[samtools.git] / misc / bamcheck.c

/* 
    Author: petr.danecek@sanger
    gcc -Wall -Winline -g -O2 -I ~/git/samtools bamcheck.c -o bamcheck -lm -lz -L ~/git/samtools -lbam

    Assumptions, approximations and other issues:
        - GC-depth graph does not split reads, the starting position determines which bin is incremented.
            There are small overlaps between bins (max readlen-1). However, the bins are big (20k).
        - coverage distribution ignores softclips and deletions
        - some stats require sorted BAMs
        - GC content graph can have an untidy, step-like pattern when BAM contains multiple read lengths.
        - The whole reads are used with -t, no splicing is done, no indels or soft clips are 
            considered, even small overlap is good enough to include the read in the stats.
*/

#define BAMCHECK_VERSION "2012-04-04"

#define _ISOC99_SOURCE
#include <stdio.h>
#include <stdlib.h>
#include <stdarg.h>
#include <string.h>
#include <math.h>
#include <ctype.h>
#include <getopt.h>
#include <errno.h>
#include "faidx.h"
#include "khash.h"
#include "sam.h"
#include "razf.h"

#define BWA_MIN_RDLEN 35
#define IS_PAIRED(bam) ((bam)->core.flag&BAM_FPAIRED && !((bam)->core.flag&BAM_FUNMAP) && !((bam)->core.flag&BAM_FMUNMAP))
#define IS_UNMAPPED(bam) ((bam)->core.flag&BAM_FUNMAP)
#define IS_REVERSE(bam) ((bam)->core.flag&BAM_FREVERSE)
#define IS_MATE_REVERSE(bam) ((bam)->core.flag&BAM_FMREVERSE)
#define IS_READ1(bam) ((bam)->core.flag&BAM_FREAD1)
#define IS_READ2(bam) ((bam)->core.flag&BAM_FREAD2)
#define IS_DUP(bam) ((bam)->core.flag&BAM_FDUP)

typedef struct 
{
    int32_t line_len, line_blen;
    int64_t len;
    uint64_t offset;
} 
faidx1_t;
KHASH_MAP_INIT_STR(s, faidx1_t)
KHASH_MAP_INIT_STR(str, int)
struct __faidx_t {
    RAZF *rz;
    int n, m;
    char **name;
    khash_t(s) *hash;
};

typedef struct
{
    float gc;
    uint32_t depth;
}
gc_depth_t;

// For coverage distribution, a simple pileup
typedef struct 
{
    int64_t pos;
    int size, start;
    int *buffer;
} 
round_buffer_t;

typedef struct { uint32_t from, to; } pos_t;
typedef struct
{
    int npos,mpos,cpos;
    pos_t *pos;
}
regions_t;

typedef struct
{
    // Parameters
    int trim_qual;      // bwa trim quality
    int rmdup;          // Exclude reads marked as duplicates from the stats

    // Dimensions of the quality histogram holder (quals_1st,quals_2nd), GC content holder (gc_1st,gc_2nd),
    //  insert size histogram holder
    int nquals;         // The number of quality bins 
    int nbases;         // The maximum sequence length the allocated array can hold
    int nisize;         // The maximum insert size that the allocated array can hold
    int ngc;            // The size of gc_1st and gc_2nd
    int nindels;        // The maximum indel length for indel distribution

    // Arrays for the histogram data
    uint64_t *quals_1st, *quals_2nd;
    uint64_t *gc_1st, *gc_2nd;
    uint64_t *isize_inward, *isize_outward, *isize_other;
    uint64_t *acgt_cycles;
    uint64_t *read_lengths;
    uint64_t *insertions, *deletions;
    uint64_t *ins_cycles, *del_cycles;

    // The extremes encountered
    int max_len;            // Maximum read length
    int max_qual;           // Maximum quality
    float isize_main_bulk;  // There are always some unrealistically big insert sizes, report only the main part
    int is_sorted;

    // Summary numbers
    uint64_t total_len;
    uint64_t total_len_dup;
    uint64_t nreads_1st;
    uint64_t nreads_2nd;
    uint64_t nreads_dup;
    uint64_t nreads_unmapped;
    uint64_t nreads_unpaired;
    uint64_t nreads_paired;
    uint64_t nreads_mq0;
    uint64_t nbases_mapped;
    uint64_t nbases_mapped_cigar;
    uint64_t nbases_trimmed;  // bwa trimmed bases
    uint64_t nmismatches;

    // GC-depth related data
    uint32_t ngcd, igcd;        // The maximum number of GC depth bins and index of the current bin
    gc_depth_t *gcd;            // The GC-depth bins holder
    int gcd_bin_size;           // The size of GC-depth bin
    int32_t tid, gcd_pos;       // Position of the current bin
    int32_t pos;                // Position of the last read

    // Coverage distribution related data
    int ncov;                       // The number of coverage bins
    uint64_t *cov;                  // The coverage frequencies
    int cov_min,cov_max,cov_step;   // Minimum, maximum coverage and size of the coverage bins
    round_buffer_t cov_rbuf;        // Pileup round buffer

    // Mismatches by read cycle 
    uint8_t *rseq_buf;             // A buffer for reference sequence to check the mismatches against
    int nref_seq;               // The size of the buffer
    int32_t rseq_pos;           // The coordinate of the first base in the buffer
    int32_t rseq_len;           // The used part of the buffer
    uint64_t *mpc_buf;          // Mismatches per cycle

    // Filters
    int filter_readlen;

    // Target regions
    int nregions;
    regions_t *regions;

    // Auxiliary data
    double sum_qual;            // For calculating average quality value 
    samfile_t *sam;             // Unused
    faidx_t *fai;               // Reference sequence for GC-depth graph
    int argc;                   // Command line arguments to be printed on the output
    char **argv;
}
stats_t;

void error(const char *format, ...);

// Coverage distribution methods
inline int coverage_idx(int min, int max, int n, int step, int depth)
{
    if ( depth < min )
        return 0;

    if ( depth > max )
        return n-1;

    return 1 + (depth - min) / step;
}

inline int round_buffer_lidx2ridx(int offset, int size, int64_t refpos, int64_t pos)
{
    return (offset + (pos-refpos) % size) % size;
}

void round_buffer_flush(stats_t *stats, int64_t pos)
{
    int ibuf,idp;

    if ( pos==stats->cov_rbuf.pos ) 
        return;

    int64_t new_pos = pos;
    if ( pos==-1 || pos - stats->cov_rbuf.pos >= stats->cov_rbuf.size )
    {
        // Flush the whole buffer, but in sequential order, 
        pos = stats->cov_rbuf.pos + stats->cov_rbuf.size - 1;
    }

    if ( pos < stats->cov_rbuf.pos ) 
        error("Expected coordinates in ascending order, got %ld after %ld\n", pos,stats->cov_rbuf.pos);

    int ifrom = stats->cov_rbuf.start;
    int ito = round_buffer_lidx2ridx(stats->cov_rbuf.start,stats->cov_rbuf.size,stats->cov_rbuf.pos,pos-1);
    if ( ifrom>ito )
    {
        for (ibuf=ifrom; ibuf<stats->cov_rbuf.size; ibuf++)
        {
            if ( !stats->cov_rbuf.buffer[ibuf] )
                continue;
            idp = coverage_idx(stats->cov_min,stats->cov_max,stats->ncov,stats->cov_step,stats->cov_rbuf.buffer[ibuf]);
            stats->cov[idp]++;
            stats->cov_rbuf.buffer[ibuf] = 0;
        }
        ifrom = 0;
    }
    for (ibuf=ifrom; ibuf<=ito; ibuf++)
    {
        if ( !stats->cov_rbuf.buffer[ibuf] )
            continue;
        idp = coverage_idx(stats->cov_min,stats->cov_max,stats->ncov,stats->cov_step,stats->cov_rbuf.buffer[ibuf]);
        stats->cov[idp]++;
        stats->cov_rbuf.buffer[ibuf] = 0;
    }
    stats->cov_rbuf.start = (new_pos==-1) ? 0 : round_buffer_lidx2ridx(stats->cov_rbuf.start,stats->cov_rbuf.size,stats->cov_rbuf.pos,pos);
    stats->cov_rbuf.pos   = new_pos;
}

void round_buffer_insert_read(round_buffer_t *rbuf, int64_t from, int64_t to)
{
    if ( to-from >= rbuf->size )
        error("The read length too big (%d), please increase the buffer length (currently %d)\n", to-from+1,rbuf->size);
    if ( from < rbuf->pos )
        error("The reads are not sorted (%ld comes after %ld).\n", from,rbuf->pos);

    int ifrom,ito,ibuf;
    ifrom = round_buffer_lidx2ridx(rbuf->start,rbuf->size,rbuf->pos,from);
    ito   = round_buffer_lidx2ridx(rbuf->start,rbuf->size,rbuf->pos,to);
    if ( ifrom>ito )
    {
        for (ibuf=ifrom; ibuf<rbuf->size; ibuf++)
            rbuf->buffer[ibuf]++;
        ifrom = 0;
    }
    for (ibuf=ifrom; ibuf<=ito; ibuf++)
        rbuf->buffer[ibuf]++;
}

// Calculate the number of bases in the read trimmed by BWA
int bwa_trim_read(int trim_qual, uint8_t *quals, int len, int reverse) 
{
    if ( len<BWA_MIN_RDLEN ) return 0;

    // Although the name implies that the read cannot be trimmed to more than BWA_MIN_RDLEN,
    //  the calculation can in fact trim it to (BWA_MIN_RDLEN-1). (bwa_trim_read in bwa/bwaseqio.c).
    int max_trimmed = len - BWA_MIN_RDLEN + 1;
    int l, sum=0, max_sum=0, max_l=0;

    for (l=0; l<max_trimmed; l++)
    {
        sum += trim_qual - quals[ reverse ? l : len-1-l ];
        if ( sum<0 ) break;
        if ( sum>max_sum )
        {
            max_sum = sum;
            // This is the correct way, but bwa clips from some reason one base less
            // max_l   = l+1;
            max_l   = l;
        }
    }
    return max_l;
}


void count_indels(stats_t *stats,bam1_t *bam_line) 
{
    int is_fwd = IS_REVERSE(bam_line) ? 0 : 1;
    int icig;
    int icycle = 0;
    int read_len = bam_line->core.l_qseq;
    for (icig=0; icig<bam_line->core.n_cigar; icig++) 
    {
        // Conversion from uint32_t to MIDNSHP
        //  0123456
        //  MIDNSHP
        int cig  = bam1_cigar(bam_line)[icig] & BAM_CIGAR_MASK;
        int ncig = bam1_cigar(bam_line)[icig] >> BAM_CIGAR_SHIFT;

        if ( cig==1 )
        {
            int idx = is_fwd ? icycle : read_len-icycle;
            if ( idx<0 ) error("FIXME: read_len=%d vs icycle=%d\n", read_len,icycle);
            if ( idx >= stats->nbases || idx<0 ) error("FIXME: %d vs %d\n", idx,stats->nbases);
            stats->ins_cycles[idx]++;
            icycle += ncig;
            if ( ncig<=stats->nindels )
                stats->insertions[ncig-1]++;
            continue;
        }
        if ( cig==2 )
        {
            int idx = is_fwd ? icycle-1 : read_len-icycle-1;
            if ( idx<0 ) continue;  // discard meaningless deletions
            if ( idx >= stats->nbases ) error("FIXME: %d vs %d\n", idx,stats->nbases);
            stats->del_cycles[idx]++;
            if ( ncig<=stats->nindels )
                stats->deletions[ncig-1]++;
            continue;
        }
        icycle += ncig;
    }
}

void count_mismatches_per_cycle(stats_t *stats,bam1_t *bam_line) 
{
    int is_fwd = IS_REVERSE(bam_line) ? 0 : 1;
    int icig,iread=0,icycle=0;
    int iref = bam_line->core.pos - stats->rseq_pos;
    int read_len   = bam_line->core.l_qseq;
    uint8_t *read  = bam1_seq(bam_line);
    uint8_t *quals = bam1_qual(bam_line);
    uint64_t *mpc_buf = stats->mpc_buf;
    for (icig=0; icig<bam_line->core.n_cigar; icig++) 
    {
        // Conversion from uint32_t to MIDNSHP
        //  0123456
        //  MIDNSHP
        int cig  = bam1_cigar(bam_line)[icig] & BAM_CIGAR_MASK;
        int ncig = bam1_cigar(bam_line)[icig] >> BAM_CIGAR_SHIFT;
        if ( cig==1 )
        {
            iread  += ncig;
            icycle += ncig;
            continue;
        }
        if ( cig==2 )
        {
            iref += ncig;
            continue;
        }
        if ( cig==4 )
        {
            icycle += ncig;
            // Soft-clips are present in the sequence, but the position of the read marks a start of non-clipped sequence
            //   iref += ncig;
            iread  += ncig;
            continue;
        }
        if ( cig==5 )
        {
            icycle += ncig;
            continue;
        }
        if ( cig!=0 )
            error("TODO: cigar %d, %s\n", cig,bam1_qname(bam_line));
       
        if ( ncig+iref > stats->rseq_len )
            error("FIXME: %d+%d > %d, %s, %s:%d\n",ncig,iref,stats->rseq_len, bam1_qname(bam_line),stats->sam->header->target_name[bam_line->core.tid],bam_line->core.pos+1);

        int im;
        for (im=0; im<ncig; im++)
        {
            uint8_t cread = bam1_seqi(read,iread);
            uint8_t cref  = stats->rseq_buf[iref];

            // ---------------15
            // =ACMGRSVTWYHKDBN
            if ( cread==15 )
            {
                int idx = is_fwd ? icycle : read_len-icycle-1;
                if ( idx>stats->max_len )
                    error("mpc: %d>%d\n",idx,stats->max_len);
                idx = idx*stats->nquals;
                if ( idx>=stats->nquals*stats->nbases )
                    error("FIXME: mpc_buf overflow\n");
                mpc_buf[idx]++;
            }
            else if ( cref && cread && cref!=cread )
            {
                uint8_t qual = quals[iread] + 1;
                if ( qual>=stats->nquals )
                    error("TODO: quality too high %d>=%d\n", quals[iread],stats->nquals);

                int idx = is_fwd ? icycle : read_len-icycle-1;
                if ( idx>stats->max_len )
                    error("mpc: %d>%d\n",idx,stats->max_len);

                idx = idx*stats->nquals + qual;
                if ( idx>=stats->nquals*stats->nbases )
                    error("FIXME: mpc_buf overflow\n");
                mpc_buf[idx]++;
            }

            iref++;
            iread++;
            icycle++;
        }
    }
}

void read_ref_seq(stats_t *stats,int32_t tid,int32_t pos)
{
    khash_t(s) *h;
    khiter_t iter;
    faidx1_t val;
    char *chr, c;
    faidx_t *fai = stats->fai;

    h = fai->hash;
    chr = stats->sam->header->target_name[tid];

    // ID of the sequence name
    iter = kh_get(s, h, chr);
    if (iter == kh_end(h)) 
        error("No such reference sequence [%s]?\n", chr);
    val = kh_value(h, iter);

    // Check the boundaries
    if (pos >= val.len)
        error("Was the bam file mapped with the reference sequence supplied?"
              " A read mapped beyond the end of the chromosome (%s:%d, chromosome length %d).\n", chr,pos,val.len);
    int size = stats->nref_seq;
    // The buffer extends beyond the chromosome end. Later the rest will be filled with N's.
    if (size+pos > val.len) size = val.len-pos;

    // Position the razf reader
    razf_seek(fai->rz, val.offset + pos / val.line_blen * val.line_len + pos % val.line_blen, SEEK_SET);

    uint8_t *ptr = stats->rseq_buf;
    int nread = 0;
    while ( nread<size && razf_read(fai->rz,&c,1) && !fai->rz->z_err )
    {
        if ( !isgraph(c) )
            continue;

        // Conversion between uint8_t coding and ACGT
        //      -12-4---8-------
        //      =ACMGRSVTWYHKDBN
        if ( c=='A' || c=='a' )
            *ptr = 1;
        else if ( c=='C' || c=='c' )
            *ptr = 2;
        else if ( c=='G' || c=='g' )
            *ptr = 4;
        else if ( c=='T' || c=='t' )
            *ptr = 8;
        else
            *ptr = 0;
        ptr++;
        nread++;
    }
    if ( nread < stats->nref_seq )
    {
        memset(ptr,0, stats->nref_seq - nread);
        nread = stats->nref_seq;
    }
    stats->rseq_len = nread;
    stats->rseq_pos = pos;
    stats->tid      = tid;
}

float fai_gc_content(stats_t *stats)
{
    uint32_t gc,count,c;
    int i,size = stats->rseq_len;

    // Count GC content
    gc = count = 0;
    for (i=0; i<size; i++)
    {
        c = stats->rseq_buf[i];
        if ( c==2 || c==4 )
        {
            gc++;
            count++;
        }
        else if ( c==1 || c==8 )
            count++;
    }
    return count ? (float)gc/count : 0;
}


void realloc_buffers(stats_t *stats, int seq_len)
{
    int n = 2*(1 + seq_len - stats->nbases) + stats->nbases;

    stats->quals_1st = realloc(stats->quals_1st, n*stats->nquals*sizeof(uint64_t));
    if ( !stats->quals_1st )
        error("Could not realloc buffers, the sequence too long: %d (%ld)\n", seq_len,n*stats->nquals*sizeof(uint64_t));
    memset(stats->quals_1st + stats->nbases*stats->nquals, 0, (n-stats->nbases)*stats->nquals*sizeof(uint64_t));

    stats->quals_2nd = realloc(stats->quals_2nd, n*stats->nquals*sizeof(uint64_t));
    if ( !stats->quals_2nd )
        error("Could not realloc buffers, the sequence too long: %d (2x%ld)\n", seq_len,n*stats->nquals*sizeof(uint64_t));
    memset(stats->quals_2nd + stats->nbases*stats->nquals, 0, (n-stats->nbases)*stats->nquals*sizeof(uint64_t));

    if ( stats->mpc_buf )
    {
        stats->mpc_buf = realloc(stats->mpc_buf, n*stats->nquals*sizeof(uint64_t));
        if ( !stats->mpc_buf )
            error("Could not realloc buffers, the sequence too long: %d (%ld)\n", seq_len,n*stats->nquals*sizeof(uint64_t));
        memset(stats->mpc_buf + stats->nbases*stats->nquals, 0, (n-stats->nbases)*stats->nquals*sizeof(uint64_t));
    }

    stats->acgt_cycles = realloc(stats->acgt_cycles, n*4*sizeof(uint64_t));
    if ( !stats->acgt_cycles )
        error("Could not realloc buffers, the sequence too long: %d (%ld)\n", seq_len,n*4*sizeof(uint64_t));
    memset(stats->acgt_cycles + stats->nbases*4, 0, (n-stats->nbases)*4*sizeof(uint64_t));

    stats->read_lengths = realloc(stats->read_lengths, n*sizeof(uint64_t));
    if ( !stats->read_lengths )
        error("Could not realloc buffers, the sequence too long: %d (%ld)\n", seq_len,n*sizeof(uint64_t));
    memset(stats->read_lengths + stats->nbases, 0, (n-stats->nbases)*sizeof(uint64_t));

    stats->insertions = realloc(stats->insertions, n*sizeof(uint64_t));
    if ( !stats->insertions )
        error("Could not realloc buffers, the sequence too long: %d (%ld)\n", seq_len,n*sizeof(uint64_t));
    memset(stats->insertions + stats->nbases, 0, (n-stats->nbases)*sizeof(uint64_t));

    stats->deletions = realloc(stats->deletions, n*sizeof(uint64_t));
    if ( !stats->deletions )
        error("Could not realloc buffers, the sequence too long: %d (%ld)\n", seq_len,n*sizeof(uint64_t));
    memset(stats->deletions + stats->nbases, 0, (n-stats->nbases)*sizeof(uint64_t));

    stats->ins_cycles = realloc(stats->ins_cycles, (n+1)*sizeof(uint64_t));
    if ( !stats->ins_cycles )
        error("Could not realloc buffers, the sequence too long: %d (%ld)\n", seq_len,(n+1)*sizeof(uint64_t));
    memset(stats->ins_cycles + stats->nbases + 1, 0, (n+1-stats->nbases)*sizeof(uint64_t));

    stats->del_cycles = realloc(stats->del_cycles, (n+1)*sizeof(uint64_t));
    if ( !stats->del_cycles )
        error("Could not realloc buffers, the sequence too long: %d (%ld)\n", seq_len,(n+1)*sizeof(uint64_t));
    memset(stats->del_cycles + stats->nbases + 1, 0, (n+1-stats->nbases)*sizeof(uint64_t));

    stats->nbases = n;

    // Realloc the coverage distribution buffer
    int *rbuffer = calloc(sizeof(int),seq_len*5);
    n = stats->cov_rbuf.size-stats->cov_rbuf.start;
    memcpy(rbuffer,stats->cov_rbuf.buffer+stats->cov_rbuf.start,n);
    if ( stats->cov_rbuf.start>1 )
        memcpy(rbuffer+n,stats->cov_rbuf.buffer,stats->cov_rbuf.start);
    stats->cov_rbuf.start = 0;
    free(stats->cov_rbuf.buffer);
    stats->cov_rbuf.buffer = rbuffer;
    stats->cov_rbuf.size = seq_len*5;
}

void collect_stats(bam1_t *bam_line, stats_t *stats)
{
    if ( stats->rmdup && IS_DUP(bam_line) )
        return;

    int seq_len = bam_line->core.l_qseq;
    if ( !seq_len ) return;
    if ( stats->filter_readlen!=-1 && seq_len!=stats->filter_readlen ) return;
    if ( seq_len >= stats->nbases )
        realloc_buffers(stats,seq_len);
    if ( stats->max_len<seq_len )
        stats->max_len = seq_len;

    stats->read_lengths[seq_len]++;

    // Count GC and ACGT per cycle
    uint8_t base, *seq  = bam1_seq(bam_line);
    int gc_count  = 0;
    int i;
    int reverse = IS_REVERSE(bam_line);
    for (i=0; i<seq_len; i++)
    {
        // Conversion from uint8_t coding to ACGT
        //      -12-4---8-------
        //      =ACMGRSVTWYHKDBN
        //       01 2   3
        base = bam1_seqi(seq,i);
        base /= 2;
        if ( base==1 || base==2 ) gc_count++;
        else if ( base>2 ) base=3;
        if ( 4*(reverse ? seq_len-i-1 : i) + base >= stats->nbases*4 ) 
            error("FIXME: acgt_cycles\n");
        stats->acgt_cycles[ 4*(reverse ? seq_len-i-1 : i) + base ]++;
    }
    int gc_idx_min = gc_count*(stats->ngc-1)/seq_len;
    int gc_idx_max = (gc_count+1)*(stats->ngc-1)/seq_len;
    if ( gc_idx_max >= stats->ngc ) gc_idx_max = stats->ngc - 1;

    // Determine which array (1st or 2nd read) will these stats go to,
    //  trim low quality bases from end the same way BWA does, 
    //  fill GC histogram
    uint64_t *quals;
    uint8_t *bam_quals = bam1_qual(bam_line);
    if ( bam_line->core.flag&BAM_FREAD2 )
    {
        quals  = stats->quals_2nd;
        stats->nreads_2nd++;
        for (i=gc_idx_min; i<gc_idx_max; i++)
            stats->gc_2nd[i]++;
    }
    else
    {
        quals = stats->quals_1st;
        stats->nreads_1st++;
        for (i=gc_idx_min; i<gc_idx_max; i++)
            stats->gc_1st[i]++;
    }
    if ( stats->trim_qual>0 ) 
        stats->nbases_trimmed += bwa_trim_read(stats->trim_qual, bam_quals, seq_len, reverse);

    // Quality histogram and average quality
    for (i=0; i<seq_len; i++)
    {
        uint8_t qual = bam_quals[ reverse ? seq_len-i-1 : i];
        if ( qual>=stats->nquals )
            error("TODO: quality too high %d>=%d\n", quals[i],stats->nquals);
        if ( qual>stats->max_qual )
            stats->max_qual = qual;

        quals[ i*stats->nquals+qual ]++;
        stats->sum_qual += qual;
    }

    // Look at the flags and increment appropriate counters (mapped, paired, etc)
    if ( IS_UNMAPPED(bam_line) )
        stats->nreads_unmapped++;
    else
    {
        if ( !bam_line->core.qual )
            stats->nreads_mq0++;

        count_indels(stats,bam_line);

        // The insert size is tricky, because for long inserts the libraries are
        // prepared differently and the pairs point in other direction. BWA does
        // not set the paired flag for them.  Similar thing is true also for 454
        // reads. Therefore, do the insert size stats for all mapped reads.
        int32_t isize = bam_line->core.isize;
        if ( isize<0 ) isize = -isize;
        if ( IS_PAIRED(bam_line) && isize!=0 )
        {
            stats->nreads_paired++;
            if ( isize >= stats->nisize )
                isize=stats->nisize-1;

            int pos_fst = bam_line->core.mpos - bam_line->core.pos;
            int is_fst  = IS_READ1(bam_line) ? 1 : -1;
            int is_fwd  = IS_REVERSE(bam_line) ? -1 : 1;
            int is_mfwd = IS_MATE_REVERSE(bam_line) ? -1 : 1;

            if ( is_fwd*is_mfwd>0 )
                stats->isize_other[isize]++;
            else if ( is_fst*pos_fst>0 )
            {
                if ( is_fst*is_fwd>0 )
                    stats->isize_inward[isize]++;
                else
                    stats->isize_outward[isize]++;
            }
            else if ( is_fst*pos_fst<0 )
            {
                if ( is_fst*is_fwd>0 )
                    stats->isize_outward[isize]++;
                else
                    stats->isize_inward[isize]++;
            }
        }
        else
            stats->nreads_unpaired++;

        // Number of mismatches 
        uint8_t *nm = bam_aux_get(bam_line,"NM");
        if (nm) 
            stats->nmismatches += bam_aux2i(nm);

        // Number of mapped bases from cigar 
        if ( bam_line->core.n_cigar == 0) 
            error("FIXME: mapped read with no cigar?\n");
        int readlen = seq_len;
        for (i=0; i<bam_line->core.n_cigar; i++) 
        {
            // Conversion from uint32_t to MIDNSHP
            //  01--4--
            //  MIDNSHP
            if ( (bam1_cigar(bam_line)[i]&BAM_CIGAR_MASK)==0 || (bam1_cigar(bam_line)[i]&BAM_CIGAR_MASK)==1 )
                stats->nbases_mapped_cigar += bam1_cigar(bam_line)[i]>>BAM_CIGAR_SHIFT;

            if ( (bam1_cigar(bam_line)[i]&BAM_CIGAR_MASK)==2 )
                readlen += bam1_cigar(bam_line)[i]>>BAM_CIGAR_SHIFT;
        }
        stats->nbases_mapped += seq_len;

        if ( stats->tid==bam_line->core.tid && bam_line->core.pos<stats->pos )
            stats->is_sorted = 0;
        stats->pos = bam_line->core.pos;

        if ( stats->is_sorted )
        {
            if ( stats->tid==-1 || stats->tid!=bam_line->core.tid )
                round_buffer_flush(stats,-1);

            // Mismatches per cycle and GC-depth graph
            if ( stats->fai )
            {
                // First pass, new chromosome or sequence spanning beyond the end of the buffer 
                if ( stats->rseq_pos==-1 || stats->tid != bam_line->core.tid || stats->rseq_pos+stats->rseq_len < bam_line->core.pos+readlen )
                {
                    read_ref_seq(stats,bam_line->core.tid,bam_line->core.pos);

                    // Get the reference GC content for this bin. Note that in the current implementation
                    //  the GCD bins overlap by seq_len-1. Because the bin size is by default 20k and the 
                    //  expected read length only ~100bp, it shouldn't really matter.
                    stats->gcd_pos = bam_line->core.pos;
                    stats->igcd++;

                    if ( stats->igcd >= stats->ngcd )
                        error("The genome too long or the GCD bin overlaps too big [%ud]\n", stats->igcd);

                    stats->gcd[ stats->igcd ].gc = fai_gc_content(stats);
                }
                count_mismatches_per_cycle(stats,bam_line);
            }
            else if ( stats->gcd_pos==-1 || stats->tid != bam_line->core.tid || bam_line->core.pos - stats->gcd_pos > stats->gcd_bin_size )
            {
                // First pass or a new chromosome
                stats->tid     = bam_line->core.tid;
                stats->gcd_pos = bam_line->core.pos;
                stats->igcd++;

                if ( stats->igcd >= stats->ngcd )
                {
                    uint32_t n = 2*(1 + stats->ngcd);
                    stats->gcd = realloc(stats->gcd, n*sizeof(gc_depth_t));
                    if ( !stats->gcd )
                        error("Could not realloc GCD buffer, too many chromosomes or the genome too long?? [%u %u]\n", stats->ngcd,n);
                    memset(&(stats->gcd[stats->ngcd]),0,(n-stats->ngcd)*sizeof(gc_depth_t));
                }
            }

            stats->gcd[ stats->igcd ].depth++;
            // When no reference sequence is given, approximate the GC from the read (much shorter window, but otherwise OK)
            if ( !stats->fai )
                stats->gcd[ stats->igcd ].gc += (float) gc_count / seq_len;

            // Coverage distribution graph
            round_buffer_flush(stats,bam_line->core.pos);
            round_buffer_insert_read(&(stats->cov_rbuf),bam_line->core.pos,bam_line->core.pos+seq_len-1);
        }
    }

    stats->total_len += seq_len;
    if ( IS_DUP(bam_line) )
    {
        stats->total_len_dup += seq_len;
        stats->nreads_dup++;
    }
}

// Sort by GC and depth
#define GCD_t(x) ((gc_depth_t *)x)
static int gcd_cmp(const void *a, const void *b)
{
    if ( GCD_t(a)->gc < GCD_t(b)->gc ) return -1;
    if ( GCD_t(a)->gc > GCD_t(b)->gc ) return 1;
    if ( GCD_t(a)->depth < GCD_t(b)->depth ) return -1;
    if ( GCD_t(a)->depth > GCD_t(b)->depth ) return 1;
    return 0;
}
#undef GCD_t

float gcd_percentile(gc_depth_t *gcd, int N, int p)
{
    float n,d;
    int k;

    n = p*(N+1)/100;
    k = n;
    if ( k<=0 ) 
        return gcd[0].depth;
    if ( k>=N ) 
        return gcd[N-1].depth;

    d = n - k;
    return gcd[k-1].depth + d*(gcd[k].depth - gcd[k-1].depth);
}

void output_stats(stats_t *stats)
{
    // Calculate average insert size and standard deviation (from the main bulk data only)
    int isize, ibulk=0;
    uint64_t nisize=0, nisize_inward=0, nisize_outward=0, nisize_other=0;
    for (isize=1; isize<stats->nisize; isize++)
    {
        // Each pair was counted twice
        stats->isize_inward[isize] *= 0.5;
        stats->isize_outward[isize] *= 0.5;
        stats->isize_other[isize] *= 0.5;

        nisize_inward += stats->isize_inward[isize];
        nisize_outward += stats->isize_outward[isize];
        nisize_other += stats->isize_other[isize];
        nisize += stats->isize_inward[isize] + stats->isize_outward[isize] + stats->isize_other[isize];
    }

    double bulk=0, avg_isize=0, sd_isize=0;
    for (isize=1; isize<stats->nisize; isize++)
    {
        bulk += stats->isize_inward[isize] + stats->isize_outward[isize] + stats->isize_other[isize];
        avg_isize += isize * (stats->isize_inward[isize] + stats->isize_outward[isize] + stats->isize_other[isize]);

        if ( bulk/nisize > stats->isize_main_bulk )
        {
            ibulk  = isize+1;
            nisize = bulk;
            break;
        }
    }
    avg_isize /= nisize ? nisize : 1;
    for (isize=1; isize<ibulk; isize++)
        sd_isize += (stats->isize_inward[isize] + stats->isize_outward[isize] + stats->isize_other[isize]) * (isize-avg_isize)*(isize-avg_isize) / nisize;
    sd_isize = sqrt(sd_isize);


    printf("# This file was produced by bamcheck (%s)\n",BAMCHECK_VERSION);
    printf("# The command line was:  %s",stats->argv[0]);
    int i;
    for (i=1; i<stats->argc; i++)
        printf(" %s",stats->argv[i]);
    printf("\n");
    printf("# Summary Numbers. Use `grep ^SN | cut -f 2-` to extract this part.\n");
    printf("SN\tsequences:\t%ld\n", (long)(stats->nreads_1st+stats->nreads_2nd));
    printf("SN\tis paired:\t%d\n", stats->nreads_1st&&stats->nreads_2nd ? 1 : 0);
    printf("SN\tis sorted:\t%d\n", stats->is_sorted ? 1 : 0);
    printf("SN\t1st fragments:\t%ld\n", (long)stats->nreads_1st);
    printf("SN\tlast fragments:\t%ld\n", (long)stats->nreads_2nd);
    printf("SN\treads mapped:\t%ld\n", (long)(stats->nreads_paired+stats->nreads_unpaired));
    printf("SN\treads unmapped:\t%ld\n", (long)stats->nreads_unmapped);
    printf("SN\treads unpaired:\t%ld\n", (long)stats->nreads_unpaired);
    printf("SN\treads paired:\t%ld\n", (long)stats->nreads_paired);
    printf("SN\treads duplicated:\t%ld\n", (long)stats->nreads_dup);
    printf("SN\treads MQ0:\t%ld\n", (long)stats->nreads_mq0);
    printf("SN\ttotal length:\t%ld\n", (long)stats->total_len);
    printf("SN\tbases mapped:\t%ld\n", (long)stats->nbases_mapped);
    printf("SN\tbases mapped (cigar):\t%ld\n", (long)stats->nbases_mapped_cigar);
    printf("SN\tbases trimmed:\t%ld\n", (long)stats->nbases_trimmed);
    printf("SN\tbases duplicated:\t%ld\n", (long)stats->total_len_dup);
    printf("SN\tmismatches:\t%ld\n", (long)stats->nmismatches);
    printf("SN\terror rate:\t%e\n", (float)stats->nmismatches/stats->nbases_mapped_cigar);
    float avg_read_length = (stats->nreads_1st+stats->nreads_2nd)?stats->total_len/(stats->nreads_1st+stats->nreads_2nd):0;
    printf("SN\taverage length:\t%.0f\n", avg_read_length);
    printf("SN\tmaximum length:\t%d\n", stats->max_len);
    printf("SN\taverage quality:\t%.1f\n", stats->total_len?stats->sum_qual/stats->total_len:0);
    printf("SN\tinsert size average:\t%.1f\n", avg_isize);
    printf("SN\tinsert size standard deviation:\t%.1f\n", sd_isize);
    printf("SN\tinward oriented pairs:\t%ld\n", (long)nisize_inward);
    printf("SN\toutward oriented pairs:\t%ld\n", (long)nisize_outward);
    printf("SN\tpairs with other orientation:\t%ld\n", (long)nisize_other);

    int ibase,iqual;
    if ( stats->max_len<stats->nbases ) stats->max_len++;
    if ( stats->max_qual+1<stats->nquals ) stats->max_qual++;
    printf("# First Fragment Qualitites. Use `grep ^FFQ | cut -f 2-` to extract this part.\n");
    printf("# Columns correspond to qualities and rows to cycles. First column is the cycle number.\n");
    for (ibase=0; ibase<stats->max_len; ibase++)
    {
        printf("FFQ\t%d",ibase+1);
        for (iqual=0; iqual<=stats->max_qual; iqual++)
        {
            printf("\t%ld", (long)stats->quals_1st[ibase*stats->nquals+iqual]);
        }
        printf("\n");
    }
    printf("# Last Fragment Qualitites. Use `grep ^LFQ | cut -f 2-` to extract this part.\n");
    printf("# Columns correspond to qualities and rows to cycles. First column is the cycle number.\n");
    for (ibase=0; ibase<stats->max_len; ibase++)
    {
        printf("LFQ\t%d",ibase+1);
        for (iqual=0; iqual<=stats->max_qual; iqual++)
        {
            printf("\t%ld", (long)stats->quals_2nd[ibase*stats->nquals+iqual]);
        }
        printf("\n");
    }
    if ( stats->mpc_buf )
    {
        printf("# Mismatches per cycle and quality. Use `grep ^MPC | cut -f 2-` to extract this part.\n");
        printf("# Columns correspond to qualities, rows to cycles. First column is the cycle number, second\n");
        printf("# is the number of N's and the rest is the number of mismatches\n");
        for (ibase=0; ibase<stats->max_len; ibase++)
        {
            printf("MPC\t%d",ibase+1);
            for (iqual=0; iqual<=stats->max_qual; iqual++)
            {
                printf("\t%ld", (long)stats->mpc_buf[ibase*stats->nquals+iqual]);
            }
            printf("\n");
        }
    }
    printf("# GC Content of first fragments. Use `grep ^GCF | cut -f 2-` to extract this part.\n");
    int ibase_prev = 0;
    for (ibase=0; ibase<stats->ngc; ibase++)
    {
        if ( stats->gc_1st[ibase]==stats->gc_1st[ibase_prev] ) continue;
        printf("GCF\t%.2f\t%ld\n", (ibase+ibase_prev)*0.5*100./(stats->ngc-1), (long)stats->gc_1st[ibase_prev]);
        ibase_prev = ibase;
    }
    printf("# GC Content of last fragments. Use `grep ^GCL | cut -f 2-` to extract this part.\n");
    ibase_prev = 0;
    for (ibase=0; ibase<stats->ngc; ibase++)
    {
        if ( stats->gc_2nd[ibase]==stats->gc_2nd[ibase_prev] ) continue;
        printf("GCL\t%.2f\t%ld\n", (ibase+ibase_prev)*0.5*100./(stats->ngc-1), (long)stats->gc_2nd[ibase_prev]);
        ibase_prev = ibase;
    }
    printf("# ACGT content per cycle. Use `grep ^GCC | cut -f 2-` to extract this part. The columns are: cycle, and A,C,G,T counts [%%]\n");
    for (ibase=0; ibase<stats->max_len; ibase++)
    {
        uint64_t *ptr = &(stats->acgt_cycles[ibase*4]);
        uint64_t  sum = ptr[0]+ptr[1]+ptr[2]+ptr[3];
        if ( ! sum ) continue;
        printf("GCC\t%d\t%.2f\t%.2f\t%.2f\t%.2f\n", ibase,100.*ptr[0]/sum,100.*ptr[1]/sum,100.*ptr[2]/sum,100.*ptr[3]/sum);
    }
    printf("# Insert sizes. Use `grep ^IS | cut -f 2-` to extract this part. The columns are: pairs total, inward oriented pairs, outward oriented pairs, other pairs\n");
    for (isize=1; isize<ibulk; isize++)
        printf("IS\t%d\t%ld\t%ld\t%ld\t%ld\n", isize, (long)(stats->isize_inward[isize]+stats->isize_outward[isize]+stats->isize_other[isize]),
            (long)stats->isize_inward[isize], (long)stats->isize_outward[isize], (long)stats->isize_other[isize]);

    printf("# Read lengths. Use `grep ^RL | cut -f 2-` to extract this part. The columns are: read length, count\n");
    int ilen;
    for (ilen=0; ilen<stats->max_len; ilen++)
    {
        if ( stats->read_lengths[ilen]>0 )
            printf("RL\t%d\t%ld\n", ilen, (long)stats->read_lengths[ilen]);
    }

    printf("# Indel distribution. Use `grep ^ID | cut -f 2-` to extract this part. The columns are: length, number of insertions, number of deletions\n");
    for (ilen=0; ilen<stats->nindels; ilen++)
    {
        if ( stats->insertions[ilen]>0 || stats->deletions[ilen]>0 )
            printf("ID\t%d\t%ld\t%ld\n", ilen+1, (long)stats->insertions[ilen], (long)stats->deletions[ilen]);
    }

    printf("# Indels per cycle. Use `grep ^IC | cut -f 2-` to extract this part. The columns are: cycle, number of insertions, number of deletions\n");
    for (ilen=0; ilen<=stats->nbases; ilen++)
    {
        if ( stats->ins_cycles[ilen]>0 || stats->del_cycles[ilen]>0 )
            printf("IC\t%d\t%ld\t%ld\n", ilen+1, (long)stats->ins_cycles[ilen], (long)stats->del_cycles[ilen]);
    }

    printf("# Coverage distribution. Use `grep ^COV | cut -f 2-` to extract this part.\n");
    printf("COV\t[<%d]\t%d\t%ld\n",stats->cov_min,stats->cov_min-1, (long)stats->cov[0]);
    int icov;
    for (icov=1; icov<stats->ncov-1; icov++)
        printf("COV\t[%d-%d]\t%d\t%ld\n",stats->cov_min + (icov-1)*stats->cov_step, stats->cov_min + icov*stats->cov_step-1,stats->cov_min + icov*stats->cov_step-1, (long)stats->cov[icov]);
    printf("COV\t[%d<]\t%d\t%ld\n",stats->cov_min + (stats->ncov-2)*stats->cov_step-1,stats->cov_min + (stats->ncov-2)*stats->cov_step-1, (long)stats->cov[stats->ncov-1]);


    // Calculate average GC content, then sort by GC and depth
    printf("# GC-depth. Use `grep ^GCD | cut -f 2-` to extract this part. The columns are: GC%%, unique sequence percentiles, 10th, 25th, 50th, 75th and 90th depth percentile\n");
    uint32_t igcd;
    for (igcd=0; igcd<stats->igcd; igcd++)
    {
        if ( stats->fai )
            stats->gcd[igcd].gc = round(100. * stats->gcd[igcd].gc);
        else
            if ( stats->gcd[igcd].depth ) 
                stats->gcd[igcd].gc = round(100. * stats->gcd[igcd].gc / stats->gcd[igcd].depth);
    }
    qsort(stats->gcd, stats->igcd+1, sizeof(gc_depth_t), gcd_cmp);
    igcd = 0;
    while ( igcd < stats->igcd )
    {
        // Calculate percentiles (10,25,50,75,90th) for the current GC content and print
        uint32_t nbins=0, itmp=igcd;
        float gc = stats->gcd[igcd].gc;
        while ( itmp<stats->igcd && fabs(stats->gcd[itmp].gc-gc)<0.1 )
        {
            nbins++;
            itmp++;
        }
        printf("GCD\t%.1f\t%.3f\t%.3f\t%.3f\t%.3f\t%.3f\t%.3f\n", gc, (igcd+nbins+1)*100./(stats->igcd+1),
                gcd_percentile(&(stats->gcd[igcd]),nbins,10) *avg_read_length/stats->gcd_bin_size,
                gcd_percentile(&(stats->gcd[igcd]),nbins,25) *avg_read_length/stats->gcd_bin_size, 
                gcd_percentile(&(stats->gcd[igcd]),nbins,50) *avg_read_length/stats->gcd_bin_size, 
                gcd_percentile(&(stats->gcd[igcd]),nbins,75) *avg_read_length/stats->gcd_bin_size, 
                gcd_percentile(&(stats->gcd[igcd]),nbins,90) *avg_read_length/stats->gcd_bin_size 
              );
        igcd += nbins;
    }
}

void bam_init_header_hash(bam_header_t *header);

size_t getline(char **line, size_t *n, FILE *fp)
{
    if (line == NULL || n == NULL || fp == NULL)
    {
        errno = EINVAL;
        return -1;
    }
    if (*n==0 || !*line)
    {
        *line = NULL;
        *n = 0;
    }

    size_t nread=0;
    int c;
    while ((c=getc(fp))!= EOF && c!='\n')
    {
        if ( ++nread>=*n )
        {
            *n += 255;
            *line = realloc(*line, sizeof(char)*(*n));
        }
        (*line)[nread-1] = c;
    }
    if ( nread>=*n )
    {
        *n += 255;
        *line = realloc(*line, sizeof(char)*(*n));
    }
    (*line)[nread] = 0;
    return nread>0 ? nread : -1;

}

void init_regions(stats_t *stats, char *file)
{
    khiter_t iter;
    khash_t(str) *header_hash;

    bam_init_header_hash(stats->sam->header);
    header_hash = (khash_t(str)*)stats->sam->header->hash;

    FILE *fp = fopen(file,"r");
    if ( !fp ) error("%s: %s\n",file,strerror(errno));

    char *line = NULL;
    size_t len = 0;
    ssize_t nread;
    int warned = 0;
    int prev_tid=-1, prev_pos=-1;
    while ((nread = getline(&line, &len, fp)) != -1) 
    {
        if ( line[0] == '#' ) continue;

        int i = 0;
        while ( i<nread && !isspace(line[i]) ) i++;
        if ( i>=nread ) error("Could not parse the file: %s [%s]\n", file,line);
        line[i] = 0;

        iter = kh_get(str, header_hash, line);
        int tid = kh_val(header_hash, iter);
        if ( iter == kh_end(header_hash) )
        {
            if ( !warned )
                fprintf(stderr,"Warning: Some sequences not present in the BAM (%s)\n", line);
            warned = 1;
            continue;
        }

        if ( tid >= stats->nregions )
        {
            stats->regions = realloc(stats->regions,sizeof(regions_t)*(stats->nregions+100));
            int j;
            for (j=stats->nregions; j<stats->nregions+100; j++)
            {
                stats->regions[j].npos = stats->regions[j].mpos = stats->regions[j].cpos = 0;
                stats->regions[j].pos = NULL;
            }
            stats->nregions += 100;
        }
        int npos = stats->regions[tid].npos;
        if ( npos >= stats->regions[tid].mpos )
        {
            stats->regions[tid].mpos += 1000;
            stats->regions[tid].pos = realloc(stats->regions[tid].pos,sizeof(pos_t)*stats->regions[tid].mpos);
        }

        if ( (sscanf(line+i+1,"%d %d",&stats->regions[tid].pos[npos].from,&stats->regions[tid].pos[npos].to))!=2 ) error("Could not parse the region [%s]\n");
        if ( prev_tid==-1 || prev_tid!=tid )
        {
            prev_tid = tid;
            prev_pos = stats->regions[tid].pos[npos].from;
        }
        if ( prev_pos>stats->regions[tid].pos[npos].from )
            error("The positions are not in chromosomal order (%s:%d comes after %d)\n", line,stats->regions[tid].pos[npos].from,prev_pos);
        stats->regions[tid].npos++;
    }
    if (line) free(line);
    fclose(fp);
}

void destroy_regions(stats_t *stats)
{
    int i;
    for (i=0; i<stats->nregions; i++)
    {
        if ( !stats->regions[i].mpos ) continue;
        free(stats->regions[i].pos);
    }
    if ( stats->regions ) free(stats->regions);
}

static int fetch_read(const bam1_t *bam_line, void *data)
{
    collect_stats((bam1_t*)bam_line,(stats_t*)data);
    return 1;
}


void error(const char *format, ...)
{
    if ( !format )
    {
        printf("Version: %s\n", BAMCHECK_VERSION);
        printf("About: The program collects statistics from BAM files. The output can be visualized using plot-bamcheck.\n");
        printf("Usage: bamcheck [OPTIONS] file.bam\n");
        printf("       bamcheck [OPTIONS] file.bam chr:from-to\n");
        printf("Options:\n");
        printf("    -c, --coverage <int>,<int>,<int>    Coverage distribution min,max,step [1,1000,1]\n");
        printf("    -d, --remove-dups                   Exlude from statistics reads marked as duplicates\n");
        printf("    -h, --help                          This help message\n");
        printf("    -i, --insert-size <int>             Maximum insert size [8000]\n");
        printf("    -l, --read-length <int>             Include in the statistics only reads with the given read length []\n");
        printf("    -m, --most-inserts <float>          Report only the main part of inserts [0.99]\n");
        printf("    -q, --trim-quality <int>            The BWA trimming parameter [0]\n");
        printf("    -r, --ref-seq <file>                Reference sequence (required for GC-depth calculation).\n");
        printf("    -t, --target-regions <file>         Do stats in these regions only. Tab-delimited file chr,from,to, 1-based, inclusive.\n");
        printf("    -s, --sam                           Input is SAM\n");
        printf("\n");
    }
    else
    {
        va_list ap;
        va_start(ap, format);
        vfprintf(stderr, format, ap);
        va_end(ap);
    }
    exit(-1);
}

int main(int argc, char *argv[])
{
    char *targets = NULL;
    char *bam_fname = NULL;
    samfile_t *sam = NULL;
    char in_mode[5];

    stats_t *stats = calloc(1,sizeof(stats_t));
    stats->ngc    = 200;
    stats->nquals = 95;
    stats->nbases = 300;
    stats->nisize = 8000;
    stats->max_len   = 30;
    stats->max_qual  = 40;
    stats->isize_main_bulk = 0.99;   // There are always outliers at the far end
    stats->gcd_bin_size = 20000;
    stats->ngcd         = 3e5;     // 300k of 20k bins is enough to hold a genome 6Gbp big
    stats->nref_seq     = stats->gcd_bin_size;
    stats->rseq_pos     = -1;
    stats->tid = stats->gcd_pos = -1;
    stats->is_sorted = 1;
    stats->cov_min  = 1;
    stats->cov_max  = 1000;
    stats->cov_step = 1;
    stats->argc = argc;
    stats->argv = argv;
    stats->filter_readlen = -1;
    stats->nindels = stats->nbases;

    strcpy(in_mode, "rb");

    static struct option loptions[] = 
    {
        {"help",0,0,'h'},
        {"remove-dups",0,0,'d'},
        {"sam",0,0,'s'},
        {"ref-seq",0,0,'r'},
        {"coverage",0,0,'c'},
        {"read-length",0,0,'l'},
        {"insert-size",0,0,'i'},
        {"most-inserts",0,0,'m'},
        {"trim-quality",0,0,'q'},
        {"target-regions",0,0,'t'},
        {0,0,0,0}
    };
    int opt;
    while ( (opt=getopt_long(argc,argv,"?hdsr:c:l:i:t:m:q:",loptions,NULL))>0 )
    {
        switch (opt)
        {
            case 'd': stats->rmdup=1; break;
            case 's': strcpy(in_mode, "r"); break;
            case 'r': stats->fai = fai_load(optarg); 
                      if (stats->fai==0) 
                          error("Could not load faidx: %s\n", optarg); 
                      break;
            case 'c': if ( sscanf(optarg,"%d,%d,%d",&stats->cov_min,&stats->cov_max,&stats->cov_step)!= 3 ) 
                          error("Unable to parse -c %s\n", optarg); 
                      break;
            case 'l': stats->filter_readlen = atoi(optarg); break;
            case 'i': stats->nisize = atoi(optarg); break;
            case 'm': stats->isize_main_bulk = atof(optarg); break;
            case 'q': stats->trim_qual = atoi(optarg); break;
            case 't': targets = optarg; break;
            case '?': 
            case 'h': error(NULL);
            default: error("Unknown argument: %s\n", optarg);
        }
    }
    if ( optind<argc )
        bam_fname = argv[optind++];

    if ( !bam_fname )
    {
        if ( isatty(fileno((FILE *)stdin)) )
            error(NULL);
        bam_fname = "-";
    }

    // Init structures
    //  .. coverage bins and round buffer
    if ( stats->cov_step > stats->cov_max - stats->cov_min + 1 )
    {
        stats->cov_step = stats->cov_max - stats->cov_min;
        if ( stats->cov_step <= 0 )
            stats->cov_step = 1;
    }
    stats->ncov = 3 + (stats->cov_max-stats->cov_min) / stats->cov_step;
    stats->cov_max = stats->cov_min + ((stats->cov_max-stats->cov_min)/stats->cov_step +1)*stats->cov_step - 1;
    stats->cov = calloc(sizeof(uint64_t),stats->ncov);
    stats->cov_rbuf.size = stats->nbases*5;
    stats->cov_rbuf.buffer = calloc(sizeof(int32_t),stats->cov_rbuf.size);
    // .. bam
    if ((sam = samopen(bam_fname, in_mode, NULL)) == 0) 
        error("Failed to open: %s\n", bam_fname);
    stats->sam = sam;
    bam1_t *bam_line = bam_init1();
    // .. arrays
    stats->quals_1st     = calloc(stats->nquals*stats->nbases,sizeof(uint64_t));
    stats->quals_2nd     = calloc(stats->nquals*stats->nbases,sizeof(uint64_t));
    stats->gc_1st        = calloc(stats->ngc,sizeof(uint64_t));
    stats->gc_2nd        = calloc(stats->ngc,sizeof(uint64_t));
    stats->isize_inward  = calloc(stats->nisize,sizeof(uint64_t));
    stats->isize_outward = calloc(stats->nisize,sizeof(uint64_t));
    stats->isize_other   = calloc(stats->nisize,sizeof(uint64_t));
    stats->gcd           = calloc(stats->ngcd,sizeof(gc_depth_t));
    stats->rseq_buf      = calloc(stats->nref_seq,sizeof(uint8_t));
    stats->mpc_buf       = stats->fai ? calloc(stats->nquals*stats->nbases,sizeof(uint64_t)) : NULL;
    stats->acgt_cycles   = calloc(4*stats->nbases,sizeof(uint64_t));
    stats->read_lengths  = calloc(stats->nbases,sizeof(uint64_t));
    stats->insertions    = calloc(stats->nbases,sizeof(uint64_t));
    stats->deletions     = calloc(stats->nbases,sizeof(uint64_t));
    stats->ins_cycles    = calloc(stats->nbases+1,sizeof(uint64_t));
    stats->del_cycles    = calloc(stats->nbases+1,sizeof(uint64_t));
    if ( targets )
        init_regions(stats, targets);

    // Collect statistics
    if ( optind<argc )
    {
        // Collect stats in selected regions only
        bam_index_t *bam_idx = bam_index_load(bam_fname);
        if (bam_idx == 0)
            error("Random alignment retrieval only works for indexed BAM files.\n");

        int i;
        for (i=optind; i<argc; i++) 
        {
            int tid, beg, end;
            bam_parse_region(stats->sam->header, argv[i], &tid, &beg, &end);
            if ( tid < 0 ) continue;
            bam_fetch(stats->sam->x.bam, bam_idx, tid, beg, end, stats, fetch_read);
        }
        bam_index_destroy(bam_idx);
    }
    else
    {
        // Stream through the entire BAM ignoring off-target regions if -t is given
        while (samread(sam,bam_line) >= 0) 
        {
            if ( stats->regions )
            {
                if ( bam_line->core.tid >= stats->nregions || bam_line->core.tid<0 ) continue;
                if ( !stats->is_sorted ) error("The BAM must be sorted in order for -t to work.\n");

                regions_t *reg = &stats->regions[bam_line->core.tid];
                if ( reg->cpos==reg->npos ) continue;       // done for this chr

                // Find a matching interval or skip this read. No splicing of reads is done, no indels or soft clips considered, 
                //  even small overlap is enough to include the read in the stats.
                int i = reg->cpos;
                while ( i<reg->npos && reg->pos[i].to<=bam_line->core.pos ) i++;
                if ( i>=reg->npos ) { reg->cpos = reg->npos; continue; }
                if ( bam_line->core.pos + bam_line->core.l_qseq + 1 < reg->pos[i].from ) continue;
                reg->cpos = i;
            }
            collect_stats(bam_line,stats);
        }
    }
    round_buffer_flush(stats,-1);

    output_stats(stats);

    bam_destroy1(bam_line);
    samclose(stats->sam);
    if (stats->fai) fai_destroy(stats->fai);
    free(stats->cov_rbuf.buffer); free(stats->cov);
    free(stats->quals_1st); free(stats->quals_2nd); 
    free(stats->gc_1st); free(stats->gc_2nd);
    free(stats->isize_inward); free(stats->isize_outward); free(stats->isize_other);
    free(stats->gcd);
    free(stats->rseq_buf);
    free(stats->mpc_buf);
    free(stats->acgt_cycles);
    free(stats->read_lengths);
    free(stats->insertions);
    free(stats->deletions);
    free(stats->ins_cycles);
    free(stats->del_cycles);
    destroy_regions(stats);
    free(stats);

        return 0;
}