#include <ctype.h>
#include <getopt.h>
#include <errno.h>
+#include <assert.h>
#include "faidx.h"
#include "khash.h"
#include "sam.h"
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;
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
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
+ 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;
if ( cig==1 )
{
- int idx = is_fwd ? icycle : read_len-icycle;
+ 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));
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\n", cig,bam1_qname(bam_line));
+ 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->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);
+ 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 qual = quals[iread] + 1;
if ( qual>=stats->nquals )
- error("TODO: quality too high %d>=%d\n", quals[iread],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 )
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;
+ 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;
ptr++;
nread++;
}
- if ( nread < stats->nref_seq )
+ if ( nread < stats->mrseq_buf )
{
- memset(ptr,0, stats->nref_seq - nread);
- nread = stats->nref_seq;
+ memset(ptr,0, stats->mrseq_buf - nread);
+ nread = stats->mrseq_buf;
}
- stats->rseq_len = nread;
- stats->rseq_pos = pos;
- stats->tid = tid;
+ stats->nrseq_buf = nread;
+ stats->rseq_pos = pos;
+ stats->tid = tid;
}
-float fai_gc_content(stats_t *stats)
+float fai_gc_content(stats_t *stats, int pos, int len)
{
uint32_t gc,count,c;
- int i,size = stats->rseq_len;
+ int i = pos - stats->rseq_pos, ito = i + len;
+ assert( i>=0 && ito<=stats->nrseq_buf );
// Count GC content
gc = count = 0;
- for (i=0; i<size; i++)
+ for (; i<ito; i++)
{
c = stats->rseq_buf[i];
if ( c==2 || c==4 )
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)
{
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)
{
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);
+ 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;
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 )
+ if ( !IS_PAIRED(bam_line) )
+ stats->nreads_unpaired++;
+ else
{
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 ( bam_line->core.tid!=bam_line->core.mtid )
+ stats->nreads_anomalous++;
- 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 )
+ // 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 )
{
- if ( is_fst*is_fwd>0 )
- stats->isize_outward[isize]++;
- else
- stats->isize_inward[isize]++;
+ 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 ( stats->tid==-1 || stats->tid!=bam_line->core.tid )
round_buffer_flush(stats,-1);
- // Mismatches per cycle and GC-depth graph
+ // 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 )
{
- // 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 )
+ 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 )
{
- 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);
+ 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 )
- {
- 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));
- }
+ 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 )
// 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++)
+ for (isize=0; isize<stats->nisize; isize++)
{
// Each pair was counted twice
stats->isize_inward[isize] *= 0.5;
}
double bulk=0, avg_isize=0, sd_isize=0;
- for (isize=1; isize<stats->nisize; isize++)
+ 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]);
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++;
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++)
+ 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("# 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");
- printf("COV\t[<%d]\t%d\t%ld\n",stats->cov_min,stats->cov_min-1, (long)stats->cov[0]);
+ 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++)
- 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]);
-
+ 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");
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 [2e4,6e9]\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");
stats_t *stats = calloc(1,sizeof(stats_t));
stats->ngc = 200;
- stats->nquals = 95;
+ 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 = 20000;
- stats->ngcd = 3e5; // 300k of 20k bins is enough to hold a genome 6Gbp big
+ 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;
if ( sscanf(optarg,"%f,%f",&fbin,&flen)!= 2 )
error("Unable to parse --GC-depth %s\n", optarg);
stats->gcd_bin_size = fbin;
- stats->ngcd = flen/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 )
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->nref_seq = stats->gcd_bin_size;
- 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->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);
free(stats);
if ( stats->rg_hash ) kh_destroy(kh_rg, stats->rg_hash);
- return 0;
+ return 0;
}
projects / samtools.git / blobdiff