bcftools-1.14/plugins/trio-stats.c

/* The MIT License

   Copyright (c) 2018-2021 Genome Research Ltd.

   Author: Petr Danecek <pd3@sanger.ac.uk>

   Permission is hereby granted, free of charge, to any person obtaining a copy
   of this software and associated documentation files (the "Software"), to deal
   in the Software without restriction, including without limitation the rights
   to use, copy, modify, merge, publish, distribute, sublicense, and/or sell
   copies of the Software, and to permit persons to whom the Software is
   furnished to do so, subject to the following conditions:

   The above copyright notice and this permission notice shall be included in
   all copies or substantial portions of the Software.

   THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR
   IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY,
   FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE
   AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER
   LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM,
   OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN
   THE SOFTWARE.

 */

#include <stdio.h>
#include <stdlib.h>
#include <strings.h>
#include <getopt.h>
#include <unistd.h>     // for isatty
#include <inttypes.h>
#include <htslib/hts.h>
#include <htslib/vcf.h>
#include <htslib/kstring.h>
#include <htslib/kseq.h>
#include <htslib/synced_bcf_reader.h>
#include <htslib/vcfutils.h>
#include <htslib/kbitset.h>
#include <htslib/khash_str2int.h>
#include "bcftools.h"
#include "filter.h"


// Logic of the filters: include or exclude sites which match the filters?
#define FLT_INCLUDE 1
#define FLT_EXCLUDE 2

#define iCHILD  0
#define iFATHER 1
#define iMOTHER 2

#define VERBOSE_MENDEL 1
#define VERBOSE_TRANSMITTED 2

typedef struct
{
    int idx[3];     // VCF sample index for father, mother and child
    int pass;       // do all three pass the filters?
}
trio_t;

typedef struct
{
    uint32_t
        npass,          // number of genotypes passing the filter
        nnon_ref,       // number of non-reference genotypes
        nmendel_err,    // number of DNMs / mendelian errors
        nnovel,         // a singleton allele, but observed only in the child. Counted as mendel_err as well.
        nsingleton,     // het mother or father different from everyone else
        ndoubleton,     // het mother+child or father+child different from everyone else (transmitted alleles)
        nts, ntv,       // number of transitions and transversions
        ndnm_recurrent, // number of recurrent DNMs / mendelian errors (counted as GTs, not sites; in ambiguous cases the allele with smaller AF is chosen)
        ndnm_hom;       // number of homozygous DNMs / mendelian errors
}
trio_stats_t;

typedef struct
{
    trio_stats_t *stats;
    filter_t *filter;
    char *expr;
}
flt_stats_t;

typedef struct
{
    kbitset_t *sd_bset; // singleton (1) or doubleton (0) trio?
    uint32_t
        nalt,   // number of all alternate trios
        nsd,    // number of singleton or doubleton trios
        *idx;   // indexes of the singleton and doubleon trios
}
alt_trios_t;    // for one alt allele

typedef struct
{
    int max_alt_trios;      // maximum number of alternate trios [1]
    int malt_trios;
    alt_trios_t *alt_trios;
    int argc, filter_logic, regions_is_file, targets_is_file;
    int nflt_str;
    char *filter_str, **flt_str;
    char **argv, *ped_fname, *pfm, *output_fname, *fname, *regions, *targets;
    bcf_srs_t *sr;
    bcf_hdr_t *hdr;
    trio_t *trio;
    int ntrio, mtrio;
    flt_stats_t *filters;
    int nfilters;
    int32_t *gt_arr, *ac, *ac_trio, *dnm_als;
    int mgt_arr, mac, mac_trio, mdnm_als;
    int verbose;
    FILE *fp_out;
}
args_t;

args_t args;

const char *about(void)
{
    return "Calculate transmission rate and other stats in trio children.\n";
}

static const char *usage_text(void)
{
    return
        "\n"
        "About: Calculate transmission rate in trio children. Use curly brackets to scan\n"
        "       a range of values simultaneously\n"
        "Usage: bcftools +trio-stats [Plugin Options]\n"
        "Plugin options:\n"
        "   -a, --alt-trios INT         for transmission rate consider only sites with at most this\n"
        "                                   many alternate trios, 0 for unlimited [0]\n"
        "   -d, --debug TYPE            comma-separted list of features: {mendel-errors,transmitted}\n"
        "   -e, --exclude EXPR          exclude trios for which the expression is true (one matching sample invalidates a trio)\n"
        "   -i, --include EXPR          include trios for which the expression is true (one failing sample invalidates a trio)\n"
        "   -o, --output FILE           output file name [stdout]\n"
        "   -p, --ped FILE              PED file\n"
        "   -P, --pfm P,F,M             sample names of proband, father, and mother\n"
        "   -r, --regions REG           restrict to comma-separated list of regions\n"
        "   -R, --regions-file FILE     restrict to regions listed in a file\n"
        "   -t, --targets REG           similar to -r but streams rather than index-jumps\n"
        "   -T, --targets-file FILE     similar to -R but streams rather than index-jumps\n"
        "\n"
        "Example:\n"
        "   bcftools +trio-stats -p file.ped -i 'GQ>{10,20,30,40,50}' file.bcf\n"
        "\n";
}

static int cmp_trios(const void *_a, const void *_b)
{
    trio_t *a = (trio_t *) _a;
    trio_t *b = (trio_t *) _b;
    int i;
    int amin = a->idx[0];
    for (i=1; i<3; i++)
        if ( amin > a->idx[i] ) amin = a->idx[i];
    int bmin = b->idx[0];
    for (i=1; i<3; i++)
        if ( bmin > b->idx[i] ) bmin = b->idx[i];
    if ( amin < bmin ) return -1;
    if ( amin > bmin ) return 1;
    return 0;
}

static void parse_ped(args_t *args, char *fname)
{
    htsFile *fp = hts_open(fname, "r");
    if ( !fp ) error("Could not read: %s\n", fname);

    kstring_t str = {0,0,0};
    if ( hts_getline(fp, KS_SEP_LINE, &str) <= 0 ) error("Empty file: %s\n", fname);

    kstring_t tmp = {0,0,0};
    void *has_smpl = khash_str2int_init();
    void *has_trio = khash_str2int_init();

    int dup_trio_warned = 0;
    int moff = 0, *off = NULL;
    do
    {
        // familyID    sampleID paternalID maternalID sex   phenotype   population relationship   siblings   secondOrder   thirdOrder   children    comment
        // BB03    HG01884 HG01885 HG01956 2   0   ACB child   0   0   0   0
        int ncols = ksplit_core(str.s,0,&moff,&off);
        if ( ncols<4 ) error("Could not parse the ped file: %s\n", str.s);

        int father = bcf_hdr_id2int(args->hdr,BCF_DT_SAMPLE,&str.s[off[2]]);
        if ( father<0 ) continue;
        int mother = bcf_hdr_id2int(args->hdr,BCF_DT_SAMPLE,&str.s[off[3]]);
        if ( mother<0 ) continue;
        int child = bcf_hdr_id2int(args->hdr,BCF_DT_SAMPLE,&str.s[off[1]]);
        if ( child<0 ) continue;

        tmp.l = 0;
        ksprintf(&tmp,"%s %s %s",&str.s[off[1]],&str.s[off[2]],&str.s[off[3]]);
        if ( khash_str2int_has_key(has_trio,tmp.s) )
        {
            if ( !dup_trio_warned ) fprintf(stderr,"Warning: The trio \"%s\" is listed multiple times in %s, skipping. (This message is printed only once.)\n",tmp.s,fname);
            dup_trio_warned = 1;
            continue;
        }
        if ( khash_str2int_has_key(has_smpl,&str.s[off[1]]) )
            error("Error: The child \"%s\" is listed in two trios\n",&str.s[off[1]]);
        khash_str2int_inc(has_smpl,strdup(&str.s[off[1]]));
        khash_str2int_inc(has_trio,strdup(tmp.s));

        args->ntrio++;
        hts_expand0(trio_t,args->ntrio,args->mtrio,args->trio);
        trio_t *trio = &args->trio[args->ntrio-1];
        trio->idx[iFATHER] = father;
        trio->idx[iMOTHER] = mother;
        trio->idx[iCHILD]  = child;
    }
    while ( hts_getline(fp, KS_SEP_LINE, &str)>=0 );

    fprintf(stderr,"Identified %d complete trios in the VCF file\n", args->ntrio);
    if ( !args->ntrio ) error("No complete trio identified\n");

    // sort the sample by index so that they are accessed more or less sequentially
    qsort(args->trio,args->ntrio,sizeof(trio_t),cmp_trios);

    khash_str2int_destroy_free(has_smpl);
    khash_str2int_destroy_free(has_trio);
    free(tmp.s);
    free(str.s);
    free(off);
    if ( hts_close(fp)!=0 ) error("[%s] Error: close failed .. %s\n", __func__,fname);
}

static void parse_filters(args_t *args)
{
    if ( !args->filter_str ) return;
    int mflt = 1;
    args->nflt_str = 1;
    args->flt_str  = (char**) malloc(sizeof(char*));
    args->flt_str[0] = strdup(args->filter_str);
    while (1)
    {
        int i, expanded = 0;
        for (i=args->nflt_str-1; i>=0; i--)
        {
            char *exp_beg = strchr(args->flt_str[i], '{');
            if ( !exp_beg ) continue;
            char *exp_end = strchr(exp_beg+1, '}');
            if ( !exp_end ) error("Could not parse the expression: %s\n", args->filter_str);
            char *beg = exp_beg+1, *mid = beg;
            while ( mid<exp_end )
            {
                while ( mid<exp_end && *mid!=',' ) mid++;
                kstring_t tmp = {0,0,0};
                kputsn(args->flt_str[i], exp_beg - args->flt_str[i], &tmp);
                kputsn(beg, mid - beg, &tmp);
                kputs(exp_end+1, &tmp);
                args->nflt_str++;
                hts_expand(char*, args->nflt_str, mflt, args->flt_str);
                args->flt_str[args->nflt_str-1] = tmp.s;
                beg = ++mid;
            }
            expanded = 1;
            free(args->flt_str[i]);
            memmove(&args->flt_str[i], &args->flt_str[i+1], (args->nflt_str-i-1)*sizeof(*args->flt_str));
            args->nflt_str--;
            args->flt_str[args->nflt_str] = NULL;
        }
        if ( !expanded ) break;
    }

    fprintf(stderr,"Collecting data for %d filtering expressions\n", args->nflt_str);
}

static void init_data(args_t *args)
{
    args->sr = bcf_sr_init();
    if ( args->regions )
    {
        args->sr->require_index = 1;
        if ( bcf_sr_set_regions(args->sr, args->regions, args->regions_is_file)<0 ) error("Failed to read the regions: %s\n",args->regions);
    }
    if ( args->targets && bcf_sr_set_targets(args->sr, args->targets, args->targets_is_file, 0)<0 ) error("Failed to read the targets: %s\n",args->targets);
    if ( !bcf_sr_add_reader(args->sr,args->fname) ) error("Error: %s\n", bcf_sr_strerror(args->sr->errnum));
    args->hdr = bcf_sr_get_header(args->sr,0);

    if ( args->ped_fname )
        parse_ped(args, args->ped_fname);
    else
    {
        args->ntrio = 1;
        args->trio = (trio_t*) calloc(1,sizeof(trio_t));
        int ibeg, iend = 0;
        while ( args->pfm[iend] && args->pfm[iend]!=',' ) iend++;
        if ( !args->pfm[iend] ) error("Could not parse -P %s\n", args->pfm);
        args->pfm[iend] = 0;
        int child = bcf_hdr_id2int(args->hdr,BCF_DT_SAMPLE,args->pfm);
        if ( child<0 ) error("No such sample: \"%s\"\n", args->pfm);
        args->pfm[iend] = ',';
        ibeg = ++iend;
        while ( args->pfm[iend] && args->pfm[iend]!=',' ) iend++;
        if ( !args->pfm[iend] ) error("Could not parse -P %s\n", args->pfm);
        args->pfm[iend] = 0;
        int father = bcf_hdr_id2int(args->hdr,BCF_DT_SAMPLE,args->pfm+ibeg);
        if ( father<0 ) error("No such sample: \"%s\"\n", args->pfm+ibeg);
        args->pfm[iend] = ',';
        ibeg = ++iend;
        int mother = bcf_hdr_id2int(args->hdr,BCF_DT_SAMPLE,args->pfm+ibeg);
        if ( mother<0 ) error("No such sample: \"%s\"\n", args->pfm+ibeg);
        args->trio[0].idx[iFATHER] = father;
        args->trio[0].idx[iMOTHER] = mother;
        args->trio[0].idx[iCHILD]  = child;
    }
    parse_filters(args);

    int i;
    if ( !args->nflt_str )
    {
        args->filters = (flt_stats_t*) calloc(1, sizeof(flt_stats_t));
        args->nfilters = 1;
        args->filters[0].expr = strdup("all");
    }
    else
    {
        args->nfilters = args->nflt_str;
        args->filters = (flt_stats_t*) calloc(args->nfilters, sizeof(flt_stats_t));
        for (i=0; i<args->nfilters; i++)
        {
            args->filters[i].filter = filter_init(args->hdr, args->flt_str[i]);
            args->filters[i].expr   = strdup(args->flt_str[i]);

            // replace tab's with spaces so that the output stays parsable
            char *tmp = args->filters[i].expr;
            while ( *tmp )
            {
                if ( *tmp=='\t' ) *tmp = ' ';
                tmp++;
            }
        }
    }
    for (i=0; i<args->nfilters; i++)
        args->filters[i].stats = (trio_stats_t*) calloc(args->ntrio,sizeof(trio_stats_t));

    args->fp_out = !args->output_fname || !strcmp("-",args->output_fname) ? stdout : fopen(args->output_fname,"w");
    if ( !args->fp_out ) error("Could not open the file for writing: %s\n", args->output_fname);
    fprintf(args->fp_out,"# CMD line shows the command line used to generate this output\n");
    fprintf(args->fp_out,"# DEF lines define expressions for all tested thresholds\n");
    fprintf(args->fp_out,"# FLT* lines report numbers for every threshold and every trio:\n");
    i = 0;
    fprintf(args->fp_out,"#   %d) filter id\n", ++i);
    fprintf(args->fp_out,"#   %d) child\n", ++i);
    fprintf(args->fp_out,"#   %d) father\n", ++i);
    fprintf(args->fp_out,"#   %d) mother\n", ++i);
    fprintf(args->fp_out,"#   %d) number of valid trio genotypes (all trio members pass filters, all non-missing)\n", ++i);
    fprintf(args->fp_out,"#   %d) number of non-reference trio GTs (at least one trio member carries an alternate allele)\n", ++i);
    fprintf(args->fp_out,"#   %d) number of DNMs/Mendelian errors\n", ++i);
    fprintf(args->fp_out,"#   %d) number of novel singleton alleles in the child (counted also as DNM / Mendelian error)\n", ++i);
    fprintf(args->fp_out,"#   %d) number of untransmitted trio singletons (one alternate allele present in one parent)\n", ++i);
    fprintf(args->fp_out,"#   %d) number of transmitted trio singletons (one alternate allele present in one parent and the child)\n", ++i);
    fprintf(args->fp_out,"#   %d) number of transitions, all distinct ALT alleles present in the trio are considered\n", ++i);
    fprintf(args->fp_out,"#   %d) number of transversions, all distinct ALT alleles present in the trio are considered\n", ++i);
    fprintf(args->fp_out,"#   %d) overall ts/tv, all distinct ALT alleles present in the trio are considered\n", ++i);
    fprintf(args->fp_out,"#   %d) number of homozygous DNMs/Mendelian errors (likely genotyping errors)\n", ++i);
    fprintf(args->fp_out,"#   %d) number of recurrent DNMs/Mendelian errors (non-inherited alleles present in other samples; counts GTs, not sites)\n", ++i);
    fprintf(args->fp_out, "CMD\t%s", args->argv[0]);
    for (i=1; i<args->argc; i++) fprintf(args->fp_out, " %s",args->argv[i]);
    fprintf(args->fp_out, "\n");
}
static void alt_trios_reset(args_t *args, int nals)
{
    int i;
    hts_expand0(alt_trios_t, nals, args->malt_trios, args->alt_trios);
    for (i=0; i<nals; i++)
    {
        alt_trios_t *tr = &args->alt_trios[i];
        if ( !tr->idx )
        {
            tr->idx = (uint32_t*)malloc(sizeof(*tr->idx)*args->ntrio);
            tr->sd_bset = kbs_init(args->ntrio);
        }
        else
            kbs_clear(tr->sd_bset);
        tr->nsd  = 0;
        tr->nalt = 0;
    }
}
static void alt_trios_destroy(args_t *args)
{
    if ( !args->max_alt_trios ) return;
    int i;
    for (i=0; i<args->malt_trios; i++)
    {
        free(args->alt_trios[i].idx);
        kbs_destroy(args->alt_trios[i].sd_bset);
    }
    free(args->alt_trios);
}
static inline void alt_trios_add(args_t *args, int itrio, int ial, int is_singleton)
{
    alt_trios_t *tr = &args->alt_trios[ial];
    if ( is_singleton ) kbs_insert(tr->sd_bset, tr->nsd);
    tr->idx[ tr->nsd++ ] = itrio;
}
static void destroy_data(args_t *args)
{
    int i;
    for (i=0; i<args->nfilters; i++)
    {
        if ( args->filters[i].filter ) filter_destroy(args->filters[i].filter);
        free(args->filters[i].stats);
        free(args->filters[i].expr);
    }
    free(args->filters);
    for (i=0; i<args->nflt_str; i++) free(args->flt_str[i]);
    free(args->flt_str);
    bcf_sr_destroy(args->sr);
    alt_trios_destroy(args);
    free(args->trio);
    free(args->ac);
    free(args->ac_trio);
    free(args->gt_arr);
    free(args->dnm_als);
    if ( fclose(args->fp_out)!=0 ) error("Close failed: %s\n", (!args->output_fname || !strcmp("-",args->output_fname)) ? "stdout" : args->output_fname);
    free(args);
}
static void report_stats(args_t *args)
{
    int i = 0,j;
    for (i=0; i<args->nfilters; i++)
    {
        flt_stats_t *flt = &args->filters[i];
        fprintf(args->fp_out,"DEF\tFLT%d\t%s\n", i, flt->expr);
    }
    for (i=0; i<args->nfilters; i++)
    {
        flt_stats_t *flt = &args->filters[i];
        for (j=0; j<args->ntrio; j++)
        {
            fprintf(args->fp_out,"FLT%d", i);
            fprintf(args->fp_out,"\t%s",args->hdr->samples[args->trio[j].idx[iCHILD]]);
            fprintf(args->fp_out,"\t%s",args->hdr->samples[args->trio[j].idx[iFATHER]]);
            fprintf(args->fp_out,"\t%s",args->hdr->samples[args->trio[j].idx[iMOTHER]]);
            trio_stats_t *stats = &flt->stats[j];
            fprintf(args->fp_out,"\t%d", stats->npass);
            fprintf(args->fp_out,"\t%d", stats->nnon_ref);
            fprintf(args->fp_out,"\t%d", stats->nmendel_err);
            fprintf(args->fp_out,"\t%d", stats->nnovel);
            fprintf(args->fp_out,"\t%d", stats->nsingleton);
            fprintf(args->fp_out,"\t%d", stats->ndoubleton);
            fprintf(args->fp_out,"\t%d", stats->nts);
            fprintf(args->fp_out,"\t%d", stats->ntv);
            fprintf(args->fp_out,"\t%.2f", stats->ntv ? (float)stats->nts/stats->ntv : INFINITY);
            fprintf(args->fp_out,"\t%d", stats->ndnm_hom);
            fprintf(args->fp_out,"\t%d", stats->ndnm_recurrent);
            fprintf(args->fp_out,"\n");
        }
    }
}

static inline int parse_genotype(int32_t *arr, int ngt1, int idx, int als[2])
{
    int32_t *ptr = arr + ngt1 * idx;
    if ( bcf_gt_is_missing(ptr[0]) ) return -1;
    als[0] = bcf_gt_allele(ptr[0]);

    // treat haploid GTs as homozygous diploid
    if ( ngt1==1 || ptr[1]==bcf_int32_vector_end ) { als[1] = als[0]; return 0; }

    if ( bcf_gt_is_missing(ptr[1]) ) return -1;
    als[1] = bcf_gt_allele(ptr[1]);

    return 0;
}

static void process_record(args_t *args, bcf1_t *rec, flt_stats_t *flt)
{
    int i,j;

    // Find out which trios pass and if the site passes
    if ( flt->filter )
    {
        uint8_t *smpl_pass = NULL;
        int pass_site = filter_test(flt->filter, rec, (const uint8_t**) &smpl_pass);
        if ( args->filter_logic & FLT_EXCLUDE )
        {
            if ( pass_site )
            {
                if ( !smpl_pass ) return;
                pass_site = 0;
                for (i=0; i<args->ntrio; i++)
                {
                    int pass_trio = 1;
                    for (j=0; j<3; j++)
                    {
                        int idx = args->trio[i].idx[j];
                        if ( smpl_pass[idx] ) { pass_trio = 0; break; }
                    }
                    args->trio[i].pass = pass_trio;
                    if ( pass_trio ) pass_site = 1;
                }
                if ( !pass_site ) return;
            }
            else
                for (i=0; i<args->ntrio; i++) args->trio[i].pass = 1;
        }
        else if ( !pass_site ) return;
        else if ( smpl_pass )
        {
            pass_site = 0;
            for (i=0; i<args->ntrio; i++)
            {
                int pass_trio = 1;
                for (j=0; j<3; j++)
                {
                    int idx = args->trio[i].idx[j];
                    if ( !smpl_pass[idx] ) { pass_trio = 0; break; }
                }
                args->trio[i].pass = pass_trio;
                if ( pass_trio ) pass_site = 1;
            }
            if ( !pass_site ) return;
        }
        else
            for (i=0; i<args->ntrio; i++) args->trio[i].pass = 1;
    }

    // Find out the allele counts. Try to use INFO/AC, if not present, determine from the genotypes
    hts_expand(int, rec->n_allele, args->mac, args->ac);
    if ( !bcf_calc_ac(args->hdr, rec, args->ac, BCF_UN_INFO|BCF_UN_FMT) ) return;
    hts_expand(int, rec->n_allele, args->mac_trio, args->ac_trio);
    hts_expand(int, rec->n_allele, args->mdnm_als, args->dnm_als);

    // Get the genotypes
    int ngt = bcf_get_genotypes(args->hdr, rec, &args->gt_arr, &args->mgt_arr);
    if ( ngt<0 ) return;
    int ngt1 = ngt / rec->n_sample;


    // For ts/tv: numeric code of the reference allele, -1 for insertions
    int ref = !rec->d.allele[0][1] ? bcf_acgt2int(*rec->d.allele[0]) : -1;

    int star_allele = -1;
    for (i=1; i<rec->n_allele; i++)
        if ( !rec->d.allele[i][1] && rec->d.allele[i][0]=='*' ) { star_allele = i; break; }

    // number of non-reference trios
    if ( args->max_alt_trios ) alt_trios_reset(args, rec->n_allele);

    // Run the stats
    for (i=0; i<args->ntrio; i++)
    {
        if ( flt->filter && !args->trio[i].pass ) continue;
        trio_stats_t *stats = &flt->stats[i];

        // Determine the alternate allele and the genotypes, skip if any of the alleles is missing.
        // the order is: child, father, mother
        int als[6], *als_child = als, *als_father = als+2, *als_mother = als+4;
        if ( parse_genotype(args->gt_arr, ngt1, args->trio[i].idx[iCHILD], als_child) < 0 ) continue;
        if ( parse_genotype(args->gt_arr, ngt1, args->trio[i].idx[iFATHER], als_father) < 0 ) continue;
        if ( parse_genotype(args->gt_arr, ngt1, args->trio[i].idx[iMOTHER], als_mother) < 0 ) continue;

        stats->npass++;

        // Has the trio an alternate allele other than *?
        int has_star_allele = 0, has_nonref = 0;
        memset(args->ac_trio,0,rec->n_allele*sizeof(*args->ac_trio));
        for (j=0; j<6; j++)
        {
            if ( als[j]==star_allele ) { has_star_allele = 1; continue; }
            if ( als[j]!=0 ) has_nonref = 1;
            args->ac_trio[ als[j] ]++;
        }
        if ( !has_nonref ) continue;   // only ref or * in this trio

        stats->nnon_ref++;

        // Calculate ts/tv. It does the right thing and handles also HetAA genotypes
        if ( ref != -1 )
        {
            int has_ts = 0, has_tv = 0;
            for (j=0; j<6; j++)
            {
                if ( als[j]==0 || als[j]==star_allele ) continue;
                if ( als[j] >= rec->n_allele )
                    error("The GT index is out of range at %s:%"PRId64" in %s\n", bcf_seqname(args->hdr,rec),(int64_t) rec->pos+1,args->hdr->samples[args->trio[i].idx[j/2]]);
                if ( rec->d.allele[als[j]][1] ) continue;

                int alt = bcf_acgt2int(rec->d.allele[als[j]][0]);
                if ( abs(ref-alt)==2 ) has_ts = 1;
                else has_tv = 1;
            }
            if ( has_ts ) stats->nts++;
            if ( has_tv ) stats->ntv++;
        }

        // Skip some stats if the star allele is present as it was already checked at the primary record, we do not want to count the same
        // thing multiple times. There can be other alternate allele, but we ignore that for simplicity.
        if ( has_star_allele ) continue;

        // Detect mendelian errors
        int a0F = als_child[0]==als_father[0] || als_child[0]==als_father[1] ? 1 : 0;
        int a1M = als_child[1]==als_mother[0] || als_child[1]==als_mother[1] ? 1 : 0;
        if ( !a0F || !a1M )
        {
            int a0M = als_child[0]==als_mother[0] || als_child[0]==als_mother[1] ? 1 : 0;
            int a1F = als_child[1]==als_father[0] || als_child[1]==als_father[1] ? 1 : 0;
            if ( !a0M || !a1F )
            {
                stats->nmendel_err++;

                int dnm_hom = 0;
                if ( als_child[0]==als_child[1] ) { stats->ndnm_hom++; dnm_hom = 1; }

                int culprit;    // neglecting the unlikely possibility of alt het 1/2 DNM genotype
                if ( !a0F && !a0M ) culprit = als_child[0];
                else if ( !a1F && !a1M ) culprit = als_child[1];
                else if ( args->ac[als_child[0]] < args->ac[als_child[1]] ) culprit = als_child[0];
                else culprit = als_child[1];

                int dnm_recurrent = 0;
                if ( (!dnm_hom && args->ac[culprit]>1) || (dnm_hom && args->ac[culprit]>2) ) { stats->ndnm_recurrent++; dnm_recurrent = 1; }

                if ( args->verbose & VERBOSE_MENDEL )
                    fprintf(args->fp_out,"MERR\t%s\t%"PRId64"\t%s\t%s\t%s\t%s\t%s\n", bcf_seqname(args->hdr,rec),(int64_t) rec->pos+1,
                            args->hdr->samples[args->trio[i].idx[iCHILD]],
                            args->hdr->samples[args->trio[i].idx[iFATHER]],
                            args->hdr->samples[args->trio[i].idx[iMOTHER]],
                            dnm_hom ? "HOM" : "-",
                            dnm_recurrent ? "RECURRENT" : "-"
                           );
            }
        }

        // Is this a singleton, doubleton, neither?
        for (j=0; j<rec->n_allele; j++)
        {
            if ( !args->ac_trio[j] ) continue;
            if ( args->max_alt_trios ) args->alt_trios[j].nalt++;

            if ( args->ac_trio[j]==1 )  // singleton (in parent) or novel (in child)
            {
                if ( als_child[0]==j || als_child[1]==j ) stats->nnovel++;
                else
                {
                    if ( !args->max_alt_trios )
                    {
                        stats->nsingleton++;
                        if ( args->verbose & VERBOSE_TRANSMITTED )
                            fprintf(args->fp_out,"TRANSMITTED\t%s\t%"PRId64"\t%s\t%s\t%s\tNO\n", bcf_seqname(args->hdr,rec),(int64_t) rec->pos+1,
                                    args->hdr->samples[args->trio[i].idx[iCHILD]],
                                    args->hdr->samples[args->trio[i].idx[iFATHER]],
                                    args->hdr->samples[args->trio[i].idx[iMOTHER]]
                                   );
                    }
                    else alt_trios_add(args, i,j,1);
                }
            }
            else if ( args->ac_trio[j]==2 ) // possibly a doubleton
            {
                if ( (als_child[0]!=j && als_child[1]!=j) || (als_child[0]==j && als_child[1]==j) ) continue;
                if ( (als_father[0]==j && als_father[1]==j) || (als_mother[0]==j && als_mother[1]==j) ) continue;
                if ( !args->max_alt_trios )
                {
                    stats->ndoubleton++;
                    if ( args->verbose & VERBOSE_TRANSMITTED )
                        fprintf(args->fp_out,"TRANSMITTED\t%s\t%"PRId64"\t%s\t%s\t%s\tYES\n", bcf_seqname(args->hdr,rec),(int64_t) rec->pos+1,
                                args->hdr->samples[args->trio[i].idx[iCHILD]],
                                args->hdr->samples[args->trio[i].idx[iFATHER]],
                                args->hdr->samples[args->trio[i].idx[iMOTHER]]
                               );
                }
                else alt_trios_add(args, i,j,0);
            }
        }
    }
    if ( args->max_alt_trios )
    {
        for (j=0; j<rec->n_allele; j++)
        {
            alt_trios_t *tr = &args->alt_trios[j];
            if ( !tr->nsd || tr->nalt > args->max_alt_trios ) continue;
            for (i=0; i<tr->nsd; i++)
            {
                int itr = tr->idx[i];
                trio_stats_t *stats = &flt->stats[itr];
                if ( kbs_exists(tr->sd_bset,i) )
                {
                    stats->nsingleton++;
                    if ( args->verbose & VERBOSE_TRANSMITTED )
                        fprintf(args->fp_out,"TRANSMITTED\t%s\t%"PRId64"\t%s\t%s\t%s\tNO\n", bcf_seqname(args->hdr,rec),(int64_t) rec->pos+1,
                                args->hdr->samples[args->trio[itr].idx[iCHILD]],
                                args->hdr->samples[args->trio[itr].idx[iFATHER]],
                                args->hdr->samples[args->trio[itr].idx[iMOTHER]]
                               );
                }
                else
                {
                    stats->ndoubleton++;
                    if ( args->verbose & VERBOSE_TRANSMITTED )
                        fprintf(args->fp_out,"TRANSMITTED\t%s\t%"PRId64"\t%s\t%s\t%s\tYES\n", bcf_seqname(args->hdr,rec),(int64_t) rec->pos+1,
                                args->hdr->samples[args->trio[itr].idx[iCHILD]],
                                args->hdr->samples[args->trio[itr].idx[iFATHER]],
                                args->hdr->samples[args->trio[itr].idx[iMOTHER]]
                               );
                }
            }
        }
    }
}

int run(int argc, char **argv)
{
    args_t *args = (args_t*) calloc(1,sizeof(args_t));
    args->argc   = argc; args->argv = argv;
    args->output_fname = "-";
    static struct option loptions[] =
    {
        {"debug",required_argument,0,'d'},
        {"alt-trios",required_argument,0,'a'},
        {"include",required_argument,0,'i'},
        {"exclude",required_argument,0,'e'},
        {"output",required_argument,NULL,'o'},
        {"ped",required_argument,NULL,'p'},
        {"pfm",required_argument,NULL,'P'},
        {"regions",1,0,'r'},
        {"regions-file",1,0,'R'},
        {"targets",1,0,'t'},
        {"targets-file",1,0,'T'},
        {NULL,0,NULL,0}
    };
    int c, i;
    while ((c = getopt_long(argc, argv, "P:p:o:s:i:e:r:R:t:T:a:d:",loptions,NULL)) >= 0)
    {
        switch (c)
        {
            case 'd':
            {
                int n;
                char **tmp = hts_readlist(optarg, 0, &n);
                for(i=0; i<n; i++)
                {
                    if ( !strcasecmp(tmp[i],"mendel-errors") ) args->verbose |= VERBOSE_MENDEL;
                    else if ( !strcasecmp(tmp[i],"transmitted") ) args->verbose |= VERBOSE_TRANSMITTED;
                    else error("Error: The argument \"%s\" to option --debug is not recognised\n", tmp[i]);
                    free(tmp[i]);
                }
                free(tmp);
                break;
            }
            case 'a': args->max_alt_trios = atoi(optarg); break;
            case 'e':
                if ( args->filter_str ) error("Error: only one -i or -e expression can be given, and they cannot be combined\n");
                args->filter_str = optarg; args->filter_logic |= FLT_EXCLUDE; break;
            case 'i':
                if ( args->filter_str ) error("Error: only one -i or -e expression can be given, and they cannot be combined\n");
                args->filter_str = optarg; args->filter_logic |= FLT_INCLUDE; break;
            case 't': args->targets = optarg; break;
            case 'T': args->targets = optarg; args->targets_is_file = 1; break;
            case 'r': args->regions = optarg; break;
            case 'R': args->regions = optarg; args->regions_is_file = 1; break;
            case 'o': args->output_fname = optarg; break;
            case 'p': args->ped_fname = optarg; break;
            case 'P': args->pfm = optarg; break;
            case 'h':
            case '?':
            default: error("%s", usage_text()); break;
        }
    }
    if ( optind==argc )
    {
        if ( !isatty(fileno((FILE *)stdin)) ) args->fname = "-";  // reading from stdin
        else { error("%s", usage_text()); }
    }
    else if ( optind+1!=argc ) error("%s", usage_text());
    else args->fname = argv[optind];

    if ( !args->ped_fname && !args->pfm ) error("Missing the -p or -P option\n");

    init_data(args);

    while ( bcf_sr_next_line(args->sr) )
    {
        bcf1_t *rec = bcf_sr_get_line(args->sr,0);
        for (i=0; i<args->nfilters; i++)
            process_record(args, rec, &args->filters[i]);
    }

    report_stats(args);
    destroy_data(args);

    return 0;
}