diff PsiCLASS-1.0.2/samtools-0.1.19/misc/bamcheck.c @ 0:903fc43d6227 draft default tip

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author lsong10
date Fri, 26 Mar 2021 16:52:45 +0000
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--- /dev/null	Thu Jan 01 00:00:00 1970 +0000
+++ b/PsiCLASS-1.0.2/samtools-0.1.19/misc/bamcheck.c	Fri Mar 26 16:52:45 2021 +0000
@@ -0,0 +1,1521 @@
+/* 
+    Author: petr.danecek@sanger
+    gcc -Wall -Winline -g -O2 -I ~/git/samtools bamcheck.c -o bamcheck -lm -lz -L ~/git/samtools -lbam -lpthread
+
+    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.
+        - 'bases mapped' (stats->nbases_mapped) is calculated from read lengths given by BAM (core.l_qseq)
+        - With the -t option, the whole reads are used. Except for the number of mapped bases (cigar)
+            counts, 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 "2012-09-04"
+
+#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 <assert.h>
+#include "faidx.h"
+#include "khash.h"
+#include "sam.h"
+#include "sam_header.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(kh_faidx, faidx1_t)
+KHASH_MAP_INIT_STR(kh_bam_tid, int)
+KHASH_MAP_INIT_STR(kh_rg, const char *)
+struct __faidx_t {
+    RAZF *rz;
+    int n, m;
+    char **name;
+    khash_t(kh_faidx) *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
+
+    // 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_1st, *ins_cycles_2nd, *del_cycles_1st, *del_cycles_2nd;
+
+    // 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_filtered;
+    uint64_t nreads_dup;
+    uint64_t nreads_unmapped;
+    uint64_t nreads_unpaired;
+    uint64_t nreads_paired;
+    uint64_t nreads_anomalous;
+    uint64_t nreads_mq0;
+    uint64_t nbases_mapped;
+    uint64_t nbases_mapped_cigar;
+    uint64_t nbases_trimmed;  // bwa trimmed bases
+    uint64_t nmismatches;
+    uint64_t nreads_QCfailed, nreads_secondary;
+
+    // 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
+    uint32_t gcd_ref_size;      // The approximate size of the genome
+    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 mrseq_buf;                  // The size of the buffer
+    int32_t rseq_pos;               // The coordinate of the first base in the buffer
+    int32_t nrseq_buf;              // The used part of the buffer
+    uint64_t *mpc_buf;              // Mismatches per cycle
+
+    // Filters
+    int filter_readlen;
+
+    // Target regions
+    int nregions, reg_from,reg_to;
+    regions_t *regions;
+
+    // Auxiliary data
+    int flag_require, flag_filter;
+    double sum_qual;                // For calculating average quality value 
+    samfile_t *sam;             
+    khash_t(kh_rg) *rg_hash;        // Read groups to include, the array is null-terminated
+    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, ...);
+void bam_init_header_hash(bam_header_t *header);
+int is_in_regions(bam1_t *bam_line, stats_t *stats);
+
+
+// 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 is_1st = IS_READ1(bam_line) ? 1 : 0;
+    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-ncig;
+            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, %s:%d %s\n", idx,stats->nbases, stats->sam->header->target_name[bam_line->core.tid],bam_line->core.pos+1,bam1_qname(bam_line));
+            if ( is_1st ) 
+                stats->ins_cycles_1st[idx]++;
+            else
+                stats->ins_cycles_2nd[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);
+            if ( is_1st )
+                stats->del_cycles_1st[idx]++;
+            else
+                stats->del_cycles_2nd[idx]++;
+            if ( ncig<=stats->nindels )
+                stats->deletions[ncig-1]++;
+            continue;
+        }
+        if ( cig!=3 && cig!=5 )
+            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;
+        }
+        // Ignore H and N CIGARs. The letter are inserted e.g. by TopHat and often require very large
+        //  chunk of refseq in memory. Not very frequent and not noticable in the stats.
+        if ( cig==3 || cig==5 ) continue;
+        if ( cig!=0 )
+            error("TODO: cigar %d, %s:%d %s\n", cig,stats->sam->header->target_name[bam_line->core.tid],bam_line->core.pos+1,bam1_qname(bam_line));
+       
+        if ( ncig+iref > stats->nrseq_buf )
+            error("FIXME: %d+%d > %d, %s, %s:%d\n",ncig,iref,stats->nrseq_buf, 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 (%s %d %s)\n", qual,stats->nquals, stats->sam->header->target_name[bam_line->core.tid],bam_line->core.pos+1,bam1_qname(bam_line));
+
+                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(kh_faidx) *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(kh_faidx, 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->mrseq_buf;
+    // 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->mrseq_buf )
+    {
+        memset(ptr,0, stats->mrseq_buf - nread);
+        nread = stats->mrseq_buf;
+    }
+    stats->nrseq_buf = nread;
+    stats->rseq_pos  = pos;
+    stats->tid       = tid;
+}
+
+float fai_gc_content(stats_t *stats, int pos, int len)
+{
+    uint32_t gc,count,c;
+    int i = pos - stats->rseq_pos, ito = i + len;
+    assert( i>=0 && ito<=stats->nrseq_buf );
+
+    // Count GC content
+    gc = count = 0;
+    for (; i<ito; 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_rseq_buffer(stats_t *stats)
+{
+    int n = stats->nbases*10;
+    if ( stats->gcd_bin_size > n ) n = stats->gcd_bin_size;
+    if ( stats->mrseq_buf<n )
+    {
+        stats->rseq_buf = realloc(stats->rseq_buf,sizeof(uint8_t)*n);
+        stats->mrseq_buf = n;
+    }
+}
+
+void realloc_gcd_buffer(stats_t *stats, int seq_len)
+{
+    if ( seq_len >= stats->gcd_bin_size )
+        error("The --GC-depth bin size (%d) is set too low for the read length %d\n", stats->gcd_bin_size, seq_len);
+
+    int n = 1 + stats->gcd_ref_size / (stats->gcd_bin_size - seq_len);
+    if ( n <= stats->igcd )
+        error("The --GC-depth bin size is too small or reference genome too big; please decrease the bin size or increase the reference length\n");
+        
+    if ( n > 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->ngcd = n;
+    }
+
+    realloc_rseq_buffer(stats);
+}
+
+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_1st = realloc(stats->ins_cycles_1st, (n+1)*sizeof(uint64_t));
+    if ( !stats->ins_cycles_1st )
+        error("Could not realloc buffers, the sequence too long: %d (%ld)\n", seq_len,(n+1)*sizeof(uint64_t));
+    memset(stats->ins_cycles_1st + stats->nbases + 1, 0, (n-stats->nbases)*sizeof(uint64_t));
+
+    stats->ins_cycles_2nd = realloc(stats->ins_cycles_2nd, (n+1)*sizeof(uint64_t));
+    if ( !stats->ins_cycles_2nd )
+        error("Could not realloc buffers, the sequence too long: %d (%ld)\n", seq_len,(n+1)*sizeof(uint64_t));
+    memset(stats->ins_cycles_2nd + stats->nbases + 1, 0, (n-stats->nbases)*sizeof(uint64_t));
+
+    stats->del_cycles_1st = realloc(stats->del_cycles_1st, (n+1)*sizeof(uint64_t));
+    if ( !stats->del_cycles_1st )
+        error("Could not realloc buffers, the sequence too long: %d (%ld)\n", seq_len,(n+1)*sizeof(uint64_t));
+    memset(stats->del_cycles_1st + stats->nbases + 1, 0, (n-stats->nbases)*sizeof(uint64_t));
+
+    stats->del_cycles_2nd = realloc(stats->del_cycles_2nd, (n+1)*sizeof(uint64_t));
+    if ( !stats->del_cycles_2nd )
+        error("Could not realloc buffers, the sequence too long: %d (%ld)\n", seq_len,(n+1)*sizeof(uint64_t));
+    memset(stats->del_cycles_2nd + stats->nbases + 1, 0, (n-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;
+
+    realloc_rseq_buffer(stats);
+}
+
+void collect_stats(bam1_t *bam_line, stats_t *stats)
+{
+    if ( stats->rg_hash )
+    {
+        const uint8_t *rg = bam_aux_get(bam_line, "RG");
+        if ( !rg ) return; 
+        khiter_t k = kh_get(kh_rg, stats->rg_hash, (const char*)(rg + 1));
+        if ( k == kh_end(stats->rg_hash) ) return;
+    }
+    if ( stats->flag_require && (bam_line->core.flag & stats->flag_require)!=stats->flag_require )
+    {
+        stats->nreads_filtered++;
+        return;
+    }
+    if ( stats->flag_filter && (bam_line->core.flag & stats->flag_filter) )
+    {
+        stats->nreads_filtered++;
+        return;
+    }
+    if ( !is_in_regions(bam_line,stats) )
+        return;
+    if ( stats->filter_readlen!=-1 && bam_line->core.l_qseq!=stats->filter_readlen ) 
+        return;
+
+    if ( bam_line->core.flag & BAM_FQCFAIL ) stats->nreads_QCfailed++;
+    if ( bam_line->core.flag & BAM_FSECONDARY ) stats->nreads_secondary++;
+
+    int seq_len = bam_line->core.l_qseq;
+    if ( !seq_len ) 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 (%s %d %s)\n", qual,stats->nquals,stats->sam->header->target_name[bam_line->core.tid],bam_line->core.pos+1,bam1_qname(bam_line));
+        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);
+
+        if ( !IS_PAIRED(bam_line) )
+            stats->nreads_unpaired++;
+        else 
+        {
+            stats->nreads_paired++;
+
+            if ( bam_line->core.tid!=bam_line->core.mtid )
+                stats->nreads_anomalous++;
+
+            // 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. Mates mapped to different chromosomes have isize==0.
+            int32_t isize = bam_line->core.isize;
+            if ( isize<0 ) isize = -isize;
+            if ( isize >= stats->nisize )
+                isize = stats->nisize-1;
+            if ( isize>0 || bam_line->core.tid==bam_line->core.mtid )
+            {
+                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]++;
+                }
+            }
+        }
+
+        // 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 
+        // Conversion from uint32_t to MIDNSHP
+        //  012-4--
+        //  MIDNSHP
+        if ( bam_line->core.n_cigar == 0) 
+            error("FIXME: mapped read with no cigar?\n");
+        int readlen=seq_len;
+        if ( stats->regions )
+        {
+            // Count only on-target bases
+            int iref = bam_line->core.pos + 1;
+            for (i=0; i<bam_line->core.n_cigar; i++)
+            {
+                int cig  = bam1_cigar(bam_line)[i]&BAM_CIGAR_MASK;
+                int ncig = bam1_cigar(bam_line)[i]>>BAM_CIGAR_SHIFT;
+                if ( cig==2 ) readlen += ncig;
+                else if ( cig==0 ) 
+                {
+                    if ( iref < stats->reg_from ) ncig -= stats->reg_from-iref;
+                    else if ( iref+ncig-1 > stats->reg_to ) ncig -= iref+ncig-1 - stats->reg_to;
+                    if ( ncig<0 ) ncig = 0;
+                    stats->nbases_mapped_cigar += ncig;
+                    iref += bam1_cigar(bam_line)[i]>>BAM_CIGAR_SHIFT;
+                }
+                else if ( cig==1 )
+                {
+                    iref += ncig;
+                    if ( iref>=stats->reg_from && iref<=stats->reg_to )
+                        stats->nbases_mapped_cigar += ncig;
+                }
+            }
+        }
+        else
+        {
+            // Count the whole read
+            for (i=0; i<bam_line->core.n_cigar; i++) 
+            {
+                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. For simplicity, reads overlapping GCD bins
+            //  are not splitted which results in up to seq_len-1 overlaps. The default bin size is
+            //  20kbp, so the effect is negligible.
+            if ( stats->fai )
+            {
+                int inc_ref = 0, inc_gcd = 0;
+                // First pass or new chromosome
+                if ( stats->rseq_pos==-1 || stats->tid != bam_line->core.tid ) { inc_ref=1; inc_gcd=1; }
+                // Read goes beyond the end of the rseq buffer
+                else if ( stats->rseq_pos+stats->nrseq_buf < bam_line->core.pos+readlen ) { inc_ref=1; inc_gcd=1; }
+                // Read overlaps the next gcd bin
+                else if ( stats->gcd_pos+stats->gcd_bin_size < bam_line->core.pos+readlen ) 
+                { 
+                    inc_gcd = 1;
+                    if ( stats->rseq_pos+stats->nrseq_buf < bam_line->core.pos+stats->gcd_bin_size ) inc_ref = 1;
+                }
+                if ( inc_gcd )
+                {
+                    stats->igcd++;
+                    if ( stats->igcd >= stats->ngcd )
+                        realloc_gcd_buffer(stats, readlen);
+                    if ( inc_ref )
+                        read_ref_seq(stats,bam_line->core.tid,bam_line->core.pos);
+                    stats->gcd_pos = bam_line->core.pos;
+                    stats->gcd[ stats->igcd ].gc = fai_gc_content(stats, stats->gcd_pos, stats->gcd_bin_size);
+                }
+
+                count_mismatches_per_cycle(stats,bam_line);
+            }
+            // No reference and first pass, new chromosome or sequence going beyond the end of the gcd bin
+            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 )
+                    realloc_gcd_buffer(stats, readlen);
+            }
+            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=0; 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=0; 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\traw total sequences:\t%ld\n", (long)(stats->nreads_filtered+stats->nreads_1st+stats->nreads_2nd));
+    printf("SN\tfiltered sequences:\t%ld\n", (long)stats->nreads_filtered);
+    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\treads QC failed:\t%ld\n", (long)stats->nreads_QCfailed);
+    printf("SN\tnon-primary alignments:\t%ld\n", (long)stats->nreads_secondary);
+    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);
+    printf("SN\tpairs on different chromosomes:\t%ld\n", (long)stats->nreads_anomalous/2);
+
+    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=0; 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 (fwd), .. (rev) , number of deletions (fwd), .. (rev)\n");
+    for (ilen=0; ilen<=stats->nbases; ilen++)
+    {
+        // For deletions we print the index of the cycle before the deleted base (1-based) and for insertions
+        //  the index of the cycle of the first inserted base (also 1-based)
+        if ( stats->ins_cycles_1st[ilen]>0 || stats->ins_cycles_2nd[ilen]>0 || stats->del_cycles_1st[ilen]>0 || stats->del_cycles_2nd[ilen]>0 )
+            printf("IC\t%d\t%ld\t%ld\t%ld\t%ld\n", ilen+1, (long)stats->ins_cycles_1st[ilen], (long)stats->ins_cycles_2nd[ilen], (long)stats->del_cycles_1st[ilen], (long)stats->del_cycles_2nd[ilen]);
+    }
+
+    printf("# Coverage distribution. Use `grep ^COV | cut -f 2-` to extract this part.\n");
+    if  ( stats->cov[0] )
+        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++)
+        if ( stats->cov[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]);
+    if ( stats->cov[stats->ncov-1] )
+        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;
+    }
+}
+
+size_t mygetline(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(kh_bam_tid) *header_hash;
+
+    bam_init_header_hash(stats->sam->header);
+    header_hash = (khash_t(kh_bam_tid)*)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 = mygetline(&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(kh_bam_tid, 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, e.g. \"%s\". This message is printed only once.\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);
+    if ( !stats->regions ) error("Unable to map the -t sequences to the BAM sequences.\n");
+    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 reset_regions(stats_t *stats)
+{
+    int i;
+    for (i=0; i<stats->nregions; i++)
+        stats->regions[i].cpos = 0;
+}
+
+int is_in_regions(bam1_t *bam_line, stats_t *stats)
+{
+    if ( !stats->regions ) return 1;
+
+    if ( bam_line->core.tid >= stats->nregions || bam_line->core.tid<0 ) return 0;
+    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 ) return 0;       // 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; return 0; }
+    if ( bam_line->core.pos + bam_line->core.l_qseq + 1 < reg->pos[i].from ) return 0;
+    reg->cpos = i;
+    stats->reg_from = reg->pos[i].from;
+    stats->reg_to   = reg->pos[i].to;
+
+    return 1;
+}
+
+void init_group_id(stats_t *stats, char *id)
+{
+    if ( !stats->sam->header->dict )
+        stats->sam->header->dict = sam_header_parse2(stats->sam->header->text);
+    void *iter = stats->sam->header->dict;
+    const char *key, *val;
+    int n = 0;
+    stats->rg_hash = kh_init(kh_rg);
+    while ( (iter = sam_header2key_val(iter, "RG","ID","SM", &key, &val)) )
+    {
+        if ( !strcmp(id,key) || (val && !strcmp(id,val)) )
+        {
+            khiter_t k = kh_get(kh_rg, stats->rg_hash, key);
+            if ( k != kh_end(stats->rg_hash) ) 
+                fprintf(stderr, "[init_group_id] The group ID not unique: \"%s\"\n", key);
+            int ret;
+            k = kh_put(kh_rg, stats->rg_hash, key, &ret);
+            kh_value(stats->rg_hash, k) = val;
+            n++;
+        }
+    }
+    if ( !n )
+        error("The sample or read group \"%s\" not present.\n", id);
+}
+
+
+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("    -f, --required-flag <int>           Required flag, 0 for unset [0]\n");
+        printf("    -F, --filtering-flag <int>          Filtering flag, 0 for unset [0]\n");
+        printf("        --GC-depth <float,float>        Bin size for GC-depth graph and the maximum reference length [2e4,4.2e9]\n");
+        printf("    -h, --help                          This help message\n");
+        printf("    -i, --insert-size <int>             Maximum insert size [8000]\n");
+        printf("    -I, --id <string>                   Include only listed read group or sample name\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;
+    char *group_id = NULL;
+    samfile_t *sam = NULL;
+    char in_mode[5];
+
+    stats_t *stats = calloc(1,sizeof(stats_t));
+    stats->ngc    = 200;
+    stats->nquals = 256;
+    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 = 20e3;
+    stats->gcd_ref_size = 4.2e9;
+    stats->rseq_pos     = -1;
+    stats->tid = stats->gcd_pos = -1;
+    stats->igcd = 0;
+    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",1,0,'r'},
+        {"coverage",1,0,'c'},
+        {"read-length",1,0,'l'},
+        {"insert-size",1,0,'i'},
+        {"most-inserts",1,0,'m'},
+        {"trim-quality",1,0,'q'},
+        {"target-regions",0,0,'t'},
+        {"required-flag",1,0,'f'},
+        {"filtering-flag",0,0,'F'},
+        {"id",1,0,'I'},
+        {"GC-depth",1,0,1},
+        {0,0,0,0}
+    };
+    int opt;
+    while ( (opt=getopt_long(argc,argv,"?hdsr:c:l:i:t:m:q:f:F:I:1:",loptions,NULL))>0 )
+    {
+        switch (opt)
+        {
+            case 'f': stats->flag_require=strtol(optarg,0,0); break;
+            case 'F': stats->flag_filter=strtol(optarg,0,0); break;
+            case 'd': stats->flag_filter|=BAM_FDUP; 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  1 : {
+                        float flen,fbin;
+                        if ( sscanf(optarg,"%f,%f",&fbin,&flen)!= 2 ) 
+                            error("Unable to parse --GC-depth %s\n", optarg); 
+                        stats->gcd_bin_size = fbin;
+                        stats->gcd_ref_size = flen;
+                      }
+                      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 'I': group_id = 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;
+    if ( group_id ) init_group_id(stats, group_id);
+    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->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_1st = calloc(stats->nbases+1,sizeof(uint64_t));
+    stats->ins_cycles_2nd = calloc(stats->nbases+1,sizeof(uint64_t));
+    stats->del_cycles_1st = calloc(stats->nbases+1,sizeof(uint64_t));
+    stats->del_cycles_2nd = calloc(stats->nbases+1,sizeof(uint64_t));
+    realloc_rseq_buffer(stats);
+    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;
+            reset_regions(stats);
+            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) 
+            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_1st);
+    free(stats->ins_cycles_2nd);
+    free(stats->del_cycles_1st);
+    free(stats->del_cycles_2nd);
+    destroy_regions(stats);
+    free(stats);
+    if ( stats->rg_hash ) kh_destroy(kh_rg, stats->rg_hash);
+
+    return 0;
+}
+
+
+