Exemplo n.º 1
0
/*
 * Performs pileup
 * @param conf configuration for this pileup
 * @param n number of files specified in fn
 * @param fn filenames
 */
static int mpileup(mplp_conf_t *conf, int n, char **fn)
{
    extern void *bcf_call_add_rg(void *rghash, const char *hdtext, const char *list);
    extern void bcf_call_del_rghash(void *rghash);
    mplp_aux_t **data;
    int i, tid, pos, *n_plp, tid0 = -1, beg0 = 0, end0 = 1u<<29, ref_len, ref_tid = -1, max_depth, max_indel_depth;
    const bam_pileup1_t **plp;
    bam_mplp_t iter;
    bam_hdr_t *h = NULL; /* header of first file in input list */
    char *ref;
    void *rghash = NULL;
    FILE *pileup_fp = NULL;

    bcf_callaux_t *bca = NULL;
    bcf_callret1_t *bcr = NULL;
    bcf_call_t bc;
    htsFile *bcf_fp = NULL;
    bcf_hdr_t *bcf_hdr = NULL;

    bam_sample_t *sm = NULL;
    kstring_t buf;
    mplp_pileup_t gplp;

    memset(&gplp, 0, sizeof(mplp_pileup_t));
    memset(&buf, 0, sizeof(kstring_t));
    memset(&bc, 0, sizeof(bcf_call_t));
    data = calloc(n, sizeof(mplp_aux_t*));
    plp = calloc(n, sizeof(bam_pileup1_t*));
    n_plp = calloc(n, sizeof(int));
    sm = bam_smpl_init();

    if (n == 0) {
        fprintf(stderr,"[%s] no input file/data given\n", __func__);
        exit(1);
    }

    // read the header of each file in the list and initialize data
    for (i = 0; i < n; ++i) {
        bam_hdr_t *h_tmp;
        data[i] = calloc(1, sizeof(mplp_aux_t));
        data[i]->fp = sam_open(fn[i], "rb");
        if ( !data[i]->fp )
        {
            fprintf(stderr, "[%s] failed to open %s: %s\n", __func__, fn[i], strerror(errno));
            exit(1);
        }
        hts_set_fai_filename(data[i]->fp, conf->fai_fname);
        data[i]->conf = conf;
        h_tmp = sam_hdr_read(data[i]->fp);
        if ( !h_tmp ) {
            fprintf(stderr,"[%s] fail to read the header of %s\n", __func__, fn[i]);
            exit(1);
        }
        data[i]->h = i? h : h_tmp; // for i==0, "h" has not been set yet
        bam_smpl_add(sm, fn[i], (conf->flag&MPLP_IGNORE_RG)? 0 : h_tmp->text);
        // Collect read group IDs with PL (platform) listed in pl_list (note: fragile, strstr search)
        rghash = bcf_call_add_rg(rghash, h_tmp->text, conf->pl_list);
        if (conf->reg) {
            hts_idx_t *idx = sam_index_load(data[i]->fp, fn[i]);
            if (idx == 0) {
                fprintf(stderr, "[%s] fail to load index for %s\n", __func__, fn[i]);
                exit(1);
            }
            if ( (data[i]->iter=sam_itr_querys(idx, data[i]->h, conf->reg)) == 0) {
                fprintf(stderr, "[E::%s] fail to parse region '%s'\n", __func__, conf->reg);
                exit(1);
            }
            if (i == 0) tid0 = data[i]->iter->tid, beg0 = data[i]->iter->beg, end0 = data[i]->iter->end;
            hts_idx_destroy(idx);
        }
        if (i == 0) h = h_tmp; /* save the header of first file in list */
        else {
            // FIXME: to check consistency
            bam_hdr_destroy(h_tmp);
        }
    }
    // allocate data storage proportionate to number of samples being studied sm->n
    gplp.n = sm->n;
    gplp.n_plp = calloc(sm->n, sizeof(int));
    gplp.m_plp = calloc(sm->n, sizeof(int));
    gplp.plp = calloc(sm->n, sizeof(bam_pileup1_t*));

    fprintf(stderr, "[%s] %d samples in %d input files\n", __func__, sm->n, n);
    // write the VCF header
    if (conf->flag & MPLP_BCF)
    {
        const char *mode;
        if ( conf->flag & MPLP_VCF )
            mode = (conf->flag&MPLP_NO_COMP)? "wu" : "wz";   // uncompressed VCF or compressed VCF
        else
            mode = (conf->flag&MPLP_NO_COMP)? "wub" : "wb";  // uncompressed BCF or compressed BCF

        bcf_fp = bcf_open(conf->output_fname? conf->output_fname : "-", mode);
        if (bcf_fp == NULL) {
            fprintf(stderr, "[%s] failed to write to %s: %s\n", __func__, conf->output_fname? conf->output_fname : "standard output", strerror(errno));
            exit(1);
        }

        bcf_hdr = bcf_hdr_init("w");
        kstring_t str = {0,0,0};

        ksprintf(&str, "##samtoolsVersion=%s+htslib-%s\n",samtools_version(),hts_version());
        bcf_hdr_append(bcf_hdr, str.s);

        str.l = 0;
        ksprintf(&str, "##samtoolsCommand=samtools mpileup");
        for (i=1; i<conf->argc; i++) ksprintf(&str, " %s", conf->argv[i]);
        kputc('\n', &str);
        bcf_hdr_append(bcf_hdr, str.s);

        if (conf->fai_fname)
        {
            str.l = 0;
            ksprintf(&str, "##reference=file://%s\n", conf->fai_fname);
            bcf_hdr_append(bcf_hdr, str.s);
        }

        // todo: use/write new BAM header manipulation routines, fill also UR, M5
        for (i=0; i<h->n_targets; i++)
        {
            str.l = 0;
            ksprintf(&str, "##contig=<ID=%s,length=%d>", h->target_name[i], h->target_len[i]);
            bcf_hdr_append(bcf_hdr, str.s);
        }
        free(str.s);
        bcf_hdr_append(bcf_hdr,"##ALT=<ID=X,Description=\"Represents allele(s) other than observed.\">");
        bcf_hdr_append(bcf_hdr,"##INFO=<ID=INDEL,Number=0,Type=Flag,Description=\"Indicates that the variant is an INDEL.\">");
        bcf_hdr_append(bcf_hdr,"##INFO=<ID=IDV,Number=1,Type=Integer,Description=\"Maximum number of reads supporting an indel\">");
        bcf_hdr_append(bcf_hdr,"##INFO=<ID=IMF,Number=1,Type=Float,Description=\"Maximum fraction of reads supporting an indel\">");
        bcf_hdr_append(bcf_hdr,"##INFO=<ID=DP,Number=1,Type=Integer,Description=\"Raw read depth\">");
        bcf_hdr_append(bcf_hdr,"##INFO=<ID=VDB,Number=1,Type=Float,Description=\"Variant Distance Bias for filtering splice-site artefacts in RNA-seq data (bigger is better)\",Version=\"3\">");
        bcf_hdr_append(bcf_hdr,"##INFO=<ID=RPB,Number=1,Type=Float,Description=\"Mann-Whitney U test of Read Position Bias (bigger is better)\">");
        bcf_hdr_append(bcf_hdr,"##INFO=<ID=MQB,Number=1,Type=Float,Description=\"Mann-Whitney U test of Mapping Quality Bias (bigger is better)\">");
        bcf_hdr_append(bcf_hdr,"##INFO=<ID=BQB,Number=1,Type=Float,Description=\"Mann-Whitney U test of Base Quality Bias (bigger is better)\">");
        bcf_hdr_append(bcf_hdr,"##INFO=<ID=MQSB,Number=1,Type=Float,Description=\"Mann-Whitney U test of Mapping Quality vs Strand Bias (bigger is better)\">");
#if CDF_MWU_TESTS
        bcf_hdr_append(bcf_hdr,"##INFO=<ID=RPB2,Number=1,Type=Float,Description=\"Mann-Whitney U test of Read Position Bias [CDF] (bigger is better)\">");
        bcf_hdr_append(bcf_hdr,"##INFO=<ID=MQB2,Number=1,Type=Float,Description=\"Mann-Whitney U test of Mapping Quality Bias [CDF] (bigger is better)\">");
        bcf_hdr_append(bcf_hdr,"##INFO=<ID=BQB2,Number=1,Type=Float,Description=\"Mann-Whitney U test of Base Quality Bias [CDF] (bigger is better)\">");
        bcf_hdr_append(bcf_hdr,"##INFO=<ID=MQSB2,Number=1,Type=Float,Description=\"Mann-Whitney U test of Mapping Quality vs Strand Bias [CDF] (bigger is better)\">");
#endif
        bcf_hdr_append(bcf_hdr,"##INFO=<ID=SGB,Number=1,Type=Float,Description=\"Segregation based metric.\">");
        bcf_hdr_append(bcf_hdr,"##INFO=<ID=MQ0F,Number=1,Type=Float,Description=\"Fraction of MQ0 reads (smaller is better)\">");
        bcf_hdr_append(bcf_hdr,"##INFO=<ID=I16,Number=16,Type=Float,Description=\"Auxiliary tag used for calling, see description of bcf_callret1_t in bam2bcf.h\">");
        bcf_hdr_append(bcf_hdr,"##INFO=<ID=QS,Number=R,Type=Float,Description=\"Auxiliary tag used for calling\">");
        bcf_hdr_append(bcf_hdr,"##FORMAT=<ID=PL,Number=G,Type=Integer,Description=\"List of Phred-scaled genotype likelihoods\">");
        if ( conf->fmt_flag&B2B_FMT_DP )
            bcf_hdr_append(bcf_hdr,"##FORMAT=<ID=DP,Number=1,Type=Integer,Description=\"Number of high-quality bases\">");
        if ( conf->fmt_flag&B2B_FMT_DV )
            bcf_hdr_append(bcf_hdr,"##FORMAT=<ID=DV,Number=1,Type=Integer,Description=\"Number of high-quality non-reference bases\">");
        if ( conf->fmt_flag&B2B_FMT_DPR )
            bcf_hdr_append(bcf_hdr,"##FORMAT=<ID=DPR,Number=R,Type=Integer,Description=\"Number of high-quality bases observed for each allele\">");
        if ( conf->fmt_flag&B2B_INFO_DPR )
            bcf_hdr_append(bcf_hdr,"##INFO=<ID=DPR,Number=R,Type=Integer,Description=\"Number of high-quality bases observed for each allele\">");
        if ( conf->fmt_flag&B2B_FMT_DP4 )
            bcf_hdr_append(bcf_hdr,"##FORMAT=<ID=DP4,Number=4,Type=Integer,Description=\"Number of high-quality ref-fwd, ref-reverse, alt-fwd and alt-reverse bases\">");
        if ( conf->fmt_flag&B2B_FMT_SP )
            bcf_hdr_append(bcf_hdr,"##FORMAT=<ID=SP,Number=1,Type=Integer,Description=\"Phred-scaled strand bias P-value\">");

        for (i=0; i<sm->n; i++)
            bcf_hdr_add_sample(bcf_hdr, sm->smpl[i]);
        bcf_hdr_add_sample(bcf_hdr, NULL);
        bcf_hdr_write(bcf_fp, bcf_hdr);

        bca = bcf_call_init(-1., conf->min_baseQ);
        bcr = calloc(sm->n, sizeof(bcf_callret1_t));
        bca->rghash = rghash;
        bca->openQ = conf->openQ, bca->extQ = conf->extQ, bca->tandemQ = conf->tandemQ;
        bca->min_frac = conf->min_frac;
        bca->min_support = conf->min_support;
        bca->per_sample_flt = conf->flag & MPLP_PER_SAMPLE;

        bc.bcf_hdr = bcf_hdr;
        bc.n = sm->n;
        bc.PL = malloc(15 * sm->n * sizeof(*bc.PL));
        if (conf->fmt_flag)
        {
            assert( sizeof(float)==sizeof(int32_t) );
            bc.DP4 = malloc(sm->n * sizeof(int32_t) * 4);
            bc.fmt_arr = malloc(sm->n * sizeof(float)); // all fmt_flag fields
            if ( conf->fmt_flag&(B2B_INFO_DPR|B2B_FMT_DPR) )
            {
                // first B2B_MAX_ALLELES fields for total numbers, the rest per-sample
                bc.DPR = malloc((sm->n+1)*B2B_MAX_ALLELES*sizeof(int32_t));
                for (i=0; i<sm->n; i++)
                    bcr[i].DPR = bc.DPR + (i+1)*B2B_MAX_ALLELES;
            }
        }
    }
    else {
        pileup_fp = conf->output_fname? fopen(conf->output_fname, "w") : stdout;

        if (pileup_fp == NULL) {
            fprintf(stderr, "[%s] failed to write to %s: %s\n", __func__, conf->output_fname, strerror(errno));
            exit(1);
        }
    }

    if (tid0 >= 0 && conf->fai) { // region is set
        ref = faidx_fetch_seq(conf->fai, h->target_name[tid0], 0, 0x7fffffff, &ref_len);
        ref_tid = tid0;
        for (i = 0; i < n; ++i) data[i]->ref = ref, data[i]->ref_id = tid0;
    } else ref_tid = -1, ref = 0;

    // begin pileup
    iter = bam_mplp_init(n, mplp_func, (void**)data);
    if ( conf->flag & MPLP_SMART_OVERLAPS ) bam_mplp_init_overlaps(iter);
    max_depth = conf->max_depth;
    if (max_depth * sm->n > 1<<20)
        fprintf(stderr, "(%s) Max depth is above 1M. Potential memory hog!\n", __func__);
    if (max_depth * sm->n < 8000) {
        max_depth = 8000 / sm->n;
        fprintf(stderr, "<%s> Set max per-file depth to %d\n", __func__, max_depth);
    }
    max_indel_depth = conf->max_indel_depth * sm->n;
    bam_mplp_set_maxcnt(iter, max_depth);
    bcf1_t *bcf_rec = bcf_init1();
    int ret;
    while ( (ret=bam_mplp_auto(iter, &tid, &pos, n_plp, plp)) > 0) {
        if (conf->reg && (pos < beg0 || pos >= end0)) continue; // out of the region requested
        if (conf->bed && tid >= 0 && !bed_overlap(conf->bed, h->target_name[tid], pos, pos+1)) continue;
        if (tid != ref_tid) {
            free(ref); ref = 0;
            if (conf->fai) ref = faidx_fetch_seq(conf->fai, h->target_name[tid], 0, 0x7fffffff, &ref_len);
            for (i = 0; i < n; ++i) data[i]->ref = ref, data[i]->ref_id = tid;
            ref_tid = tid;
        }
        if (conf->flag & MPLP_BCF) {
            int total_depth, _ref0, ref16;
            for (i = total_depth = 0; i < n; ++i) total_depth += n_plp[i];
            group_smpl(&gplp, sm, &buf, n, fn, n_plp, plp, conf->flag & MPLP_IGNORE_RG);
            _ref0 = (ref && pos < ref_len)? ref[pos] : 'N';
            ref16 = seq_nt16_table[_ref0];
            bcf_callaux_clean(bca, &bc);
            for (i = 0; i < gplp.n; ++i)
                bcf_call_glfgen(gplp.n_plp[i], gplp.plp[i], ref16, bca, bcr + i);
            bc.tid = tid; bc.pos = pos;
            bcf_call_combine(gplp.n, bcr, bca, ref16, &bc);
            bcf_clear1(bcf_rec);
            bcf_call2bcf(&bc, bcf_rec, bcr, conf->fmt_flag, 0, 0);
            bcf_write1(bcf_fp, bcf_hdr, bcf_rec);
            // call indels; todo: subsampling with total_depth>max_indel_depth instead of ignoring?
            if (!(conf->flag&MPLP_NO_INDEL) && total_depth < max_indel_depth && bcf_call_gap_prep(gplp.n, gplp.n_plp, gplp.plp, pos, bca, ref, rghash) >= 0)
            {
                bcf_callaux_clean(bca, &bc);
                for (i = 0; i < gplp.n; ++i)
                    bcf_call_glfgen(gplp.n_plp[i], gplp.plp[i], -1, bca, bcr + i);
                if (bcf_call_combine(gplp.n, bcr, bca, -1, &bc) >= 0) {
                    bcf_clear1(bcf_rec);
                    bcf_call2bcf(&bc, bcf_rec, bcr, conf->fmt_flag, bca, ref);
                    bcf_write1(bcf_fp, bcf_hdr, bcf_rec);
                }
            }
        } else {
            fprintf(pileup_fp, "%s\t%d\t%c", h->target_name[tid], pos + 1, (ref && pos < ref_len)? ref[pos] : 'N');
            for (i = 0; i < n; ++i) {
                int j, cnt;
                for (j = cnt = 0; j < n_plp[i]; ++j) {
                    const bam_pileup1_t *p = plp[i] + j;
                    if (bam_get_qual(p->b)[p->qpos] >= conf->min_baseQ) ++cnt;
                }
                fprintf(pileup_fp, "\t%d\t", cnt);
                if (n_plp[i] == 0) {
                    fputs("*\t*", pileup_fp);
                    if (conf->flag & MPLP_PRINT_MAPQ) fputs("\t*", pileup_fp);
                    if (conf->flag & MPLP_PRINT_POS) fputs("\t*", pileup_fp);
                } else {
                    for (j = 0; j < n_plp[i]; ++j) {
                        const bam_pileup1_t *p = plp[i] + j;
                        if (bam_get_qual(p->b)[p->qpos] >= conf->min_baseQ)
                            pileup_seq(pileup_fp, plp[i] + j, pos, ref_len, ref);
                    }
                    putc('\t', pileup_fp);
                    for (j = 0; j < n_plp[i]; ++j) {
                        const bam_pileup1_t *p = plp[i] + j;
                        int c = bam_get_qual(p->b)[p->qpos];
                        if (c >= conf->min_baseQ) {
                            c = c + 33 < 126? c + 33 : 126;
                            putc(c, pileup_fp);
                        }
                    }
                    if (conf->flag & MPLP_PRINT_MAPQ) {
                        putc('\t', pileup_fp);
                        for (j = 0; j < n_plp[i]; ++j) {
                            const bam_pileup1_t *p = plp[i] + j;
                            int c = bam_get_qual(p->b)[p->qpos];
                            if ( c < conf->min_baseQ ) continue;
                            c = plp[i][j].b->core.qual + 33;
                            if (c > 126) c = 126;
                            putc(c, pileup_fp);
                        }
                    }
                    if (conf->flag & MPLP_PRINT_POS) {
                        putc('\t', pileup_fp);
                        for (j = 0; j < n_plp[i]; ++j) {
                            if (j > 0) putc(',', pileup_fp);
                            fprintf(pileup_fp, "%d", plp[i][j].qpos + 1); // FIXME: printf() is very slow...
                        }
                    }
                }
            }
            putc('\n', pileup_fp);
        }
    }

    // clean up
    free(bc.tmp.s);
    bcf_destroy1(bcf_rec);
    if (bcf_fp)
    {
        hts_close(bcf_fp);
        bcf_hdr_destroy(bcf_hdr);
        bcf_call_destroy(bca);
        free(bc.PL);
        free(bc.DP4);
        free(bc.DPR);
        free(bc.fmt_arr);
        free(bcr);
    }
    if (pileup_fp && conf->output_fname) fclose(pileup_fp);
    bam_smpl_destroy(sm); free(buf.s);
    for (i = 0; i < gplp.n; ++i) free(gplp.plp[i]);
    free(gplp.plp); free(gplp.n_plp); free(gplp.m_plp);
    bcf_call_del_rghash(rghash);
    bam_mplp_destroy(iter);
    bam_hdr_destroy(h);
    for (i = 0; i < n; ++i) {
        sam_close(data[i]->fp);
        if (data[i]->iter) hts_itr_destroy(data[i]->iter);
        free(data[i]);
    }
    free(data); free(plp); free(ref); free(n_plp);
    return ret;
}
Exemplo n.º 2
0
static int mpileup(mplp_conf_t *conf)
{
    if (conf->nfiles == 0) {
        fprintf(stderr,"[%s] no input file/data given\n", __func__);
        exit(EXIT_FAILURE);
    }

    mplp_ref_t mp_ref = MPLP_REF_INIT;
    conf->gplp = (mplp_pileup_t *) calloc(1,sizeof(mplp_pileup_t));
    conf->mplp_data = (mplp_aux_t**) calloc(conf->nfiles, sizeof(mplp_aux_t*));
    conf->plp = (const bam_pileup1_t**) calloc(conf->nfiles, sizeof(bam_pileup1_t*));
    conf->n_plp = (int*) calloc(conf->nfiles, sizeof(int));

    // Allow to run mpileup on multiple regions in one go. This comes at cost: the bai index
    // must be kept in the memory for the whole time which can be a problem with many bams.
    // Therefore if none or only one region is requested, we initialize the bam iterator as
    // before and free the index. Only when multiple regions are queried, we keep the index.
    int nregs = 0;
    if ( conf->reg_fname )
    {
        if ( conf->reg_is_file )
        {
            conf->reg = regidx_init(conf->reg_fname,NULL,NULL,0,NULL);
            if ( !conf->reg ) {
                fprintf(stderr,"Could not parse the regions: %s\n", conf->reg_fname);
                exit(EXIT_FAILURE);
            }
        }
        else
        {
            conf->reg = regidx_init(NULL,regidx_parse_reg,NULL,sizeof(char*),NULL);
            if ( regidx_insert_list(conf->reg,conf->reg_fname,',') !=0 ) {
                fprintf(stderr,"Could not parse the regions: %s\n", conf->reg_fname);
                exit(EXIT_FAILURE);
            }
        }
        nregs = regidx_nregs(conf->reg);
        conf->reg_itr = regitr_init(conf->reg);
        regitr_loop(conf->reg_itr);   // region iterator now positioned at the first region
    }

    // read the header of each file in the list and initialize data
    // beware: mpileup has always assumed that tid's are consistent in the headers, add sanity check at least!
    bam_hdr_t *hdr = NULL;      // header of first file in input list
    int i;
    for (i = 0; i < conf->nfiles; ++i) {
        bam_hdr_t *h_tmp;
        conf->mplp_data[i] = (mplp_aux_t*) calloc(1, sizeof(mplp_aux_t));
        conf->mplp_data[i]->fp = sam_open(conf->files[i], "rb");
        if ( !conf->mplp_data[i]->fp )
        {
            fprintf(stderr, "[%s] failed to open %s: %s\n", __func__, conf->files[i], strerror(errno));
            exit(EXIT_FAILURE);
        }
        if (hts_set_opt(conf->mplp_data[i]->fp, CRAM_OPT_DECODE_MD, 0)) {
            fprintf(stderr, "Failed to set CRAM_OPT_DECODE_MD value\n");
            exit(EXIT_FAILURE);
        }
        if (conf->fai_fname && hts_set_fai_filename(conf->mplp_data[i]->fp, conf->fai_fname) != 0) {
            fprintf(stderr, "[%s] failed to process %s: %s\n",
                    __func__, conf->fai_fname, strerror(errno));
            exit(EXIT_FAILURE);
        }
        conf->mplp_data[i]->conf = conf;
        conf->mplp_data[i]->ref = &mp_ref;
        h_tmp = sam_hdr_read(conf->mplp_data[i]->fp);
        if ( !h_tmp ) {
            fprintf(stderr,"[%s] fail to read the header of %s\n", __func__, conf->files[i]);
            exit(EXIT_FAILURE);
        }
        conf->mplp_data[i]->h = i ? hdr : h_tmp; // for j==0, "h" has not been set yet
        conf->mplp_data[i]->bam_id = bam_smpl_add_bam(conf->bsmpl,h_tmp->text,conf->files[i]);
        if ( conf->mplp_data[i]->bam_id<0 )
        {
            // no usable readgroups in this bam, it can be skipped
            sam_close(conf->mplp_data[i]->fp);
            free(conf->mplp_data[i]);
            bam_hdr_destroy(h_tmp);
            free(conf->files[i]);
            if ( i+1<conf->nfiles ) memmove(&conf->files[i],&conf->files[i+1],sizeof(*conf->files)*(conf->nfiles-i-1));
            conf->nfiles--;
            i--;
            continue;
        }
        if (conf->reg) {
            hts_idx_t *idx = sam_index_load(conf->mplp_data[i]->fp, conf->files[i]);
            if (idx == NULL) {
                fprintf(stderr, "[%s] fail to load index for %s\n", __func__, conf->files[i]);
                exit(EXIT_FAILURE);
            }
            conf->buf.l = 0;
            ksprintf(&conf->buf,"%s:%u-%u",conf->reg_itr->seq,conf->reg_itr->beg+1,conf->reg_itr->end+1);
            conf->mplp_data[i]->iter = sam_itr_querys(idx, conf->mplp_data[i]->h, conf->buf.s);
            if ( !conf->mplp_data[i]->iter ) 
            {
                conf->mplp_data[i]->iter = sam_itr_querys(idx, conf->mplp_data[i]->h, conf->reg_itr->seq);
                if ( conf->mplp_data[i]->iter ) {
                    fprintf(stderr,"[E::%s] fail to parse region '%s'\n", __func__, conf->buf.s);
                    exit(EXIT_FAILURE);
                }
                fprintf(stderr,"[E::%s] the sequence \"%s\" not found: %s\n",__func__,conf->reg_itr->seq,conf->files[i]);
                exit(EXIT_FAILURE);
            }
            if ( nregs==1 ) // no need to keep the index in memory
               hts_idx_destroy(idx);
            else
                conf->mplp_data[i]->idx = idx;
        }

        if ( !hdr ) hdr = h_tmp; /* save the header of first file in list */
        else {
            // FIXME: check consistency between h and h_tmp
            bam_hdr_destroy(h_tmp);

            // we store only the first file's header; it's (alleged to be)
            // compatible with the i-th file's target_name lookup needs
            conf->mplp_data[i]->h = hdr;
        }
    }
    // allocate data storage proportionate to number of samples being studied sm->n
    bam_smpl_get_samples(conf->bsmpl, &conf->gplp->n);
    conf->gplp->n_plp = (int*) calloc(conf->gplp->n, sizeof(int));
    conf->gplp->m_plp = (int*) calloc(conf->gplp->n, sizeof(int));
    conf->gplp->plp = (bam_pileup1_t**) calloc(conf->gplp->n, sizeof(bam_pileup1_t*));  

    fprintf(stderr, "[%s] %d samples in %d input files\n", __func__, conf->gplp->n, conf->nfiles);
    // write the VCF header
    conf->bcf_fp = hts_open(conf->output_fname?conf->output_fname:"-", hts_bcf_wmode(conf->output_type));
    if (conf->bcf_fp == NULL) {
        fprintf(stderr, "[%s] failed to write to %s: %s\n", __func__, conf->output_fname? conf->output_fname : "standard output", strerror(errno));
        exit(EXIT_FAILURE);
    }
    if ( conf->n_threads ) hts_set_threads(conf->bcf_fp, conf->n_threads);

    // BCF header creation
    conf->bcf_hdr = bcf_hdr_init("w");
    conf->buf.l = 0;

    if (conf->record_cmd_line)
    {
        ksprintf(&conf->buf, "##bcftoolsVersion=%s+htslib-%s\n",bcftools_version(),hts_version());
        bcf_hdr_append(conf->bcf_hdr, conf->buf.s);

        conf->buf.l = 0;
        ksprintf(&conf->buf, "##bcftoolsCommand=mpileup");
        for (i=1; i<conf->argc; i++) ksprintf(&conf->buf, " %s", conf->argv[i]);
        kputc('\n', &conf->buf);
        bcf_hdr_append(conf->bcf_hdr, conf->buf.s);
    }

    if (conf->fai_fname)
    {
        conf->buf.l = 0;
        ksprintf(&conf->buf, "##reference=file://%s\n", conf->fai_fname);
        bcf_hdr_append(conf->bcf_hdr, conf->buf.s);
    }

    // Translate BAM @SQ tags to BCF ##contig tags
    // todo: use/write new BAM header manipulation routines, fill also UR, M5
    for (i=0; i<hdr->n_targets; i++)
    {
        conf->buf.l = 0;
        ksprintf(&conf->buf, "##contig=<ID=%s,length=%d>", hdr->target_name[i], hdr->target_len[i]);
        bcf_hdr_append(conf->bcf_hdr, conf->buf.s);
    }
    conf->buf.l = 0;

    bcf_hdr_append(conf->bcf_hdr,"##ALT=<ID=*,Description=\"Represents allele(s) other than observed.\">");
    bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=INDEL,Number=0,Type=Flag,Description=\"Indicates that the variant is an INDEL.\">");
    bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=IDV,Number=1,Type=Integer,Description=\"Maximum number of reads supporting an indel\">");
    bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=IMF,Number=1,Type=Float,Description=\"Maximum fraction of reads supporting an indel\">");
    bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=DP,Number=1,Type=Integer,Description=\"Raw read depth\">");
    bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=VDB,Number=1,Type=Float,Description=\"Variant Distance Bias for filtering splice-site artefacts in RNA-seq data (bigger is better)\",Version=\"3\">");
    bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=RPB,Number=1,Type=Float,Description=\"Mann-Whitney U test of Read Position Bias (bigger is better)\">");
    bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=MQB,Number=1,Type=Float,Description=\"Mann-Whitney U test of Mapping Quality Bias (bigger is better)\">");
    bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=BQB,Number=1,Type=Float,Description=\"Mann-Whitney U test of Base Quality Bias (bigger is better)\">");
    bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=MQSB,Number=1,Type=Float,Description=\"Mann-Whitney U test of Mapping Quality vs Strand Bias (bigger is better)\">");
#if CDF_MWU_TESTS
    bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=RPB2,Number=1,Type=Float,Description=\"Mann-Whitney U test of Read Position Bias [CDF] (bigger is better)\">");
    bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=MQB2,Number=1,Type=Float,Description=\"Mann-Whitney U test of Mapping Quality Bias [CDF] (bigger is better)\">");
    bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=BQB2,Number=1,Type=Float,Description=\"Mann-Whitney U test of Base Quality Bias [CDF] (bigger is better)\">");
    bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=MQSB2,Number=1,Type=Float,Description=\"Mann-Whitney U test of Mapping Quality vs Strand Bias [CDF] (bigger is better)\">");
#endif
    bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=SGB,Number=1,Type=Float,Description=\"Segregation based metric.\">");
    bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=MQ0F,Number=1,Type=Float,Description=\"Fraction of MQ0 reads (smaller is better)\">");
    bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=I16,Number=16,Type=Float,Description=\"Auxiliary tag used for calling, see description of bcf_callret1_t in bam2bcf.h\">");
    bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=QS,Number=R,Type=Float,Description=\"Auxiliary tag used for calling\">");
    bcf_hdr_append(conf->bcf_hdr,"##FORMAT=<ID=PL,Number=G,Type=Integer,Description=\"List of Phred-scaled genotype likelihoods\">");
    if ( conf->fmt_flag&B2B_FMT_DP )
        bcf_hdr_append(conf->bcf_hdr,"##FORMAT=<ID=DP,Number=1,Type=Integer,Description=\"Number of high-quality bases\">");
    if ( conf->fmt_flag&B2B_FMT_DV )
        bcf_hdr_append(conf->bcf_hdr,"##FORMAT=<ID=DV,Number=1,Type=Integer,Description=\"Number of high-quality non-reference bases\">");
    if ( conf->fmt_flag&B2B_FMT_DPR )
        bcf_hdr_append(conf->bcf_hdr,"##FORMAT=<ID=DPR,Number=R,Type=Integer,Description=\"Number of high-quality bases observed for each allele\">");
    if ( conf->fmt_flag&B2B_INFO_DPR )
        bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=DPR,Number=R,Type=Integer,Description=\"Number of high-quality bases observed for each allele\">");
    if ( conf->fmt_flag&B2B_FMT_DP4 )
        bcf_hdr_append(conf->bcf_hdr,"##FORMAT=<ID=DP4,Number=4,Type=Integer,Description=\"Number of high-quality ref-fwd, ref-reverse, alt-fwd and alt-reverse bases\">");
    if ( conf->fmt_flag&B2B_FMT_SP )
        bcf_hdr_append(conf->bcf_hdr,"##FORMAT=<ID=SP,Number=1,Type=Integer,Description=\"Phred-scaled strand bias P-value\">");
    if ( conf->fmt_flag&B2B_FMT_AD )
        bcf_hdr_append(conf->bcf_hdr,"##FORMAT=<ID=AD,Number=R,Type=Integer,Description=\"Allelic depths\">");
    if ( conf->fmt_flag&B2B_FMT_ADF )
        bcf_hdr_append(conf->bcf_hdr,"##FORMAT=<ID=ADF,Number=R,Type=Integer,Description=\"Allelic depths on the forward strand\">");
    if ( conf->fmt_flag&B2B_FMT_ADR )
        bcf_hdr_append(conf->bcf_hdr,"##FORMAT=<ID=ADR,Number=R,Type=Integer,Description=\"Allelic depths on the reverse strand\">");
    if ( conf->fmt_flag&B2B_INFO_AD )
        bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=AD,Number=R,Type=Integer,Description=\"Total allelic depths\">");
    if ( conf->fmt_flag&B2B_INFO_ADF )
        bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=ADF,Number=R,Type=Integer,Description=\"Total allelic depths on the forward strand\">");
    if ( conf->fmt_flag&B2B_INFO_ADR )
        bcf_hdr_append(conf->bcf_hdr,"##INFO=<ID=ADR,Number=R,Type=Integer,Description=\"Total allelic depths on the reverse strand\">");
    if ( conf->gvcf )
        gvcf_update_header(conf->gvcf, conf->bcf_hdr);

    int nsmpl;
    const char **smpl = bam_smpl_get_samples(conf->bsmpl, &nsmpl);
    for (i=0; i<nsmpl; i++)
        bcf_hdr_add_sample(conf->bcf_hdr, smpl[i]);
    bcf_hdr_write(conf->bcf_fp, conf->bcf_hdr);

    conf->bca = bcf_call_init(-1., conf->min_baseQ);
    conf->bcr = (bcf_callret1_t*) calloc(nsmpl, sizeof(bcf_callret1_t));
    conf->bca->openQ = conf->openQ, conf->bca->extQ = conf->extQ, conf->bca->tandemQ = conf->tandemQ;
    conf->bca->min_frac = conf->min_frac;
    conf->bca->min_support = conf->min_support;
    conf->bca->per_sample_flt = conf->flag & MPLP_PER_SAMPLE;

    conf->bc.bcf_hdr = conf->bcf_hdr;
    conf->bc.n  = nsmpl;
    conf->bc.PL = (int32_t*) malloc(15 * nsmpl * sizeof(*conf->bc.PL));
    if (conf->fmt_flag)
    {
        assert( sizeof(float)==sizeof(int32_t) );
        conf->bc.DP4 = (int32_t*) malloc(nsmpl * sizeof(int32_t) * 4);
        conf->bc.fmt_arr = (uint8_t*) malloc(nsmpl * sizeof(float)); // all fmt_flag fields, float and int32
        if ( conf->fmt_flag&(B2B_INFO_DPR|B2B_FMT_DPR|B2B_INFO_AD|B2B_INFO_ADF|B2B_INFO_ADR|B2B_FMT_AD|B2B_FMT_ADF|B2B_FMT_ADR) )
        {
            // first B2B_MAX_ALLELES fields for total numbers, the rest per-sample
            conf->bc.ADR = (int32_t*) malloc((nsmpl+1)*B2B_MAX_ALLELES*sizeof(int32_t));
            conf->bc.ADF = (int32_t*) malloc((nsmpl+1)*B2B_MAX_ALLELES*sizeof(int32_t));
            for (i=0; i<nsmpl; i++)
            {
                conf->bcr[i].ADR = conf->bc.ADR + (i+1)*B2B_MAX_ALLELES;
                conf->bcr[i].ADF = conf->bc.ADF + (i+1)*B2B_MAX_ALLELES;
            }
        }
    }

    // init mpileup
    conf->iter = bam_mplp_init(conf->nfiles, mplp_func, (void**)conf->mplp_data);
    if ( conf->flag & MPLP_SMART_OVERLAPS ) bam_mplp_init_overlaps(conf->iter);
    if ( (double)conf->max_depth * conf->nfiles > 1<<20)
        fprintf(stderr, "Warning: Potential memory hog, up to %.0fM reads in the pileup!\n", (double)conf->max_depth*conf->nfiles);
    if ( (double)conf->max_depth * conf->nfiles / nsmpl < 250 )
        fprintf(stderr, "Note: The maximum per-sample depth with -d %d is %.1fx\n", conf->max_depth,(double)conf->max_depth * conf->nfiles / nsmpl);
    bam_mplp_set_maxcnt(conf->iter, conf->max_depth);
    conf->max_indel_depth = conf->max_indel_depth * nsmpl;
    conf->bcf_rec = bcf_init1();
    bam_mplp_constructor(conf->iter, pileup_constructor);

    // Run mpileup for multiple regions
    if ( nregs )
    {
        int ireg = 0;
        do 
        {
            // first region is already positioned
            if ( ireg++ > 0 )
            {
                conf->buf.l = 0;
                ksprintf(&conf->buf,"%s:%u-%u",conf->reg_itr->seq,conf->reg_itr->beg,conf->reg_itr->end);

                for (i=0; i<conf->nfiles; i++) 
                {
                    hts_itr_destroy(conf->mplp_data[i]->iter);
                    conf->mplp_data[i]->iter = sam_itr_querys(conf->mplp_data[i]->idx, conf->mplp_data[i]->h, conf->buf.s);
                    if ( !conf->mplp_data[i]->iter ) 
                    {
                        conf->mplp_data[i]->iter = sam_itr_querys(conf->mplp_data[i]->idx, conf->mplp_data[i]->h, conf->reg_itr->seq);
                        if ( conf->mplp_data[i]->iter ) {
                            fprintf(stderr,"[E::%s] fail to parse region '%s'\n", __func__, conf->buf.s);
                            exit(EXIT_FAILURE);
                        }
                        fprintf(stderr,"[E::%s] the sequence \"%s\" not found: %s\n",__func__,conf->reg_itr->seq,conf->files[i]);
                        exit(EXIT_FAILURE);
                    }
                    bam_mplp_reset(conf->iter);
                }
            }
            mpileup_reg(conf,conf->reg_itr->beg,conf->reg_itr->end);
        }
        while ( regitr_loop(conf->reg_itr) );
    }
    else
        mpileup_reg(conf,0,0);

    flush_bcf_records(conf, conf->bcf_fp, conf->bcf_hdr, NULL);

    // clean up
    free(conf->bc.tmp.s);
    bcf_destroy1(conf->bcf_rec);
    if (conf->bcf_fp)
    {
        hts_close(conf->bcf_fp);
        bcf_hdr_destroy(conf->bcf_hdr);
        bcf_call_destroy(conf->bca);
        free(conf->bc.PL);
        free(conf->bc.DP4);
        free(conf->bc.ADR);
        free(conf->bc.ADF);
        free(conf->bc.fmt_arr);
        free(conf->bcr);
    }
    if ( conf->gvcf ) gvcf_destroy(conf->gvcf);
    free(conf->buf.s);
    for (i = 0; i < conf->gplp->n; ++i) free(conf->gplp->plp[i]);
    free(conf->gplp->plp); free(conf->gplp->n_plp); free(conf->gplp->m_plp); free(conf->gplp);
    bam_mplp_destroy(conf->iter);
    bam_hdr_destroy(hdr);
    for (i = 0; i < conf->nfiles; ++i) {
        if ( nregs>1 ) hts_idx_destroy(conf->mplp_data[i]->idx);
        sam_close(conf->mplp_data[i]->fp);
        if ( conf->mplp_data[i]->iter) hts_itr_destroy(conf->mplp_data[i]->iter);
        free(conf->mplp_data[i]);
    }
    if ( conf->reg_itr ) regitr_destroy(conf->reg_itr);
    free(conf->mplp_data); free(conf->plp); free(conf->n_plp);
    free(mp_ref.ref[0]);
    free(mp_ref.ref[1]);
    return 0;
}
Exemplo n.º 3
0
void extractCalls(Config *config) {
    bam_hdr_t *hdr = sam_hdr_read(config->fp);
    bam_mplp_t iter;
    int ret, tid, pos, i, seqlen, type, rv, o = 0;
    int beg0 = 0, end0 = 1u<<29;
    int n_plp; //This will need to be modified for multiple input files
    int ctid = -1; //The tid of the contig whose sequence is stored in "seq"
    int idxBED = 0, strand;
    uint32_t nmethyl = 0, nunmethyl = 0;
    const bam_pileup1_t **plp = NULL;
    char *seq = NULL, base;
    mplp_data *data = NULL;
    struct lastCall *lastCpG = NULL;
    struct lastCall *lastCHG = NULL;

    if(config->merge) {
        if(config->keepCpG) {
            lastCpG = calloc(1, sizeof(struct lastCall));
            assert(lastCpG);
            lastCpG->tid = -1;
        }
        if(config->keepCHG) {
            lastCHG = calloc(1, sizeof(struct lastCall));
            assert(lastCHG);
            lastCHG->tid = -1;
        }
    }

    data = calloc(1,sizeof(mplp_data));
    if(data == NULL) {
        fprintf(stderr, "Couldn't allocate space for the data structure in extractCalls()!\n");
        return;
    }
    data->config = config;
    data->hdr = hdr;
    if (config->reg) {
        if((data->iter = sam_itr_querys(config->bai, hdr, config->reg)) == 0) {
            fprintf(stderr, "failed to parse regions %s", config->reg);
            return;
        }
    }
    if(config->bedName) {
        config->bed = parseBED(config->bedName, hdr);
        if(config->bed == NULL) return;
    }

    plp = calloc(1, sizeof(bam_pileup1_t *)); //This will have to be modified for multiple input files
    if(plp == NULL) {
        fprintf(stderr, "Couldn't allocate space for the plp structure in extractCalls()!\n");
        return;
    }

    //Start the pileup
    iter = bam_mplp_init(1, filter_func, (void **) &data);
    bam_mplp_init_overlaps(iter);
    bam_mplp_set_maxcnt(iter, config->maxDepth);
    while((ret = cust_mplp_auto(iter, &tid, &pos, &n_plp, plp)) > 0) {
        //Do we need to process this position?
        if (config->reg) {
            beg0 = data->iter->beg, end0 = data->iter->end;
            if ((pos < beg0 || pos >= end0)) continue; // out of the region requested
        }
        if(tid != ctid) {
            if(seq != NULL) free(seq);
            seq = faidx_fetch_seq(config->fai, hdr->target_name[tid], 0, faidx_seq_len(config->fai, hdr->target_name[tid]), &seqlen);
            if(seqlen < 0) {
                fprintf(stderr, "faidx_fetch_seq returned %i while trying to fetch the sequence for tid %i (%s)!\n",\
                        seqlen, tid, hdr->target_name[tid]);
                fprintf(stderr, "Note that the output will be truncated!\n");
                return;
            }
            ctid = tid;
        }

        if(config->bed) { //Handle -l
            while((o = posOverlapsBED(tid, pos, config->bed, idxBED)) == -1) idxBED++;
            if(o == 0) continue; //Wrong strand
        }

        if(isCpG(seq, pos, seqlen)) {
            if(!config->keepCpG) continue;
            type = 0;
        } else if(isCHG(seq, pos, seqlen)) {
            if(!config->keepCHG) continue;
            type = 1;
        } else if(isCHH(seq, pos, seqlen)) {
            if(!config->keepCHH) continue;
            type = 2;
        } else {
            continue;
        }

        nmethyl = nunmethyl = 0;
        base = *(seq+pos);
        for(i=0; i<n_plp; i++) {
            if(plp[0][i].is_del) continue;
            if(plp[0][i].is_refskip) continue;
            if(config->bed) if(!readStrandOverlapsBED(plp[0][i].b, config->bed->region[idxBED])) continue;
            strand = getStrand((plp[0]+i)->b);
            if(strand & 1) {
                if(base != 'C' && base != 'c') continue;
            } else {
                if(base != 'G' && base != 'g') continue;
            }
            rv = updateMetrics(config, plp[0]+i);
            if(rv > 0) nmethyl++;
            else if(rv<0) nunmethyl++;
        }

        if(nmethyl+nunmethyl==0) continue;
        if(!config->merge || type==2) {
            writeCall(config->output_fp[type], config, hdr->target_name[tid], pos, 1, nmethyl, nunmethyl);
        } else {
            //Merge into per-CpG/CHG metrics
            if(type==0) {
                if(base=='G' || base=='g') pos--;
                processLast(config->output_fp[0], config, lastCpG, hdr, tid, pos, 2, nmethyl, nunmethyl);
            } else {
                if(base=='G' || base=='g') pos-=2;
                processLast(config->output_fp[1], config, lastCHG, hdr, tid, pos, 3, nmethyl, nunmethyl);
            }
        }
    }

    //Don't forget the last CpG/CHG
    if(config->merge) {
        if(config->keepCpG && lastCpG->tid != -1) {
            processLast(config->output_fp[0], config, lastCpG, hdr, tid, pos, 2, nmethyl, nunmethyl);
        }
        if(config->keepCHG && lastCHG->tid != -1) {
            processLast(config->output_fp[1], config, lastCHG, hdr, tid, pos, 3, nmethyl, nunmethyl);
        }
    }

    bam_hdr_destroy(hdr);
    if(data->iter) hts_itr_destroy(data->iter);
    bam_mplp_destroy(iter);
    free(data);
    free(plp);
    if(seq != NULL) free(seq);
}