Mercurial > repos > lsong10 > psiclass
diff PsiCLASS-1.0.2/samtools-0.1.19/misc/bamcheck.c @ 0:903fc43d6227 draft default tip
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| author | lsong10 |
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| 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; +} + + +