint8_t *s = bam_get_seq(q);
        for (int i = 0; i < q->core.l_qseq; ++i){

            bam1_seq_seti(s, i, seq_nt16_table[seq->seq.s[i]]);
        s = bam_get_qual(q);
        for (int i = 0; i < seq->qual.l; ++i) {
            s[i] = seq->qual.s[i];
        int ret = bam_write1(fp, q);
        printf("return value: %d\n", ret);
    kseq_destroy(seq); // free seq 
    gzclose(f_rd); // close fastq file
    bgzf_close(fp); // close bam file

    // validate bam file
    BGZF *fp_new = bgzf_open(fn_out,"r");
    bam1_t *q = bam_init1();
    int ret = bam_read1(fp_new, q);
    printf("%d\n", ret);
    printf("%d, %d\n", (char *)bam_get_aux(q) - (char *)q->data , q->l_data);
    return 0;

the function dump_read() is used to print a bam record to see if I have successfully create a valid bam1_t structure, and the code is copied from here: with some minor modification. For printing the sequence the original code is printf("%c",seq_nt16_str[seq_nt16_table[bam_seqi(bam_get_seq(b),i)]]); . but I didnt get the point of jointly using seq_nt16_table[] and seq_nt16_str[], since bam_seqi() returns the 4 bit representation of a base and seq_nt16_str convert it to str so these two would be sufficient.

The basic structure of the code is:

open fq file and bam file ->

write bam header (I am not sure if I have done it correctly) ->

iterate through each fastq reads, convert it to a bam1_t structure ->

use dump_read() to check if the bam1_t is correct. ->

write bam1_t to the bam file. check the return to see if the writing is successful.

So far everything looks fine. the dump_read() print the same seq and quality strings as the fastq reads. bam_write1() gives positive returns. And I generated a bam file. But when I try samtools view -h new.bam, it gives an error [main_samview] truncated file. Then I hacked the code and find when sam_read1() < -1 it will print this err msg. So I reopened my bam file and calls bam_read1(fp_new, q);, the return is -4, I checked the source code to see when it will returns -4 and stuck. I don't know which part of my code is wrong.

Also I wonder if there are any toturial on how to use htslib, or any examples so I can start from there. I usually work on Python and I am not familiar with C. Now I just read the source code of htslib but it is a bit messy, with a lot of uncomment blocks.

htslib samtools bam fastq • 3.1k views
ADD COMMENTlink modified 4.3 years ago • written 4.4 years ago by Luyi Tian110

Sorry I missed this the first time. Have you tried replacing bgzf_open(fn_out,"w"); and the associated close() with the actual BAM (sam_open()/sam_close()) functions? Those should write the magic bits at the end, which won't be there in generic bgzf files.

ADD REPLYlink written 4.3 years ago by Devon Ryan96k
gravatar for Luyi Tian
4.3 years ago by
Luyi Tian110
Luyi Tian110 wrote:

finally I solved the problem. Yes bgzf_open(fn_out,"w") is a lower level functions and one should use sam_open(fn_out,"wb") to write to a binary sam file (which is bam). But just change the open function does not fix everything. I also need to add these lines to set some variables to "NULL".

q->core.tid = -1;
q->core.pos = -1;
q->core.mtid = -1;
q->core.mpos = -1;

as an example, tid is the chromosome index in a char array which start at zero. So for unaligned bam where we have no chromosome, we need to set it to -1.

Thank you @Devon Ryan.

ADD COMMENTlink written 4.3 years ago by Luyi Tian110

Good catch and thanks for replying with the fix!

ADD REPLYlink written 4.3 years ago by Devon Ryan96k
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