196d206a199ee47dc303b5ddf54170f89e4daa75 hiram Thu Nov 3 17:13:25 2016 -0700 correctly counting matches and mis matches refs #13673 diff --git src/lib/bamFile.c src/lib/bamFile.c index beeb94c..001705c 100644 --- src/lib/bamFile.c +++ src/lib/bamFile.c @@ -1,670 +1,675 @@ /* bamFile -- interface to binary alignment format files using Heng Li's samtools lib. */ /* Copyright (C) 2014 The Regents of the University of California * See README in this or parent directory for licensing information. */ #include "common.h" #include "portable.h" #include "bamFile.h" #include "htmshell.h" #include "udc.h" #include "psl.h" // If KNETFILE_HOOKS is used (as recommended!), then we can simply call bam_index_load // without worrying about the samtools lib creating local cache files in cgi-bin: static bam_index_t *bamOpenIdx(samfile_t *sam, char *fileOrUrl) /* If fileOrUrl has a valid accompanying .bai file, parse and return the index; * otherwise return NULL. */ { if (sam->format.format == cram) return sam_index_load(sam, fileOrUrl); // assume that index is a .bai file char indexName[4096]; safef(indexName, sizeof indexName, "%s.bai", fileOrUrl); return sam_index_load2(sam, fileOrUrl, indexName); } static void bamCloseIdx(bam_index_t **pIdx) /* Free unless already NULL. */ { if (pIdx != NULL && *pIdx != NULL) { free(*pIdx); // Not freeMem, freez etc -- sam just uses malloc/calloc. *pIdx = NULL; } } boolean bamFileExists(char *fileOrUrl) /* Return TRUE if we can successfully open the bam file and its index file. * NOTE: this doesn't give enough diagnostics */ { char *bamFileName = fileOrUrl; samfile_t *fh = samopen(bamFileName, "rb", NULL); if (fh != NULL) { bam_index_t *idx = bamOpenIdx(fh, bamFileName); samclose(fh); if (idx == NULL) { warn("bamFileExists: failed to read index corresponding to %s", bamFileName); return FALSE; } bamCloseIdx(&idx); bamClose(&fh); return TRUE; } return FALSE; } samfile_t *bamOpen(char *fileOrUrl, char **retBamFileName) /* Return an open bam file or errAbort (should be named bamMustOpen). * Return parameter if NON-null will return the file name (vestigial; long ago * there was a plan to use udcFuse filenames instead of URLs) */ { char *bamFileName = fileOrUrl; if (retBamFileName != NULL) *retBamFileName = bamFileName; #ifdef BAM_VERSION // suppress too verbose messages in samtools >= 0.1.18; see redmine #6491 // This variable didn't exist in older versions of samtools (where BAM_VERSION wasn't defined). bam_verbose = 1; #endif samfile_t *fh = samopen(bamFileName, "rb", NULL); // Check both fh and fh->header; non-NULL fh can have NULL header if header doesn't parse! if (fh == NULL) { boolean usingUrl = (strstr(fileOrUrl, "tp://") || strstr(fileOrUrl, "https://")); struct dyString *urlWarning = dyStringNew(0); if (usingUrl && fh == NULL) { dyStringAppend(urlWarning, ". If you are able to access the URL with your web browser, " "please try reloading this page."); } errAbort("Failed to open %s%s", fileOrUrl, urlWarning->string); } return fh; } samfile_t *bamMustOpenLocal(char *fileName, char *mode, void *extraHeader) /* Open up sam or bam file or die trying. The mode parameter is * "r" - open SAM to read * "rb" - open BAM to read * "w" - open SAM to write * "wb" - open BAM to write * The extraHeader is generally NULL in the read case, and the write case * contains a pointer to a bam_header_t with information about the header. * The implementation is just a wrapper around samopen from the samtools library * that aborts with error message if there's a problem with the open. */ { samfile_t *sf = samopen(fileName, mode, extraHeader); if (sf == NULL) errnoAbort("Couldn't open %s.\n", fileName); return sf; } void bamClose(samfile_t **pSamFile) /* Close down a samfile_t */ { if (pSamFile != NULL) { samclose(*pSamFile); *pSamFile = NULL; } } void bamFileAndIndexMustExist(char *fileOrUrl) /* Open both a bam file and its accompanying index or errAbort; this is what it * takes for diagnostic info to propagate up through errCatches in calling code. */ { samfile_t *bamF = bamOpen(fileOrUrl, NULL); bam_index_t *idx = bamOpenIdx(bamF, fileOrUrl); if (idx == NULL) errAbort("failed to read index file (.bai) corresponding to %s", fileOrUrl); bamCloseIdx(&idx); bamClose(&bamF); } void bamFetchAlreadyOpen(samfile_t *samfile, bam_hdr_t *header, bam_index_t *idx, char *bamFileName, char *position, bam_fetch_f callbackFunc, void *callbackData) /* With the open bam file, return items the same way with the callbacks as with bamFetch() */ /* except in this case use an already-open bam file and index (use bam_index_load and free() for */ /* the index). It seems a little strange to pass the filename in with the open bam, but */ /* it's just used to report errors. */ { bam1_t *b; AllocVar(b); hts_itr_t *iter = sam_itr_querys(idx, header, position); if (iter == NULL && startsWith("chr", position)) iter = sam_itr_querys(idx, header, position + strlen("chr")); if (iter == NULL) return; int result; while ((result = sam_itr_next(samfile, iter, b)) >= 0) callbackFunc(b, callbackData, header); // if we're reading a CRAM file and the MD5 string has been set // we know there was an error finding the reference and we need // to request that it be loaded. if (samfile->format.format == cram) { char *md5String = cram_get_Md5(samfile); if (!isEmpty(md5String)) { char server[4096]; char pendingFile[4096]; char errorFile[4096]; char *refPath = cram_get_ref_url(samfile); char *cacheDir = cram_get_cache_dir(samfile); sprintf(server, refPath, md5String); sprintf(pendingFile, "%s/pending/",cacheDir); sprintf(errorFile, "%s/error/%s",cacheDir,md5String); makeDirsOnPath(pendingFile); sprintf(pendingFile, "%s/pending/%s",cacheDir,md5String); FILE *downFile; if ((downFile = fopen(errorFile, "r")) != NULL) { char errorBuf[4096]; mustGetLine(downFile, errorBuf, sizeof errorBuf); errAbort("cannot find reference %s. Error: %s\n", md5String,errorBuf); } else { if ((downFile = fopen(pendingFile, "w")) == NULL) errAbort("cannot find reference %s. Cannot create file %s.",md5String, pendingFile); fprintf(downFile, "%s\n", server); fclose(downFile); errAbort("Cannot find reference %s. Downloading from %s. Please refresh screen to check download status.",md5String, server); } } } } void bamFetchPlus(char *fileOrUrl, char *position, bam_fetch_f callbackFunc, void *callbackData, samfile_t **pSamFile, char *refUrl, char *cacheDir) /* Open the .bam file, fetch items in the seq:start-end position range, * and call callbackFunc on each bam item retrieved from the file plus callbackData. * This handles BAM files with "chr"-less sequence names, e.g. from Ensembl. * The pSamFile parameter is optional. If non-NULL it will be filled in, just for * the benefit of the callback function, with the open samFile. */ { char *bamFileName = NULL; samfile_t *fh = bamOpen(fileOrUrl, &bamFileName); if (fh->format.format == cram) { if (cacheDir == NULL) errAbort("CRAM cache dir hg.conf variable (cramRef) must exist for CRAM support"); cram_set_cache_url(fh, cacheDir, refUrl); } bam_hdr_t *header = sam_hdr_read(fh); if (pSamFile != NULL) *pSamFile = fh; bam_index_t *idx = bamOpenIdx(fh, bamFileName); if (idx == NULL) warn("bam_index_load(%s) failed.", bamFileName); else { bamFetchAlreadyOpen(fh, header, idx, bamFileName, position, callbackFunc, callbackData); bamCloseIdx(&idx); } bamClose(&fh); } void bamFetch(char *fileOrUrl, char *position, bam_fetch_f callbackFunc, void *callbackData, samfile_t **pSamFile) { bamFetchPlus(fileOrUrl, position, callbackFunc, callbackData, pSamFile, NULL, NULL); } boolean bamIsRc(const bam1_t *bam) /* Return TRUE if alignment is on - strand. */ { const bam1_core_t *core = &bam->core; return (core->flag & BAM_FREVERSE); } void bamGetSoftClipping(const bam1_t *bam, int *retLow, int *retHigh, int *retClippedQLen) /* If retLow is non-NULL, set it to the number of "soft-clipped" (skipped) bases at * the beginning of the query sequence and quality; likewise for retHigh at end. * For convenience, retClippedQLen is the original query length minus soft clipping * (and the length of the query sequence that will be returned). */ { unsigned int *cigarPacked = bam1_cigar(bam); const bam1_core_t *core = &bam->core; char op; int n = bamUnpackCigarElement(cigarPacked[0], &op); int low = (op == 'S') ? n : 0; n = bamUnpackCigarElement(cigarPacked[core->n_cigar-1], &op); int high = (op == 'S') ? n : 0; if (retLow != NULL) *retLow = low; if (retHigh != NULL) *retHigh = high; if (retClippedQLen != NULL) *retClippedQLen = (core->l_qseq - low - high); } void bamUnpackQuerySequence(const bam1_t *bam, boolean useStrand, char *qSeq) /* Fill in qSeq with the nucleotide sequence encoded in bam. The BAM format * reverse-complements query sequence when the alignment is on the - strand, * so if useStrand is given we rev-comp it back to restore the original query * sequence. */ { const bam1_core_t *core = &bam->core; int qLen = core->l_qseq; uint8_t *packedQSeq = bam1_seq(bam); int i; for (i = 0; i < qLen; i++) qSeq[i] = bam_nt16_rev_table[bam1_seqi(packedQSeq, i)]; qSeq[i] = '\0'; if (useStrand && bamIsRc(bam)) reverseComplement(qSeq, qLen); } char *bamGetQuerySequence(const bam1_t *bam, boolean useStrand) /* Allocate and return the nucleotide sequence encoded in bam. The BAM format * reverse-complements query sequence when the alignment is on the - strand, * so if useStrand is given we rev-comp it back to restore the original query * sequence. */ { const bam1_core_t *core = &bam->core; int qLen = core->l_qseq; char *qSeq = needMem(qLen+1); bamUnpackQuerySequence(bam, useStrand, qSeq); return qSeq; } UBYTE *bamGetQueryQuals(const bam1_t *bam, boolean useStrand) /* Return the base quality scores encoded in bam as an array of ubytes. */ { const bam1_core_t *core = &bam->core; int qLen = core->l_qseq; UBYTE *arr = needMem(qLen); boolean isRc = useStrand && bamIsRc(bam); UBYTE *qualStr = bam1_qual(bam); int i; for (i = 0; i < core->l_qseq; i++) { int offset = isRc ? (qLen - 1 - i) : i; arr[i] = (qualStr[0] == 255) ? 255 : qualStr[offset]; } return arr; } void bamUnpackCigar(const bam1_t *bam, struct dyString *dyCigar) /* Unpack CIGAR string into dynamic string */ { unsigned int *cigarPacked = bam1_cigar(bam); const bam1_core_t *core = &bam->core; int i; for (i = 0; i < core->n_cigar; i++) { char op; int n = bamUnpackCigarElement(cigarPacked[i], &op); dyStringPrintf(dyCigar, "%d", n); dyStringAppendC(dyCigar, op); } } char *bamGetCigar(const bam1_t *bam) /* Return a BAM-enhanced CIGAR string, decoded from the packed encoding in bam. */ { const bam1_core_t *core = &bam->core; struct dyString *dyCigar = dyStringNew(min(8, core->n_cigar*4)); bamUnpackCigar(bam, dyCigar); return dyStringCannibalize(&dyCigar); } void bamShowCigarEnglish(const bam1_t *bam) /* Print out cigar in English e.g. "20 (mis)Match, 1 Deletion, 3 (mis)Match" */ { unsigned int *cigarPacked = bam1_cigar(bam); const bam1_core_t *core = &bam->core; int i; for (i = 0; i < core->n_cigar; i++) { char op; int n = bamUnpackCigarElement(cigarPacked[i], &op); if (i > 0) printf(", "); switch (op) { case 'M': // match or mismatch (gapless aligned block) printf("%d (mis)Match", n); break; case '=': // match printf("%d Match", n); break; case 'X': // mismatch printf("%d Mismatch", n); break; case 'I': // inserted in query printf("%d Insertion", n); break; case 'S': // skipped query bases at beginning or end ("soft clipping") printf("%d Skipped", n); break; case 'D': // deleted from query printf("%d Deletion", n); break; case 'N': // long deletion from query (intron as opposed to small del) printf("%d deletioN", n); break; case 'H': // skipped query bases not stored in record's query sequence ("hard clipping") printf("%d Hard clipped query", n); break; case 'P': // P="silent deletion from padded reference sequence" printf("%d Padded / silent deletion", n); break; default: errAbort("bamShowCigarEnglish: unrecognized CIGAR op %c -- update me", op); } } } static void descFlag(unsigned flag, unsigned bitMask, char *desc, boolean makeRed, boolean *retFirst) /* Describe a flag bit (or multi-bit mask) if it is set in flag. */ { if ((flag & bitMask) == bitMask) // *all* bits in bitMask are set in flag { if (!*retFirst) printf(" | "); printf("(0x%02x) %s", (makeRed ? " style='color: red'" : ""), bitMask, desc); *retFirst = FALSE; } } void bamShowFlagsEnglish(const bam1_t *bam) /* Print out flags in English, e.g. "Mate is on '-' strand; Properly paired". */ { const bam1_core_t *core = &bam->core; unsigned flag = core->flag; boolean first = TRUE; descFlag(flag, BAM_FDUP, "Optical or PCR duplicate", TRUE, &first); descFlag(flag, BAM_FQCFAIL, "QC failure", TRUE, &first); descFlag(flag, BAM_FSECONDARY, "Not primary alignment", TRUE, &first); descFlag(flag, BAM_FREAD2, "Read 2 of pair", FALSE, &first); descFlag(flag, BAM_FREAD1, "Read 1 of pair", FALSE, &first); descFlag(flag, BAM_FMREVERSE, "Mate is on '-' strand", FALSE, &first); descFlag(flag, BAM_FREVERSE, "Read is on '-' strand", FALSE, &first); descFlag(flag, BAM_FMUNMAP, "Mate is unmapped", TRUE, &first); if (flag & BAM_FUNMAP) errAbort("Read is unmapped (what is it doing here?!?)"); descFlag(flag, (BAM_FPROPER_PAIR | BAM_FPAIRED), "Properly paired", FALSE, &first); if ((flag & BAM_FPAIRED) && !(flag & BAM_FPROPER_PAIR)) descFlag(flag, BAM_FPAIRED, "Not properly paired", TRUE, &first); } int bamGetTargetLength(const bam1_t *bam) /* Tally up the alignment's length on the reference sequence from * bam's packed-int CIGAR representation. */ { unsigned int *cigarPacked = bam1_cigar(bam); const bam1_core_t *core = &bam->core; int tLength=0; int i; for (i = 0; i < core->n_cigar; i++) { char op; int n = bamUnpackCigarElement(cigarPacked[i], &op); switch (op) { case 'M': // match or mismatch (gapless aligned block) case '=': // match case 'X': // mismatch tLength += n; break; case 'I': // inserted in query break; case 'D': // deleted from query case 'N': // long deletion from query (intron as opposed to small del) tLength += n; break; case 'S': // skipped query bases at beginning or end ("soft clipping") case 'H': // skipped query bases not stored in record's query sequence ("hard clipping") case 'P': // P="silent deletion from padded reference sequence" -- ignore these. break; default: errAbort("bamGetTargetLength: unrecognized CIGAR op %c -- update me", op); } } return tLength; } bam1_t *bamClone(const bam1_t *bam) /* Return a newly allocated copy of bam. */ { // Using typecasts to get around compiler complaints about bam being const: bam1_t *newBam = cloneMem((void *)bam, sizeof(*bam)); newBam->data = cloneMem((void *)bam->data, bam->data_len*sizeof(bam->data[0])); return newBam; } void bamShowTags(const bam1_t *bam) /* Print out tags in HTML: bold key, no type indicator for brevity. */ { // adapted from part of bam.c bam_format1: uint8_t *s = bam1_aux(bam); while (s < bam->data + bam->data_len) { uint8_t type, key[2]; key[0] = s[0]; key[1] = s[1]; s += 2; type = *s; ++s; printf(" %c%c:", key[0], key[1]); if (type == 'A') { printf("%c", *s); ++s; } else if (type == 'C') { printf("%u", *s); ++s; } else if (type == 'c') { printf("%d", *(int8_t*)s); ++s; } else if (type == 'S') { printf("%u", *(uint16_t*)s); s += 2; } else if (type == 's') { printf("%d", *(int16_t*)s); s += 2; } else if (type == 'I') { printf("%u", *(uint32_t*)s); s += 4; } else if (type == 'i') { printf("%d", *(int32_t*)s); s += 4; } else if (type == 'f') { printf("%g", *(float*)s); s += 4; } else if (type == 'd') { printf("%lg", *(double*)s); s += 8; } else if (type == 'Z' || type == 'H') { htmTextOut(stdout, (char *)s); s += strlen((char *)s) + 1; } } putc('\n', stdout); } char *bamGetTagString(const bam1_t *bam, char *tag, char *buf, size_t bufSize) /* If bam's tags include the given 2-character tag, place the value into * buf (zero-terminated, trunc'd if nec) and return a pointer to buf, * or NULL if tag is not present. */ { if (tag == NULL) errAbort("NULL tag passed to bamGetTagString"); if (! (isalpha(tag[0]) && isalnum(tag[1]) && tag[2] == '\0')) errAbort("bamGetTagString: invalid tag '%s'", htmlEncode(tag)); char *val = NULL; // adapted from part of bam.c bam_format1: uint8_t *s = bam1_aux(bam); while (s < bam->data + bam->data_len) { uint8_t type, key[2]; key[0] = s[0]; key[1] = s[1]; s += 2; type = *s; ++s; if (key[0] == tag[0] && key[1] == tag[1]) { if (type == 'A') { snprintf(buf, bufSize, "%c", *s);} else if (type == 'C') { snprintf(buf, bufSize, "%u", *s); } else if (type == 'c') { snprintf(buf, bufSize, "%d", *s); } else if (type == 'S') { snprintf(buf, bufSize, "%u", *(uint16_t*)s); } else if (type == 's') { snprintf(buf, bufSize, "%d", *(int16_t*)s); } else if (type == 'I') { snprintf(buf, bufSize, "%u", *(uint32_t*)s); } else if (type == 'i') { snprintf(buf, bufSize, "%d", *(int32_t*)s); } else if (type == 'f') { snprintf(buf, bufSize, "%g", *(float*)s); } else if (type == 'd') { snprintf(buf, bufSize, "%lg", *(double*)s); } else if (type == 'Z' || type == 'H') strncpy(buf, (char *)s, bufSize); else buf[0] = '\0'; buf[bufSize-1] = '\0'; // TODO: is this nec?? see man pages val = buf; break; } else { if (type == 'A' || type == 'C' || type == 'c') { ++s; } else if (type == 'S' || type == 's') { s += 2; } else if (type == 'I' || type == 'i' || type == 'f') { s += 4; } else if (type == 'd') { s += 8; } else if (type == 'Z' || type == 'H') { while (*s++); } } } return val; } void bamUnpackAux(const bam1_t *bam, struct dyString *dy) /* Unpack the tag:type:val part of bam into dy */ { // adapted from part of bam.c bam_format1: uint8_t *s = bam1_aux(bam); boolean firstTime = TRUE; while (s < bam->data + bam->data_len) { if (firstTime) firstTime = FALSE; else dyStringAppendC(dy, '\t'); dyStringAppendC(dy, *s++); dyStringAppendC(dy, *s++); dyStringAppendC(dy, ':'); dyStringAppendC(dy, s[0]); dyStringAppendC(dy, ':'); uint8_t type = *s++; if (type == 'A') { dyStringPrintf(dy, "%c", *s); ++s; } else if (type == 'C') { dyStringPrintf(dy, "%u", *s); ++s; } else if (type == 'c') { dyStringPrintf(dy, "%d", *s); ++s; } else if (type == 'S') { dyStringPrintf(dy, "%u", *(uint16_t*)s); s += 2; } else if (type == 's') { dyStringPrintf(dy, "%d", *(int16_t*)s); s += 2; } else if (type == 'I') { dyStringPrintf(dy, "%u", *(uint32_t*)s); s += 4; } else if (type == 'i') { dyStringPrintf(dy, "%d", *(int32_t*)s); s += 4; } else if (type == 'f') { dyStringPrintf(dy, "%g", *(float*)s); s += 4; } else if (type == 'd') { dyStringPrintf(dy, "%lg", *(double*)s); s += 8; } else if (type == 'Z' || type == 'H') { dyStringAppend(dy, (char *)s); s += strlen((char *)s) + 1; } } } static void bamChromInfoFree(struct bamChromInfo **pInfo) /* Free up one chromInfo */ { struct bamChromInfo *info = *pInfo; if (info != NULL) { freeMem(info->name); freez(pInfo); } } void bamChromInfoFreeList(struct bamChromInfo **pList) /* Free a list of dynamically allocated bamChromInfo's */ { struct bamChromInfo *el, *next; for (el = *pList; el != NULL; el = next) { next = el->next; bamChromInfoFree(&el); } *pList = NULL; } struct psl *bamToPslUnscored(const bam1_t *bam, const bam_hdr_t *hdr) /* Translate BAM's numeric CIGAR encoding into PSL sufficient for cds.c (just coords, * no scoring info) */ { const bam1_core_t *core = &bam->core; struct psl *psl; AllocVar(psl); boolean isRc = (core->flag & BAM_FREVERSE); psl->strand[0] = isRc ? '-' : '+'; psl->qName = cloneString(bam1_qname(bam)); psl->tName = cloneString(hdr->target_name[core->tid]); unsigned blockCount = 0; unsigned *blockSizes, *qStarts, *tStarts; AllocArray(blockSizes, core->n_cigar); AllocArray(qStarts, core->n_cigar); AllocArray(tStarts, core->n_cigar); int tPos = core->pos, qPos = 0, qLength = 0; unsigned int *cigar = bam1_cigar(bam); int i; for (i = 0; i < core->n_cigar; i++) { char op; int n = bamUnpackCigarElement(cigar[i], &op); switch (op) { case 'X': // mismatch (gapless aligned block) case '=': // match (gapless aligned block) case 'M': // match or mismatch (gapless aligned block) blockSizes[blockCount] = n; qStarts[blockCount] = qPos; tStarts[blockCount] = tPos; blockCount++; tPos += n; qPos += n; qLength += n; + if (op == 'X') + psl->misMatch += n; + else + psl->match += n; break; case 'I': // inserted in query qPos += n; qLength += n; break; case 'D': // deleted from query case 'N': // long deletion from query (intron as opposed to small del) tPos += n; break; case 'S': // skipped query bases at beginning or end ("soft clipping") qPos += n; qLength += n; break; case 'H': // skipped query bases not stored in record's query sequence ("hard clipping") case 'P': // P="silent deletion from padded reference sequence" -- ignore these. break; default: errAbort("bamToPsl: unrecognized CIGAR op %c -- update me", op); } } if (blockCount == 0) { pslFree(&psl); return NULL; } psl->tSize = hdr->target_len[core->tid]; psl->tStart = tStarts[0]; psl->tEnd = tStarts[blockCount-1] + blockSizes[blockCount-1]; psl->qSize = qLength; psl->qStart = qStarts[0]; psl->qEnd = qStarts[blockCount-1] + blockSizes[blockCount-1]; if (isRc) reverseIntRange(&psl->qStart, &psl->qEnd, psl->qSize); psl->blockCount = blockCount; psl->blockSizes = blockSizes; psl->qStarts = qStarts; psl->tStarts = tStarts; +pslComputeInsertCounts(psl); return psl; }