/* bam_sort.c -- sorting and merging. Copyright (C) 2008-2022 Genome Research Ltd. Portions copyright (C) 2009-2012 Broad Institute. Author: Heng Li Author: Martin Pollard Permission is hereby granted, free of charge, to any person obtaining a copy of this software and associated documentation files (the "Software"), to deal in the Software without restriction, including without limitation the rights to use, copy, modify, merge, publish, distribute, sublicense, and/or sell copies of the Software, and to permit persons to whom the Software is furnished to do so, subject to the following conditions: The above copyright notice and this permission notice shall be included in all copies or substantial portions of the Software. THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM, OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE SOFTWARE. */ #include #include #include #include #include #include #include #include #include #include #include #include #include #include #include #include "htslib/ksort.h" #include "htslib/hts_os.h" #include "htslib/khash.h" #include "htslib/klist.h" #include "htslib/kstring.h" #include "htslib/sam.h" #include "htslib/hts_endian.h" #include "htslib/cram.h" #include "htslib/thread_pool.h" #include "sam_opts.h" #include "samtools.h" #include "bedidx.h" #include "bam.h" #define BAM_BLOCK_SIZE 2*1024*1024 #define MAX_TMP_FILES 64 // Struct which contains the sorting key for TemplateCoordinate sort. typedef struct { int tid1; int tid2; hts_pos_t pos1; hts_pos_t pos2; bool neg1; bool neg2; const char *library; char *mid; char *name; bool is_upper_of_pair; } template_coordinate_key_t; // Struct to store fixed buffers of template coordinate keys typedef struct { size_t n; // the # of keys stored size_t m; // the # of buffers allocated size_t buffer_size; // # the fixed size of each buffer template_coordinate_key_t **buffers; // the list of buffers } template_coordinate_keys_t; // Gets the idx'th key; does not OOB check static template_coordinate_key_t* template_coordinate_keys_get(template_coordinate_keys_t *keys, size_t idx) { size_t buffer_idx = idx / keys->buffer_size; // the index of the buffer to retrieve in buffer size_t buffer_offset = idx % keys->buffer_size; // the offset into the given buffer to retrieve //assert(buffer_idx < keys->m); //assert(buffer_offset < keys->buffer_size); return &keys->buffers[buffer_idx][buffer_offset]; } // Rellocates the buffers to hold at least max_k entries static int template_coordinate_keys_realloc(template_coordinate_keys_t *keys, int max_k) { size_t cur_m = keys->m; keys->m += 0x100; //assert(keys->m > cur_m); //assert(keys->m * keys->buffer_size >= max_k); if ((keys->buffers = realloc(keys->buffers, keys->m * sizeof(template_coordinate_key_t*))) == NULL) { print_error("sort", "couldn't reallocate memory for template coordinate key buffers"); return -1; } // allocate space for new buffers int j; for (j = cur_m; j < keys->m; ++j) { if ((keys->buffers[j]= malloc(sizeof(template_coordinate_key_t) * keys->buffer_size)) == NULL) { print_error("sort", "couldn't allocate memory for template coordinate key buffer"); return -1; } } return 0; } // Struct which contains the a record, and the pointer to the sort tag (if any) or // a combined ref / position / strand. // Used to speed up sorts (coordinate, by-tag, and template-coordinate). typedef struct bam1_tag { bam1_t *bam_record; union { const uint8_t *tag; uint8_t pos_tid[12]; template_coordinate_key_t *key; } u; } bam1_tag; /* Minimum memory required in megabytes before sort will attempt to run. This is to prevent accidents where failing to use the -m option correctly results in the creation of a temporary file for each read in the input file. Don't forget to update the man page if you change this. */ const size_t SORT_MIN_MEGS_PER_THREAD = 1; /* Default per-thread memory for sort. Must be >= SORT_MIN_MEGS_PER_THREAD. Don't forget to update the man page if you change this. */ const size_t SORT_DEFAULT_MEGS_PER_THREAD = 768; #if !defined(__DARWIN_C_LEVEL) || __DARWIN_C_LEVEL < 900000L #define NEED_MEMSET_PATTERN4 #endif #ifdef NEED_MEMSET_PATTERN4 void memset_pattern4(void *target, const void *pattern, size_t size) { uint32_t* target_iter = target; size_t loops = size/4; size_t i; for (i = 0; i < loops; ++i) { memcpy(target_iter, pattern, 4); ++target_iter; } if (size%4 != 0) memcpy(target_iter, pattern, size%4); } #endif KHASH_INIT(c2c, char*, char*, 1, kh_str_hash_func, kh_str_hash_equal) KHASH_INIT(cset, char*, char, 0, kh_str_hash_func, kh_str_hash_equal) KHASH_MAP_INIT_STR(c2i, int) KHASH_MAP_INIT_STR(const_c2c, char *) #define hdrln_free_char(p) KLIST_INIT(hdrln, char*, hdrln_free_char) static template_coordinate_key_t* template_coordinate_key(bam1_t *b, template_coordinate_key_t *key, sam_hdr_t *hdr, khash_t(const_c2c) *lib_lookup); typedef enum {Coordinate, QueryName, TagCoordinate, TagQueryName, MinHash, TemplateCoordinate} SamOrder; static SamOrder g_sam_order = Coordinate; static char g_sort_tag[2] = {0,0}; #define is_digit(c) ((c)<='9' && (c)>='0') static int strnum_cmp(const char *_a, const char *_b) { const unsigned char *a = (const unsigned char*)_a, *b = (const unsigned char*)_b; const unsigned char *pa = a, *pb = b; while (*pa && *pb) { if (!is_digit(*pa) || !is_digit(*pb)) { if (*pa != *pb) return (int)*pa - (int)*pb; ++pa; ++pb; } else { // skip leading zeros while (*pa == '0') ++pa; while (*pb == '0') ++pb; // skip matching digits while (is_digit(*pa) && *pa == *pb) pa++, pb++; // Now mismatching, so see which ends the number sooner int diff = (int)*pa - (int)*pb; while (is_digit(*pa) && is_digit(*pb)) pa++, pb++; if (is_digit(*pa)) return 1; // pa still going, so larger else if (is_digit(*pb)) return -1; // pb still going, so larger else if (diff) return diff; // same length, so earlier diff } } return *pa? 1 : *pb? -1 : 0; } #define HEAP_EMPTY (UINT64_MAX >> 1) typedef struct { int i; uint32_t tid; uint64_t pos:63, rev:1, idx; bam1_tag entry; } heap1_t; static inline int bam1_cmp_by_tag(const bam1_tag a, const bam1_tag b); static inline int bam1_cmp_by_minhash(const bam1_tag a, const bam1_tag b); static inline int bam1_cmp_template_coordinate(const bam1_tag a, const bam1_tag b); static khash_t(const_c2c) * lookup_libraries(sam_hdr_t *header); static void lib_lookup_destroy(khash_t(const_c2c) *lib_lookup); // Function to compare reads in the heap and determine which one is < the other // Note, unlike the bam1_cmp_by_X functions which return <0, 0, >0 this // is strictly 0 or 1 only. static inline int heap_lt(const heap1_t a, const heap1_t b) { if (!a.entry.bam_record) return 1; if (!b.entry.bam_record) return 0; int t, fa, fb; switch (g_sam_order) { case Coordinate: if (a.tid != b.tid) return a.tid > b.tid; if (a.pos != b.pos) return a.pos > b.pos; if (a.rev != b.rev) return a.rev > b.rev; break; case QueryName: t = strnum_cmp(bam_get_qname(a.entry.bam_record), bam_get_qname(b.entry.bam_record)); if (t != 0) return t > 0; fa = a.entry.bam_record->core.flag & 0xc0; fb = b.entry.bam_record->core.flag & 0xc0; if (fa != fb) return fa > fb; break; case TagQueryName: case TagCoordinate: t = bam1_cmp_by_tag(a.entry, b.entry); if (t != 0) return t > 0; break; case MinHash: t = bam1_cmp_by_minhash(a.entry, b.entry); if (t != 0) return t > 0; break; case TemplateCoordinate: t = bam1_cmp_template_coordinate(a.entry, b.entry); if (t != 0) return t > 0; break; default: print_error("heap_lt", "unknown sort order: %d", g_sam_order); break; } // This compares by position in the input file(s) if (a.i != b.i) return a.i > b.i; return a.idx > b.idx; } KSORT_INIT(heap, heap1_t, heap_lt) typedef struct merged_header { sam_hdr_t *hdr; kstring_t out_rg; kstring_t out_pg; kstring_t out_co; char **target_name; uint32_t *target_len; size_t n_targets; size_t targets_sz; khash_t(c2i) *sq_tids; khash_t(cset) *rg_ids; khash_t(cset) *pg_ids; bool have_hd; } merged_header_t; typedef struct trans_tbl { int32_t n_targets; int* tid_trans; kh_c2c_t* rg_trans; kh_c2c_t* pg_trans; bool lost_coord_sort; } trans_tbl_t; static void trans_tbl_destroy(trans_tbl_t *tbl) { khiter_t iter; free(tbl->tid_trans); /* * The values for the tbl->rg_trans and tbl->pg_trans hashes are pointers * to keys in the rg_ids and pg_ids sets of the merged_header_t, so * they should not be freed here. * * The keys are unique to each hash entry, so they do have to go. */ for (iter = kh_begin(tbl->rg_trans); iter != kh_end(tbl->rg_trans); ++iter) { if (kh_exist(tbl->rg_trans, iter)) { free(kh_key(tbl->rg_trans, iter)); } } for (iter = kh_begin(tbl->pg_trans); iter != kh_end(tbl->pg_trans); ++iter) { if (kh_exist(tbl->pg_trans, iter)) { free(kh_key(tbl->pg_trans, iter)); } } kh_destroy(c2c,tbl->rg_trans); kh_destroy(c2c,tbl->pg_trans); } /* * Create a merged_header_t struct. */ static merged_header_t * init_merged_header() { merged_header_t *merged_hdr; merged_hdr = calloc(1, sizeof(*merged_hdr)); if (merged_hdr == NULL) return NULL; merged_hdr->hdr = sam_hdr_init(); if (!merged_hdr->hdr) goto fail; merged_hdr->targets_sz = 16; merged_hdr->target_name = malloc(merged_hdr->targets_sz * sizeof(*merged_hdr->target_name)); if (NULL == merged_hdr->target_name) goto fail; merged_hdr->target_len = malloc(merged_hdr->targets_sz * sizeof(*merged_hdr->target_len)); if (NULL == merged_hdr->target_len) goto fail; merged_hdr->sq_tids = kh_init(c2i); if (merged_hdr->sq_tids == NULL) goto fail; merged_hdr->rg_ids = kh_init(cset); if (merged_hdr->rg_ids == NULL) goto fail; merged_hdr->pg_ids = kh_init(cset); if (merged_hdr->pg_ids == NULL) goto fail; return merged_hdr; fail: perror("[init_merged_header]"); kh_destroy(cset, merged_hdr->pg_ids); kh_destroy(cset, merged_hdr->rg_ids); kh_destroy(c2i, merged_hdr->sq_tids); free(merged_hdr->target_name); free(merged_hdr->target_len); sam_hdr_destroy(merged_hdr->hdr); free(merged_hdr); return NULL; } /* Some handy kstring manipulating functions */ // Append char range to kstring static inline int range_to_ks(const char *src, int from, int to, kstring_t *dest) { return kputsn(src + from, to - from, dest) != to - from; } // Append a kstring to a kstring static inline int ks_to_ks(kstring_t *src, kstring_t *dest) { return kputsn(ks_str(src), ks_len(src), dest) != ks_len(src); } /* * Generate a unique ID by appending a random suffix to a given prefix. * existing_ids is the set of IDs that are already in use. * If always_add_suffix is true, the suffix will always be included. * If false, prefix will be returned unchanged if it isn't in existing_ids. */ static int gen_unique_id(char *prefix, khash_t(cset) *existing_ids, bool always_add_suffix, kstring_t *dest) { khiter_t iter; if (!always_add_suffix) { // Try prefix on its own first iter = kh_get(cset, existing_ids, prefix); if (iter == kh_end(existing_ids)) { // prefix isn't used yet dest->l = 0; if (kputs(prefix, dest) == EOF) return -1; return 0; } } do { dest->l = 0; ksprintf(dest, "%s-%0lX", prefix, lrand48()); iter = kh_get(cset, existing_ids, ks_str(dest)); } while (iter != kh_end(existing_ids)); return 0; } /* * Add the @HD line to the new header * In practice the @HD line will come from the first input header. */ static int trans_tbl_add_hd(merged_header_t* merged_hdr, sam_hdr_t *translate) { kstring_t hd_line = { 0, 0, NULL }; int res; // TODO: handle case when @HD needs merging. if (merged_hdr->have_hd) return 0; res = sam_hdr_find_hd(translate, &hd_line); if (res < -1) { print_error("merge", "failed to get @HD line from header"); return -1; } if (res < 0) // Not found return 0; if (sam_hdr_add_lines(merged_hdr->hdr, hd_line.s, hd_line.l) < 0) { print_error("merge", "failed to add @HD line to new header"); free(hd_line.s); return -1; } free(hd_line.s); merged_hdr->have_hd = true; return 0; } /* * Add @SQ records to the translation table. * * Go through the target list for the input header. Any new targets found * are added to the output header target list. At the same time, a mapping * from the input to output target ids is stored in tbl. * * If any new targets are found, the header text is scanned to find the * corresponding @SQ records. They are then copied into the * merged_hdr->out_text kstring (which will eventually become the * output header text). * * Returns 0 on success, -1 on failure. */ static int trans_tbl_add_sq(merged_header_t* merged_hdr, sam_hdr_t *translate, trans_tbl_t* tbl) { int32_t i; int min_tid = -1, res; kstring_t sq_line = { 0, 0, NULL }, sq_sn = { 0, 0, NULL }; // Fill in the tid part of the translation table, adding new targets // to the merged header as we go. for (i = 0; i < sam_hdr_nref(translate); ++i) { int trans_tid; sq_sn.l = 0; res = sam_hdr_find_tag_pos(translate, "SQ", i, "SN", &sq_sn); if (res < 0) { print_error("merge", "failed to get @SQ SN #%d from header", i + 1); goto fail; } trans_tid = sam_hdr_name2tid(merged_hdr->hdr, sq_sn.s); if (trans_tid < -1) { print_error("merge", "failed to lookup ref"); goto fail; } if (trans_tid < 0) { // Append missing entries to out_hdr sq_line.l = 0; res = sam_hdr_find_line_id(translate, "SQ", "SN", sq_sn.s, &sq_line); if (res < 0) { print_error("merge", "failed to get @SQ SN:%s from header", sq_sn.s); goto fail; } trans_tid = sam_hdr_nref(merged_hdr->hdr); res = sam_hdr_add_lines(merged_hdr->hdr, sq_line.s, sq_line.l); if (res < 0) { print_error("merge", "failed to add @SQ SN:%s to new header", sq_sn.s); goto fail; } } tbl->tid_trans[i] = trans_tid; if (tbl->tid_trans[i] > min_tid) { min_tid = tbl->tid_trans[i]; } else { tbl->lost_coord_sort = true; } } free(sq_line.s); free(sq_sn.s); return 0; fail: free(sq_line.s); free(sq_sn.s); return -1; } /* * Common code for setting up RG and PG record ID tag translation. * * is_rg is true for RG translation, false for PG. * translate is the input bam header * merge is true if tags with the same ID are to be merged. * known_ids is the set of IDs already in the output header. * id_map is the translation map from input header IDs to output header IDs * If override is set, it will be used to replace the existing ID (RG only) * * known_ids and id_map have entries for the new IDs added to them. * * Return value is a linked list of header lines with the translated IDs, * or NULL if something went wrong (probably out of memory). * */ static klist_t(hdrln) * trans_rg_pg(bool is_rg, sam_hdr_t *translate, bool merge, khash_t(cset)* known_ids, khash_t(c2c)* id_map, char *override) { khiter_t iter; int num_ids, i; const char *rec_type = is_rg ? "RG" : "PG"; klist_t(hdrln) *hdr_lines; hdr_lines = kl_init(hdrln); // Search through translate's header num_ids = sam_hdr_count_lines(translate, rec_type); if (num_ids < 0) goto fail; for (i = 0; i < num_ids; i++) { kstring_t orig_id = { 0, 0, NULL }; // ID in original header kstring_t transformed_id = { 0, 0, NULL }; // ID in output header char *map_value; // Value to store in id_map bool id_changed; // Have we changed the ID? bool not_found_in_output; // ID isn't in the output header (yet) if (sam_hdr_find_tag_pos(translate, rec_type, i, "ID", &orig_id) < 0) goto fail; // is our matched ID in our output ID set already? iter = kh_get(cset, known_ids, ks_str(&orig_id)); not_found_in_output = (iter == kh_end(known_ids)); if (override) { // Override original ID (RG only) #ifdef OVERRIDE_DOES_NOT_MERGE if (gen_unique_id(override, known_ids, false, &transformed_id)) goto memfail; not_found_in_output = true; // As ID now unique #else if (kputs(override, &transformed_id) == EOF) goto memfail; // Know about override already? iter = kh_get(cset, known_ids, ks_str(&transformed_id)); not_found_in_output = (iter == kh_end(known_ids)); #endif id_changed = true; } else { if ( not_found_in_output || merge) { // Not in there or merging so can add it as 1-1 mapping if (ks_to_ks(&orig_id, &transformed_id)) goto memfail; id_changed = false; } else { // It's in there so we need to transform it by appending // a random number to the id if (gen_unique_id(ks_str(&orig_id), known_ids, true, &transformed_id)) goto memfail; id_changed = true; not_found_in_output = true; // As ID now unique } } // Does this line need to go into our output header? if (not_found_in_output) { // Take matched line and replace ID with transformed_id kstring_t new_hdr_line = { 0, 0, NULL }; if (sam_hdr_find_line_id(translate, rec_type, "ID", ks_str(&orig_id), &new_hdr_line) < 0){ goto fail; } if (id_changed) { char *idp = strstr(ks_str(&new_hdr_line), "\tID:"), *id_end; ptrdiff_t id_offset, id_len; if (!idp) { print_error("merge", "failed to find ID in \"%s\"\n", ks_str(&new_hdr_line)); goto fail; } idp += 4; for (id_end = idp; *id_end >= '\n'; id_end++) {} id_offset = idp - new_hdr_line.s; id_len = id_end - idp; if (id_len < transformed_id.l) { if (ks_resize(&new_hdr_line, new_hdr_line.l + transformed_id.l - id_len + 1/*nul*/)) goto fail; } if (id_len != transformed_id.l) { memmove(new_hdr_line.s + id_offset + transformed_id.l, new_hdr_line.s + id_offset + id_len, new_hdr_line.l - id_offset - id_len + 1); } memcpy(new_hdr_line.s + id_offset, transformed_id.s, transformed_id.l); } // append line to output linked list char** ln = kl_pushp(hdrln, hdr_lines); *ln = ks_release(&new_hdr_line); // Give away to linked list // Need to add it to known_ids set int in_there = 0; iter = kh_put(cset, known_ids, ks_str(&transformed_id), &in_there); if (in_there < 0) goto memfail; assert(in_there > 0); // Should not already be in the map map_value = ks_release(&transformed_id); } else { // Use existing string in id_map assert(kh_exist(known_ids, iter)); map_value = kh_key(known_ids, iter); free(ks_release(&transformed_id)); } // Insert it into our translation map int in_there = 0; iter = kh_put(c2c, id_map, ks_release(&orig_id), &in_there); kh_value(id_map, iter) = map_value; } // If there are no RG lines in the file and we are overriding add one if (is_rg && override && hdr_lines->size == 0) { kstring_t new_id = {0, 0, NULL}; kstring_t line = {0, 0, NULL}; kstring_t empty = {0, 0, NULL}; int in_there = 0; char** ln; // Get the new ID if (gen_unique_id(override, known_ids, false, &new_id)) goto memfail; // Make into a header line and add to linked list ksprintf(&line, "@RG\tID:%s", ks_str(&new_id)); ln = kl_pushp(hdrln, hdr_lines); *ln = ks_release(&line); // Put into known_ids set iter = kh_put(cset, known_ids, ks_str(&new_id), &in_there); if (in_there < 0) goto memfail; assert(in_there > 0); // Should be a new entry // Put into translation map (key is empty string) if (kputs("", &empty) == EOF) goto memfail; iter = kh_put(c2c, id_map, ks_release(&empty), &in_there); if (in_there < 0) goto memfail; assert(in_there > 0); // Should be a new entry kh_value(id_map, iter) = ks_release(&new_id); } return hdr_lines; memfail: perror(__func__); fail: if (hdr_lines) kl_destroy(hdrln, hdr_lines); return NULL; } /* * Common code for completing RG and PG record translation. * * Input is a list of header lines, and the mapping from input to * output @PG record IDs. * * RG and PG records can contain tags that cross-reference to other @PG * records. This fixes the tags to contain the new IDs before adding * them to the output header text. */ static int finish_rg_pg(bool is_rg, klist_t(hdrln) *hdr_lines, khash_t(c2c)* pg_map, kstring_t *out_text) { const char *search = is_rg ? "\tPG:" : "\tPP:"; khiter_t idx; char *line = NULL; while ((kl_shift(hdrln, hdr_lines, &line)) == 0) { char *id = strstr(line, search); // Look for tag to fix int pos1 = 0, pos2 = 0; char *new_id = NULL; if (id) { // Found a tag. Look up the value in the translation map // to see what it should be changed to in the output file. char *end, tmp; id += 4; // Point to value end = strchr(id, '\t'); // Find end of tag if (!end) end = id + strlen(id); tmp = *end; *end = '\0'; // Temporarily get the value on its own. // Look-up in translation table idx = kh_get(c2c, pg_map, id); if (idx == kh_end(pg_map)) { // Not found, warn. fprintf(stderr, "[W::%s] Tag %s%s not found in @PG records\n", __func__, search + 1, id); } else { // Remember new id and splice points on original string new_id = kh_value(pg_map, idx); pos1 = id - line; pos2 = end - line; } *end = tmp; // Restore string } // Copy line to output: // line[0..pos1), new_id (if not NULL), line[pos2..end), '\n' if (pos1 && range_to_ks(line, 0, pos1, out_text)) goto memfail; if (new_id && kputs(new_id, out_text) == EOF) goto memfail; if (kputs(line + pos2, out_text) == EOF) goto memfail; if (kputc('\n', out_text) == EOF) goto memfail; free(line); // No longer needed line = NULL; } return 0; memfail: perror(__func__); free(line); // Prevent leakage as no longer on list return -1; } /* * Build the translation table for an input *am file. This stores mappings * which allow IDs to be converted from those used in the input file * to the ones which will be used in the output. The mappings are for: * Reference sequence IDs (for @SQ records) * @RG record ID tags * @PG record ID tags * * At the same time, new header text is built up by copying records * from the input bam file. This will eventually become the header for * the output file. When copied, the ID tags for @RG and @PG records * are replaced with their values. The @PG PP: and @RG PG: tags * are also modified if necessary. * * merged_hdr holds state on the output header (which IDs are present, etc.) * translate is the input header * tbl is the translation table that gets filled in. * merge_rg controls merging of @RG records * merge_pg controls merging of @PG records * If rg_override is not NULL, it will be used to replace the existing @RG ID * * Returns 0 on success, -1 on failure. */ static int trans_tbl_init(merged_header_t* merged_hdr, sam_hdr_t* translate, trans_tbl_t* tbl, bool merge_rg, bool merge_pg, bool copy_co, char* rg_override) { kstring_t lines = { 0, 0, NULL }; klist_t(hdrln) *rg_list = NULL; klist_t(hdrln) *pg_list = NULL; tbl->n_targets = sam_hdr_nref(translate); tbl->rg_trans = tbl->pg_trans = NULL; tbl->tid_trans = (int*)calloc(tbl->n_targets ? tbl->n_targets : 1, sizeof(int)); if (tbl->tid_trans == NULL) goto memfail; tbl->rg_trans = kh_init(c2c); if (tbl->rg_trans == NULL) goto memfail; tbl->pg_trans = kh_init(c2c); if (tbl->pg_trans == NULL) goto memfail; tbl->lost_coord_sort = false; // Get the @HD record (if not there already). if (trans_tbl_add_hd(merged_hdr, translate)) goto fail; // Fill in map and add header lines for @SQ records if (trans_tbl_add_sq(merged_hdr, translate, tbl)) goto fail; // Get translated header lines and fill in map for @RG records rg_list = trans_rg_pg(true, translate, merge_rg, merged_hdr->rg_ids, tbl->rg_trans, rg_override); if (!rg_list) goto fail; // Get translated header lines and fill in map for @PG records pg_list = trans_rg_pg(false, translate, merge_pg, merged_hdr->pg_ids, tbl->pg_trans, NULL); if (!pg_list) goto fail; // Fix-up PG: tags in the new @RG records and add to output if (finish_rg_pg(true, rg_list, tbl->pg_trans, &merged_hdr->out_rg)) goto fail; // Fix-up PP: tags in the new @PG records and add to output lines.l = 0; if (finish_rg_pg(false, pg_list, tbl->pg_trans, &merged_hdr->out_pg)) goto fail; kl_destroy(hdrln, rg_list); rg_list = NULL; kl_destroy(hdrln, pg_list); pg_list = NULL; if (copy_co) { // Just append @CO headers without translation int num_co = sam_hdr_count_lines(translate, "CO"), i; if (num_co < 0) goto fail; for (i = 0; i < num_co; i++) { if (sam_hdr_find_line_pos(translate, "CO", i, &lines) < 0) goto fail; if (ks_to_ks(&lines, &merged_hdr->out_co)) goto fail; if (kputc('\n', &merged_hdr->out_co) < 0) goto fail; } } free(lines.s); return 0; memfail: perror(__func__); fail: trans_tbl_destroy(tbl); if (rg_list) kl_destroy(hdrln, rg_list); if (pg_list) kl_destroy(hdrln, pg_list); free(lines.s); return -1; } static int finish_merged_header(merged_header_t *merged_hdr) { if (sam_hdr_add_lines(merged_hdr->hdr, ks_c_str(&merged_hdr->out_rg), ks_len(&merged_hdr->out_rg)) < 0) return -1; if (sam_hdr_add_lines(merged_hdr->hdr, ks_c_str(&merged_hdr->out_pg), ks_len(&merged_hdr->out_pg)) < 0) return -1; if (sam_hdr_add_lines(merged_hdr->hdr, ks_c_str(&merged_hdr->out_co), ks_len(&merged_hdr->out_co)) < 0) return -1; return 0; } /* * Free a merged_header_t struct and all associated data. * * Note that the keys to the rg_ids and pg_ids sets are also used as * values in the translation tables. This function should therefore not * be called until the translation tables are no longer needed. */ static void free_merged_header(merged_header_t *merged_hdr) { size_t i; khiter_t iter; if (!merged_hdr) return; free(ks_release(&merged_hdr->out_rg)); free(ks_release(&merged_hdr->out_pg)); free(ks_release(&merged_hdr->out_co)); if (merged_hdr->target_name) { for (i = 0; i < merged_hdr->n_targets; i++) { free(merged_hdr->target_name[i]); } free(merged_hdr->target_name); } free(merged_hdr->target_len); kh_destroy(c2i, merged_hdr->sq_tids); if (merged_hdr->rg_ids) { for (iter = kh_begin(merged_hdr->rg_ids); iter != kh_end(merged_hdr->rg_ids); ++iter) { if (kh_exist(merged_hdr->rg_ids, iter)) free(kh_key(merged_hdr->rg_ids, iter)); } kh_destroy(cset, merged_hdr->rg_ids); } if (merged_hdr->pg_ids) { for (iter = kh_begin(merged_hdr->pg_ids); iter != kh_end(merged_hdr->pg_ids); ++iter) { if (kh_exist(merged_hdr->pg_ids, iter)) free(kh_key(merged_hdr->pg_ids, iter)); } kh_destroy(cset, merged_hdr->pg_ids); } free(merged_hdr); } static void bam_translate(bam1_t* b, trans_tbl_t* tbl) { // Update target id if not unmapped tid if ( b->core.tid >= 0 ) { b->core.tid = tbl->tid_trans[b->core.tid]; } if ( b->core.mtid >= 0 ) { b->core.mtid = tbl->tid_trans[b->core.mtid]; } // If we have a RG update it uint8_t *rg = bam_aux_get(b, "RG"); if (rg) { char* decoded_rg = bam_aux2Z(rg); khiter_t k = kh_get(c2c, tbl->rg_trans, decoded_rg); if (k != kh_end(tbl->rg_trans)) { char* translate_rg = kh_value(tbl->rg_trans,k); bam_aux_del(b, rg); if (translate_rg) { bam_aux_append(b, "RG", 'Z', strlen(translate_rg) + 1, (uint8_t*)translate_rg); } } else { char *tmp = strdup(decoded_rg); fprintf(stderr, "[bam_translate] RG tag \"%s\" on read \"%s\" encountered " "with no corresponding entry in header, tag lost. " "Unknown tags are only reported once per input file for " "each tag ID.\n", decoded_rg, bam_get_qname(b)); bam_aux_del(b, rg); // Prevent future whinges if (tmp) { int in_there = 0; k = kh_put(c2c, tbl->rg_trans, tmp, &in_there); if (in_there > 0) kh_value(tbl->rg_trans, k) = NULL; } } } // If we have a PG update it uint8_t *pg = bam_aux_get(b, "PG"); if (pg) { char* decoded_pg = bam_aux2Z(pg); khiter_t k = kh_get(c2c, tbl->pg_trans, decoded_pg); if (k != kh_end(tbl->pg_trans)) { char* translate_pg = kh_value(tbl->pg_trans,k); bam_aux_del(b, pg); if (translate_pg) { bam_aux_append(b, "PG", 'Z', strlen(translate_pg) + 1, (uint8_t*)translate_pg); } } else { char *tmp = strdup(decoded_pg); fprintf(stderr, "[bam_translate] PG tag \"%s\" on read \"%s\" encountered " "with no corresponding entry in header, tag lost. " "Unknown tags are only reported once per input file for " "each tag ID.\n", decoded_pg, bam_get_qname(b)); bam_aux_del(b, pg); // Prevent future whinges if (tmp) { int in_there = 0; k = kh_put(c2c, tbl->pg_trans, tmp, &in_there); if (in_there > 0) kh_value(tbl->pg_trans, k) = NULL; } } } } int* rtrans_build(int n, int n_targets, trans_tbl_t* translation_tbl) { // Create reverse translation table for tids int* rtrans = (int*)malloc(sizeof(int32_t)*n*n_targets); const int32_t NOTID = INT32_MIN; if (!rtrans) return NULL; memset_pattern4((void*)rtrans, &NOTID, sizeof(int32_t)*n*n_targets); int i; for (i = 0; i < n; ++i) { int j; for (j = 0; j < (translation_tbl+i)->n_targets; ++j) { if ((translation_tbl+i)->tid_trans[j] != -1) { rtrans[i*n_targets + (translation_tbl+i)->tid_trans[j]] = j; } } } return rtrans; } #define MERGE_RG 1 // Attach RG tag based on filename #define MERGE_UNCOMP 2 // Generate uncompressed BAM #define MERGE_LEVEL1 4 // Compress the BAM at level 1 (fast) mode #define MERGE_FORCE 8 // Overwrite output BAM if it exists #define MERGE_COMBINE_RG 16 // Combine RG tags frather than redefining them #define MERGE_COMBINE_PG 32 // Combine PG tags frather than redefining them #define MERGE_FIRST_CO 64 // Use only first file's @CO headers (sort cmd only) static hts_reglist_t *duplicate_reglist(const hts_reglist_t *rl, int rn) { if (!rl) return NULL; hts_reglist_t *new_rl = calloc(rn, sizeof(hts_reglist_t)); if (!new_rl) return NULL; int i; for (i=0; i < rn; i++) { new_rl[i].tid = rl[i].tid; new_rl[i].count = rl[i].count; new_rl[i].min_beg = rl[i].min_beg; new_rl[i].max_end = rl[i].max_end; new_rl[i].reg = rl[i].reg; new_rl[i].intervals = malloc(new_rl[i].count * sizeof(hts_pair_pos_t)); if (!new_rl[i].intervals) { hts_reglist_free(new_rl, i); return NULL; } memcpy(new_rl[i].intervals, rl[i].intervals, new_rl[i].count * sizeof(hts_pair_pos_t)); } return new_rl; } /* * How merging is handled * * If a header is defined use we will use that as our output header * otherwise we use the first header from the first input file. * * Now go through each file and create a translation table for that file for: * -RG * -tid * -PG tags * * Then whenever we read a record from a bam we translate that read before * stashing it in the hash. * * In the actual merge, a read is read from each input file, translated and * stashed in the hash. This assumes that all input files are sorted in the * same way. Next we just extract the next position ordered read from the * hash, and replace it if there are still reads left in it's source input * file. Finally we write our chosen read it to the output file. */ /*! @abstract Merge multiple sorted BAM. @param sam_order the order in which the data was sorted @param sort_tag if non-null, the tag that data was sorted by @param out output BAM file name @param mode sam_open() mode to be used to create the final output file (overrides level settings from UNCOMP and LEVEL1 flags) @param headers name of SAM file from which to copy '@' header lines, or NULL to copy them from the first file to be merged @param n number of files to be merged @param fn names of files to be merged @param flag flags that control how the merge is undertaken @param reg region to merge @param n_threads number of threads to use (passed to htslib) @param cmd command name (used in print_error() etc) @param in_fmt format options for input files @param out_fmt output file format and options @param write_index create the index, together with the output file @param arg_list command string for PG line @param no_pg if 1, do not add a new PG line @discussion Padding information may NOT correctly maintained. This function is NOT thread safe. */ int bam_merge_core2(SamOrder sam_order, char* sort_tag, const char *out, const char *mode, const char *headers, int n, char * const *fn, char * const *fn_idx, const char *fn_bed, int flag, const char *reg, int n_threads, const char *cmd, const htsFormat *in_fmt, const htsFormat *out_fmt, int write_index, char *arg_list, int no_pg) { samFile *fpout, **fp = NULL; heap1_t *heap = NULL; sam_hdr_t *hout = NULL; sam_hdr_t *hin = NULL; int i, j, *RG_len = NULL; uint64_t idx = 0; char **RG = NULL; hts_itr_t **iter = NULL; sam_hdr_t **hdr = NULL; trans_tbl_t *translation_tbl = NULL; int *rtrans = NULL; char *out_idx_fn = NULL; void *hreg = NULL; hts_reglist_t *lreg = NULL; merged_header_t *merged_hdr = init_merged_header(); if (!merged_hdr) return -1; refs_t *refs = NULL; template_coordinate_keys_t *keys = NULL; khash_t(const_c2c) *lib_lookup = NULL; // Is there a specified pre-prepared header to use for output? if (headers) { samFile* fpheaders = sam_open(headers, "r"); if (fpheaders == NULL) { print_error_errno(cmd, "cannot open \"%s\"", headers); return -1; } hin = sam_hdr_read(fpheaders); sam_close(fpheaders); if (hin == NULL) { print_error(cmd, "couldn't read headers from \"%s\"", headers); goto mem_fail; } } g_sam_order = sam_order; if (sam_order == TagQueryName || sam_order == TagCoordinate) { g_sort_tag[0] = sort_tag[0]; g_sort_tag[1] = sort_tag[0] ? sort_tag[1] : '\0'; } fp = (samFile**)calloc(n, sizeof(samFile*)); if (!fp) goto mem_fail; heap = (heap1_t*)calloc(n, sizeof(heap1_t)); if (!heap) goto mem_fail; iter = (hts_itr_t**)calloc(n, sizeof(hts_itr_t*)); if (!iter) goto mem_fail; hdr = (sam_hdr_t**)calloc(n, sizeof(sam_hdr_t*)); if (!hdr) goto mem_fail; translation_tbl = (trans_tbl_t*)calloc(n, sizeof(trans_tbl_t)); if (!translation_tbl) goto mem_fail; RG = (char**)calloc(n, sizeof(char*)); if (!RG) goto mem_fail; // prepare RG tag from file names if (flag & MERGE_RG) { RG_len = (int*)calloc(n, sizeof(int)); if (!RG_len) goto mem_fail; for (i = 0; i != n; ++i) { int l = strlen(fn[i]); const char *s = fn[i]; if (l > 4 && (strcmp(s + l - 4, ".bam") == 0 || strcmp(s + l - 4, ".sam") == 0)) l -= 4; if (l > 5 && strcmp(s + l - 5, ".cram") == 0) l -= 5; for (j = l - 1; j >= 0; --j) if (s[j] == '/') break; ++j; l -= j; RG[i] = (char*)calloc(l + 1, 1); if (!RG[i]) goto mem_fail; RG_len[i] = l; strncpy(RG[i], s + j, l); } } if (hin) { // Populate merged_hdr from the pre-prepared header trans_tbl_t dummy; int res; res = trans_tbl_init(merged_hdr, hin, &dummy, flag & MERGE_COMBINE_RG, flag & MERGE_COMBINE_PG, true, NULL); trans_tbl_destroy(&dummy); if (res) return -1; // FIXME: memory leak } // open and read the header from each file for (i = 0; i < n; ++i) { sam_hdr_t *hin; fp[i] = sam_open_format(fn[i], "r", in_fmt); if (fp[i] == NULL) { print_error_errno(cmd, "fail to open \"%s\"", fn[i]); goto fail; } hts_set_opt(fp[i], HTS_OPT_BLOCK_SIZE, BAM_BLOCK_SIZE); hin = sam_hdr_read(fp[i]); if (hin == NULL) { print_error(cmd, "failed to read header from \"%s\"", fn[i]); goto fail; } if (trans_tbl_init(merged_hdr, hin, translation_tbl+i, flag & MERGE_COMBINE_RG, flag & MERGE_COMBINE_PG, (flag & MERGE_FIRST_CO)? (i == 0) : true, RG[i])) goto fail; hdr[i] = hin; int order_ok = 1; if ((translation_tbl+i)->lost_coord_sort && (sam_order == Coordinate || sam_order == MinHash)) { fprintf(stderr, "[bam_merge_core] Order of targets in file %s caused coordinate sort to be lost\n", fn[i]); order_ok = 0; } if (!refs) refs = cram_get_refs(fp[i]); if (order_ok && refs && hts_set_opt(fp[i], CRAM_OPT_SHARED_REF, refs)) goto fail; } // Did we get an @HD line? if (!merged_hdr->have_hd) { fprintf(stderr, "[W::%s] No @HD tag found.\n", __func__); /* FIXME: Should we add an @HD line here, and if so what should we put in it? Ideally we want a way of getting htslib to tell us the SAM version number to assume given no @HD line. Is it also safe to assume that the output is coordinate sorted? SO: is optional so we don't have to have it.*/ /* ksprintf(&merged_hdr->out_hd, "@HD\tVN:1.5\tSO:coordinate\n"); */ } // Transform the header into standard form if (finish_merged_header(merged_hdr) < 0) goto fail; hout = merged_hdr->hdr; if (!hout) goto fail; // If we're only merging a specified region move our iters to start at that point int tid, nreg; hts_pos_t beg, end; if (fn_bed) { hreg = bed_read(fn_bed); if (!hreg) { fprintf(stderr, "[%s] Could not read BED file: \"%s\"\n", __func__, fn_bed); goto fail; } bed_unify(hreg); lreg = bed_reglist(hreg, ALL, &nreg); if (!lreg || !nreg) { fprintf(stderr, "[%s] Null or empty region list\n", __func__); goto fail; } } else if (reg) { rtrans = rtrans_build(n, sam_hdr_nref(hout), translation_tbl); if (!rtrans) goto mem_fail; if (!sam_parse_region(hout, reg, &tid, &beg, &end, 0)) { fprintf(stderr, "[%s] Badly formatted region or unknown reference name: \"%s\"\n", __func__, reg); goto fail; } } if (reg || fn_bed) { hts_idx_t *reg_idx = NULL; for (i = 0; i < n; ++i) { // If index filename has not been specified, look in the BAM folder if (fn_idx != NULL) { reg_idx = sam_index_load2(fp[i], fn[i], fn_idx[i]); } else { reg_idx = sam_index_load(fp[i], fn[i]); } if (reg_idx == NULL) { fprintf(stderr, "[%s] failed to load index for %s. Random alignment retrieval only works for indexed BAM or CRAM files.\n", __func__, fn[i]); free(rtrans); rtrans = NULL; goto fail; } int mapped_tid = INT32_MIN; if (fn_bed) { hts_reglist_t *rl = duplicate_reglist(lreg, nreg); iter[i] = sam_itr_regions(reg_idx, hdr[i], rl, nreg); } else { // (rtrans[i*n+tid]) Look up what hout tid translates to in input tid space mapped_tid = rtrans[i*sam_hdr_nref(hout)+tid]; if (mapped_tid != INT32_MIN) { iter[i] = sam_itr_queryi(reg_idx, mapped_tid, beg, end); } else { iter[i] = sam_itr_queryi(reg_idx, HTS_IDX_NONE, 0, 0); } } if (iter[i] == NULL) { if (fn_bed) { fprintf(stderr, "[%s] failed to get multi-region iterator " "{%s, %s}\n", __func__, fn[i], fn_bed); } else { if (mapped_tid != INT32_MIN) { fprintf(stderr, "[%s] failed to get iterator over " "{%s, %d, %"PRIhts_pos", %"PRIhts_pos"}\n", __func__, fn[i], mapped_tid, beg, end); } else { fprintf(stderr, "[%s] failed to get iterator over " "{%s, HTS_IDX_NONE, 0, 0}\n", __func__, fn[i]); } } hts_idx_destroy(reg_idx); free(rtrans); rtrans = NULL; goto fail; } hts_idx_destroy(reg_idx); } free(rtrans); rtrans = NULL; } // Make sure that there's enough memory for template coordinate keys, one per file to read if (sam_order == TemplateCoordinate) { if ((keys = malloc(sizeof(template_coordinate_keys_t))) == NULL) { print_error("sort", "could not allocate memory for the top-level keys"); goto mem_fail; } keys->n = 0; keys->m = 0; keys->buffer_size = 0x10000; keys->buffers = NULL; // Make sure that there's enough memory for template coordinate keys, one per file to read if (keys->n + n >= keys->m * keys->buffer_size) { if (template_coordinate_keys_realloc(keys, keys->n + n) < 0) goto mem_fail; } lib_lookup = lookup_libraries(hout); if (!lib_lookup) { goto mem_fail; } } // Load the first read from each file into the heap for (i = 0; i < n; ++i) { heap1_t *h = heap + i; int res; h->i = i; h->entry.bam_record = bam_init1(); h->entry.u.tag = NULL; if (!h->entry.bam_record) goto mem_fail; res = iter[i] ? sam_itr_next(fp[i], iter[i], h->entry.bam_record) : sam_read1(fp[i], hdr[i], h->entry.bam_record); if (res >= 0) { bam_translate(h->entry.bam_record, translation_tbl + i); h->tid = h->entry.bam_record->core.tid; h->pos = (uint64_t)(h->entry.bam_record->core.pos + 1); h->rev = bam_is_rev(h->entry.bam_record); h->idx = idx++; if (g_sam_order == TagQueryName || g_sam_order == TagCoordinate) { h->entry.u.tag = bam_aux_get(h->entry.bam_record, g_sort_tag); } else if (g_sam_order == TemplateCoordinate) { template_coordinate_key_t *key = template_coordinate_keys_get(keys, i); // get the next key to use h->entry.u.key = template_coordinate_key(heap->entry.bam_record, key, hout, lib_lookup); // update the key if (heap->entry.u.key == NULL) goto mem_fail; // key could not be created, error out } else { h->entry.u.tag = NULL; } } else if (res == -1 && (!iter[i] || iter[i]->finished)) { h->pos = HEAP_EMPTY; bam_destroy1(h->entry.bam_record); h->entry.bam_record = NULL; h->entry.u.tag = NULL; h->entry.u.key = NULL; } else { print_error(cmd, "failed to read first record from \"%s\"", fn[i]); goto fail; } } // Open output file and write header if ((fpout = sam_open_format(out, mode, out_fmt)) == 0) { print_error_errno(cmd, "failed to create \"%s\"", out); return -1; } hts_set_opt(fpout, HTS_OPT_BLOCK_SIZE, BAM_BLOCK_SIZE); if (!no_pg && sam_hdr_add_pg(hout, "samtools", "VN", samtools_version(), arg_list ? "CL": NULL, arg_list ? arg_list : NULL, NULL)) { print_error(cmd, "failed to add PG line to the header of \"%s\"", out); sam_close(fpout); return -1; } if (sam_hdr_write(fpout, hout) != 0) { print_error_errno(cmd, "failed to write header to \"%s\"", out); sam_close(fpout); return -1; } if (write_index) { if (!(out_idx_fn = auto_index(fpout, out, hout))){ sam_close(fpout); return -1; } } if (!(flag & MERGE_UNCOMP)) hts_set_threads(fpout, n_threads); if (refs && hts_set_opt(fpout, CRAM_OPT_SHARED_REF, refs)) goto fail; // Begin the actual merge ks_heapmake(heap, n, heap); while (heap->pos != HEAP_EMPTY) { bam1_t *b = heap->entry.bam_record; if (flag & MERGE_RG) { uint8_t *rg = bam_aux_get(b, "RG"); if (rg) bam_aux_del(b, rg); bam_aux_append(b, "RG", 'Z', RG_len[heap->i] + 1, (uint8_t*)RG[heap->i]); } if (sam_write1(fpout, hout, b) < 0) { print_error_errno(cmd, "failed writing to \"%s\"", out); sam_close(fpout); free(out_idx_fn); return -1; } if ((j = (iter[heap->i]? sam_itr_next(fp[heap->i], iter[heap->i], b) : sam_read1(fp[heap->i], hdr[heap->i], b))) >= 0) { bam_translate(b, translation_tbl + heap->i); heap->tid = b->core.tid; heap->pos = (uint64_t)(b->core.pos + 1); heap->rev = bam_is_rev(b); heap->idx = idx++; if (g_sam_order == TagQueryName || g_sam_order == TagCoordinate) { heap->entry.u.tag = bam_aux_get(heap->entry.bam_record, g_sort_tag); } else if (g_sam_order == TemplateCoordinate) { template_coordinate_key_t *key = template_coordinate_keys_get(keys, heap->i); // get the next key to use heap->entry.u.key = template_coordinate_key(heap->entry.bam_record, key, hout, lib_lookup); // update the key if (heap->entry.u.key == NULL) goto mem_fail; // key could not be created, error out } else { heap->entry.u.tag = NULL; } } else if (j == -1 && (!iter[heap->i] || iter[heap->i]->finished)) { heap->pos = HEAP_EMPTY; bam_destroy1(heap->entry.bam_record); heap->entry.bam_record = NULL; heap->entry.u.tag = NULL; } else { print_error(cmd, "\"%s\" is truncated", fn[heap->i]); goto fail; } ks_heapadjust(heap, 0, n, heap); } if (write_index) { if (sam_idx_save(fpout) < 0) { print_error_errno("merge", "writing index failed"); goto fail; } } free(out_idx_fn); // Clean up and close if (flag & MERGE_RG) { for (i = 0; i != n; ++i) free(RG[i]); free(RG_len); } for (i = 0; i < n; ++i) { trans_tbl_destroy(translation_tbl + i); hts_itr_destroy(iter[i]); sam_hdr_destroy(hdr[i]); sam_close(fp[i]); } sam_hdr_destroy(hin); sam_hdr_destroy(hout); free_merged_header(merged_hdr); hts_reglist_free(lreg, nreg); bed_destroy(hreg); free(RG); free(translation_tbl); free(fp); free(heap); free(iter); free(hdr); if (sam_close(fpout) < 0) { print_error(cmd, "error closing output file"); return -1; } return 0; mem_fail: print_error(cmd, "Out of memory"); fail: if (flag & MERGE_RG) { if (RG) { for (i = 0; i != n; ++i) free(RG[i]); } free(RG_len); } for (i = 0; i < n; ++i) { if (translation_tbl && translation_tbl[i].tid_trans) trans_tbl_destroy(translation_tbl + i); if (iter && iter[i]) hts_itr_destroy(iter[i]); if (hdr && hdr[i]) sam_hdr_destroy(hdr[i]); if (fp && fp[i]) sam_close(fp[i]); if (heap && heap[i].entry.bam_record) bam_destroy1(heap[i].entry.bam_record); } if (hout) sam_hdr_destroy(hout); free(RG); free(translation_tbl); free(hdr); hts_reglist_free(lreg, nreg); bed_destroy(hreg); free(iter); free(heap); free(fp); free(rtrans); free(out_idx_fn); if (keys != NULL) { for (i = 0; i < keys->m; ++i) { free(keys->buffers[i]); } free(keys->buffers); free(keys); } lib_lookup_destroy(lib_lookup); return -1; } // Unused here but may be used by legacy samtools-using third-party code int bam_merge_core(int by_qname, const char *out, const char *headers, int n, char * const *fn, int flag, const char *reg) { char mode[12]; strcpy(mode, "wb"); if (flag & MERGE_UNCOMP) strcat(mode, "0"); else if (flag & MERGE_LEVEL1) strcat(mode, "1"); SamOrder sam_order = by_qname ? QueryName : Coordinate; return bam_merge_core2(sam_order, NULL, out, mode, headers, n, fn, NULL, NULL, flag, reg, 0, "merge", NULL, NULL, 0, NULL, 1); } static void merge_usage(FILE *to) { fprintf(to, "Usage: samtools merge [options] -o [options] ... \n" " or: samtools merge [options] ... \n" "\n" "Options:\n" " -n Input files are sorted by read name\n" " -t TAG Input files are sorted by TAG value\n" " -r Attach RG tag (inferred from file names)\n" " -u Uncompressed BAM output\n" " -f Overwrite the output BAM if exist\n" " -o FILE Specify output file via option instead of argument\n" " -1 Compress level 1\n" " -l INT Compression level, from 0 to 9 [-1]\n" " -R STR Merge file in the specified region STR [all]\n" " -h FILE Copy the header in FILE to [in1.bam]\n" " -c Combine @RG headers with colliding IDs [alter IDs to be distinct]\n" " -p Combine @PG headers with colliding IDs [alter IDs to be distinct]\n" " -s VALUE Override random seed\n" " -b FILE List of input BAM filenames, one per line [null]\n" " -X Use customized index files\n" " -L FILE Specify a BED file for multiple region filtering [null]\n" " --no-PG do not add a PG line\n" " --template-coordinate Input files are sorted by template-coordinate\n"); sam_global_opt_help(to, "-.O..@.."); } int bam_merge(int argc, char *argv[]) { int c, flag = 0, ret = 0, level = -1, has_index_file = 0; char *fn_headers = NULL, *reg = NULL, mode[12]; char *sort_tag = NULL, *fnout = NULL, *arg_list = NULL; long random_seed = (long)time(NULL); char** fn = NULL; char** fn_idx = NULL, *fn_bed = NULL; int fn_size = 0, no_pg = 0; SamOrder sam_order = Coordinate; sam_global_args ga = SAM_GLOBAL_ARGS_INIT; static const struct option lopts[] = { SAM_OPT_GLOBAL_OPTIONS('-', 0, 'O', 0, 0, '@'), { "threads", required_argument, NULL, '@' }, {"no-PG", no_argument, NULL, 1}, { "template-coordinate", no_argument, NULL, 2}, { NULL, 0, NULL, 0 } }; if (argc == 1) { merge_usage(stdout); return 0; } while ((c = getopt_long(argc, argv, "h:nru1R:o:f@:l:cps:b:O:t:XL:", lopts, NULL)) >= 0) { switch (c) { case 'r': flag |= MERGE_RG; break; case 'f': flag |= MERGE_FORCE; break; case 'h': fn_headers = optarg; break; case 'n': sam_order = QueryName; break; case 'o': fnout = optarg; break; case 't': sort_tag = optarg; break; case '1': flag |= MERGE_LEVEL1; level = 1; break; case 'u': flag |= MERGE_UNCOMP; level = 0; break; case 'R': reg = strdup(optarg); break; case 'l': level = atoi(optarg); break; case 'c': flag |= MERGE_COMBINE_RG; break; case 'p': flag |= MERGE_COMBINE_PG; break; case 's': random_seed = atol(optarg); break; case 'X': has_index_file = 1; break; // -X flag for index filename case 'L': fn_bed = optarg; break; case 'b': { // load the list of files to read if (has_index_file) { fprintf(stderr,"Error: The -b option cannot be combined with -X\n"); ret = 1; goto end; } int nfiles; char **fn_read = hts_readlines(optarg, &nfiles); if (fn_read) { // Append to end of array fn = realloc(fn, (fn_size+nfiles) * sizeof(char*)); if (fn == NULL) { ret = 1; goto end; } memcpy(fn+fn_size, fn_read, nfiles * sizeof(char*)); fn_size += nfiles; free(fn_read); } else { print_error("merge", "Invalid file list \"%s\"", optarg); ret = 1; } break; } case 1: no_pg = 1; break; case 2: sam_order = TemplateCoordinate; break; default: if (parse_sam_global_opt(c, optarg, lopts, &ga) == 0) break; /* else fall-through */ case '?': merge_usage(stderr); return 1; } } if (sort_tag != NULL) { sam_order = sam_order == QueryName ? TagQueryName : TagCoordinate; } if (fnout == NULL && argc - optind >= 1) { fnout = argv[optind]; optind++; } if (fnout == NULL) { print_error("merge", "You must at least specify the output file"); merge_usage(stderr); return 1; } if (!no_pg && !(arg_list = stringify_argv(argc+1, argv-1))) { print_error("merge", "failed to create arg_list"); return 1; } hts_srand48(random_seed); if (!(flag & MERGE_FORCE) && strcmp(fnout, "-") != 0) { struct stat sbuf; if (stat(fnout, &sbuf) == 0 && S_ISREG(sbuf.st_mode)) { fprintf(stderr, "[%s] File '%s' exists. Please apply '-f' to overwrite. Abort.\n", __func__, fnout); ret = 1; goto end; } } int nargcfiles = 0; if (has_index_file) { // Calculate # of input BAM files if ((argc - optind) % 2 != 0) { fprintf(stderr, "Odd number of filenames detected! Each BAM file should have an index file\n"); ret = 1; goto end; } nargcfiles = (argc - optind) / 2; } else { nargcfiles = argc - optind; } if (nargcfiles > 0) { // Add argc files to end of array fn = realloc(fn, (fn_size+nargcfiles) * sizeof(char*)); if (fn == NULL) { ret = 1; goto end; } memcpy(fn+fn_size, argv + optind, nargcfiles * sizeof(char*)); if(has_index_file) { fn_idx = realloc(fn_idx, nargcfiles * sizeof(char*)); if (fn_idx == NULL) { ret = 1; goto end; } memcpy(fn_idx+fn_size, argv + nargcfiles + optind, nargcfiles * sizeof(char*)); } } if (fn_size+nargcfiles < 1) { print_error("merge", "You must specify at least one (and usually two or more) input files"); merge_usage(stderr); ret = 1; goto end; } if (reg && fn_bed) { print_error("merge", "You must specify either a BED file or a region"); ret = 1; goto end; } strcpy(mode, "wb"); sam_open_mode(mode+1, fnout, NULL); if (level >= 0) sprintf(strchr(mode, '\0'), "%d", level < 9? level : 9); if (bam_merge_core2(sam_order, sort_tag, fnout, mode, fn_headers, fn_size+nargcfiles, fn, fn_idx, fn_bed, flag, reg, ga.nthreads, "merge", &ga.in, &ga.out, ga.write_index, arg_list, no_pg) < 0) ret = 1; end: if (fn_size > 0) { int i; for (i=0; ii, res; if (i < nfiles) { // read from file res = sam_read1(fp[i], hout, heap->entry.bam_record); if (res >= 0 && g_sam_order == TemplateCoordinate) { // file read OK and TemplateCoordinate order // It is assumed that there are nfiles more keys allocated than keys->n; see allocation in bam_merge_simple template_coordinate_key_t *key = template_coordinate_keys_get(keys, keys->n + i); // get the next key to use heap->entry.u.key = template_coordinate_key(heap->entry.bam_record, key, hout, lib_lookup); // update the key if (heap->entry.u.key == NULL) res = -1; // key could not be created, error out } } else { // read from memory if (in_mem[i - nfiles].from < in_mem[i - nfiles].to) { size_t from = in_mem[i - nfiles].from; heap->entry.bam_record = buf[from].bam_record; if (g_sam_order == TemplateCoordinate) heap->entry.u.key = buf[from].u.key; in_mem[i - nfiles].from++; res = 0; } else { res = -1; } } if (res >= 0) { heap->tid = heap->entry.bam_record->core.tid; heap->pos = (uint64_t)(heap->entry.bam_record->core.pos + 1); heap->rev = bam_is_rev(heap->entry.bam_record); heap->idx = (*idx)++; if (g_sam_order == TagQueryName || g_sam_order == TagCoordinate) { heap->entry.u.tag = bam_aux_get(heap->entry.bam_record, g_sort_tag); } else if (g_sam_order != TemplateCoordinate) { heap->entry.u.tag = NULL; heap->entry.u.key = NULL; } } else if (res == -1) { heap->pos = HEAP_EMPTY; if (i < nfiles) bam_destroy1(heap->entry.bam_record); heap->entry.bam_record = NULL; heap->entry.u.tag = NULL; heap->entry.u.key = NULL; } else { return -1; } return 0; } static int bam_merge_simple(SamOrder sam_order, char *sort_tag, const char *out, const char *mode, sam_hdr_t *hout, int n, char * const *fn, int num_in_mem, buf_region *in_mem, bam1_tag *buf, template_coordinate_keys_t *keys, khash_t(const_c2c) *lib_lookup, htsThreadPool *htspool, const char *cmd, const htsFormat *in_fmt, const htsFormat *out_fmt, char *arg_list, int no_pg, int write_index) { samFile *fpout = NULL, **fp = NULL; heap1_t *heap = NULL; uint64_t idx = 0; int i, heap_size = n + num_in_mem; char *out_idx_fn = NULL; if (sam_order == TagQueryName || sam_order == TagCoordinate) { g_sort_tag[0] = sort_tag[0]; g_sort_tag[1] = sort_tag[0] ? sort_tag[1] : '\0'; } if (n > 0) { fp = (samFile**)calloc(n, sizeof(samFile*)); if (!fp) goto mem_fail; } heap = (heap1_t*)calloc(heap_size, sizeof(heap1_t)); if (!heap) goto mem_fail; // Make sure that there's enough memory for template coordinate keys, one per file to read if (keys && keys->n + n >= keys->m * keys->buffer_size) { if (template_coordinate_keys_realloc(keys, keys->n + n) < 0) goto mem_fail; } // Open each file, read the header and put the first read into the heap for (i = 0; i < heap_size; i++) { sam_hdr_t *hin; heap1_t *h = &heap[i]; if (i < n) { fp[i] = sam_open_format(fn[i], "r", in_fmt); if (fp[i] == NULL) { print_error_errno(cmd, "fail to open \"%s\"", fn[i]); goto fail; } hts_set_opt(fp[i], HTS_OPT_BLOCK_SIZE, BAM_BLOCK_SIZE); if (htspool->pool) hts_set_opt(fp[i], HTS_OPT_THREAD_POOL, htspool); // Read header ... hin = sam_hdr_read(fp[i]); if (hin == NULL) { print_error(cmd, "failed to read header from \"%s\"", fn[i]); goto fail; } // ... and throw it away as we don't really need it sam_hdr_destroy(hin); } // Get a read into the heap h->i = i; h->entry.u.tag = NULL; h->entry.u.key = NULL; if (i < n) { h->entry.bam_record = bam_init1(); if (!h->entry.bam_record) goto mem_fail; } if (heap_add_read(h, n, fp, num_in_mem, in_mem, buf, keys, &idx, hout, lib_lookup) < 0) { assert(i < n); print_error(cmd, "failed to read first record from \"%s\"", fn[i]); goto fail; } } // Open output file and write header if ((fpout = sam_open_format(out, mode, out_fmt)) == 0) { print_error_errno(cmd, "failed to create \"%s\"", out); return -1; } hts_set_opt(fpout, HTS_OPT_BLOCK_SIZE, BAM_BLOCK_SIZE); if (!no_pg && sam_hdr_add_pg(hout, "samtools", "VN", samtools_version(), arg_list ? "CL": NULL, arg_list ? arg_list : NULL, NULL)) { print_error(cmd, "failed to add PG line to the header of \"%s\"", out); sam_close(fpout); return -1; } if (htspool->pool) hts_set_opt(fpout, HTS_OPT_THREAD_POOL, htspool); if (sam_hdr_write(fpout, hout) != 0) { print_error_errno(cmd, "failed to write header to \"%s\"", out); sam_close(fpout); return -1; } if (write_index) { if (!(out_idx_fn = auto_index(fpout, out, hout))){ sam_close(fpout); return -1; } } // Now do the merge ks_heapmake(heap, heap_size, heap); while (heap->pos != HEAP_EMPTY) { bam1_t *b = heap->entry.bam_record; if (g_sam_order == MinHash && b->core.tid == -1) { // Remove the cached minhash value b->core.pos = -1; b->core.mpos = -1; b->core.isize = 0; } if (sam_write1(fpout, hout, b) < 0) { print_error_errno(cmd, "failed writing to \"%s\"", out); goto fail; } if (heap_add_read(heap, n, fp, num_in_mem, in_mem, buf, keys, &idx, hout, lib_lookup) < 0) { assert(heap->i < n); print_error(cmd, "Error reading \"%s\" : %s", fn[heap->i], strerror(errno)); goto fail; } ks_heapadjust(heap, 0, heap_size, heap); } // Clean up and close for (i = 0; i < n; i++) { if (sam_close(fp[i]) != 0) { print_error(cmd, "Error on closing \"%s\" : %s", fn[i], strerror(errno)); } } free(fp); free(heap); if (write_index) { if (sam_idx_save(fpout) < 0) { print_error_errno("merge", "writing index failed"); goto fail; } free(out_idx_fn); } if (sam_close(fpout) < 0) { print_error(cmd, "error closing output file"); return -1; } return 0; mem_fail: print_error(cmd, "Out of memory"); fail: for (i = 0; i < n; i++) { if (fp && fp[i]) sam_close(fp[i]); } for (i = 0; i < heap_size; i++) { if (heap && heap[i].i < n && heap[i].entry.bam_record) bam_destroy1(heap[i].entry.bam_record); } free(fp); free(heap); if (fpout) sam_close(fpout); free(out_idx_fn); return -1; } // Function to compare reads and determine which one is < or > the other // Handle sort-by-pos and sort-by-name. Used as the secondary sort in bam1_lt_by_tag, if reads are equivalent by tag. // Returns a value less than, equal to or greater than zero if a is less than, // equal to or greater than b, respectively. static inline int bam1_cmp_core(const bam1_tag a, const bam1_tag b) { uint64_t pa, pb; if (!a.bam_record) return 1; if (!b.bam_record) return 0; if (g_sam_order == QueryName || g_sam_order == TagQueryName) { int t = strnum_cmp(bam_get_qname(a.bam_record), bam_get_qname(b.bam_record)); if (t != 0) return t; return (int) (a.bam_record->core.flag&0xc0) - (int) (b.bam_record->core.flag&0xc0); } else { pa = a.bam_record->core.tid; pb = b.bam_record->core.tid; if (pa == pb) { pa = (uint64_t)(a.bam_record->core.pos+1); pb = (uint64_t)(b.bam_record->core.pos+1); } if (pa == pb) { pa = bam_is_rev(a.bam_record); pb = bam_is_rev(b.bam_record); } return pa < pb ? -1 : (pa > pb ? 1 : 0); } } uint8_t normalize_type(const uint8_t* aux) { if (*aux == 'c' || *aux == 'C' || *aux == 's' || *aux == 'S' || *aux == 'i' || *aux == 'I') { return 'c'; } else if (*aux == 'f' || *aux == 'd') { return 'f'; } else if (*aux == 'H' || *aux == 'Z') { return 'H'; } else { return *aux; } } // Sort record by tag, using pos or read name as a secondary key if tags are identical. Reads not carrying the tag sort first. // Tags are first sorted by the type character (in case the types differ), or by the appropriate comparator for that type if they agree. // Returns a value less than, equal to or greater than zero if a is less than, // equal to or greater than b, respectively. static inline int bam1_cmp_by_tag(const bam1_tag a, const bam1_tag b) { const uint8_t* aux_a = a.u.tag; const uint8_t* aux_b = b.u.tag; if (aux_a == NULL && aux_b != NULL) { return -1; } else if (aux_a != NULL && aux_b == NULL) { return 1; } else if (aux_a == NULL && aux_b == NULL) { return bam1_cmp_core(a,b); } // 'Normalize' the letters of the datatypes to a canonical letter, // so that comparison of different types // forms a correct total ordering. uint8_t a_type = normalize_type(aux_a); uint8_t b_type = normalize_type(aux_b); if (a_type != b_type) { // Fix int to float comparisons by using bam_aux2f() to read the int if (a_type == 'c' && b_type == 'f') { a_type = 'f'; } else if (a_type == 'f' && b_type == 'c') { b_type = 'f'; } else { // Unfixable mismatched types return a_type < b_type ? -1 : 1; } } if (a_type == 'c') { int64_t va = bam_aux2i(aux_a); int64_t vb = bam_aux2i(aux_b); if (va != vb) return va < vb ? -1 : 1; return bam1_cmp_core(a, b); } else if (a_type == 'f') { double va = bam_aux2f(aux_a); double vb = bam_aux2f(aux_b); if (va != vb) return va < vb ? -1 : 1; return bam1_cmp_core(a, b); } else if (a_type == 'A') { unsigned char va = bam_aux2A(aux_a); unsigned char vb = bam_aux2A(aux_b); if (va != vb) return va < vb ? -1 : 1; return bam1_cmp_core(a, b); } else if (a_type == 'H') { int t = strcmp(bam_aux2Z(aux_a), bam_aux2Z(aux_b)); if (t) return t; return bam1_cmp_core(a, b); } else { return bam1_cmp_core(a,b); } } // Sort by minimiser (stored in bam1_tag.u.pos). // If equal, sort by position. // // The 64-bit sort key is split over the bam pos and isize fields. // This permits it to survive writing to temporary file and coming back. static inline int bam1_cmp_by_minhash(const bam1_tag a, const bam1_tag b) { const bam1_t *A = a.bam_record; const bam1_t *B = b.bam_record; if (!A) return 1; if (!B) return 0; if (A->core.tid != -1 || B->core.tid != -1) return bam1_cmp_core(a,b); const uint64_t m_a = (((uint64_t)A->core.pos)<<32)|(uint32_t)A->core.mpos; const uint64_t m_b = (((uint64_t)B->core.pos)<<32)|(uint32_t)B->core.mpos; if (m_a < m_b) // by hash return -1; else if (m_a > m_b) return 1; else if (A->core.isize < B->core.isize) // by hash location in seq return -1; else if (A->core.isize > B->core.isize) return 1; else return bam1_cmp_core(a,b); } // compares to molecular identifiers, ignoring any trailing slash and subsequent single-character // * if mid1 is less than mid2, then -1 will be returned // * if mid1 is greater than mid2, then 1 will be returned static inline int template_coordinate_key_compare_mid(const char* mid1, const char* mid2) { size_t i = 0; size_t len1 = strlen(mid1); size_t len2 = strlen(mid2); size_t shortest; // Snip off trailing slash followed by a single character, if present if (len1 >= 2 && mid1[len1-2] == '/') len1 -= 2; if (len2 >= 2 && mid2[len2-2] == '/') len2 -= 2; shortest = len1 < len2 ? len1 : len2; // find first mismatching character while (i < shortest && mid1[i] == mid2[i]) i++; // compare last characters if (i == len1 && i < len2) return -1; // mid1 shorter if (i == len2 && i < len1) return 1; // mid2 shorter if (i == len1 && i == len2) return 0; // all characters match if (mid1[i] < mid2[i]) return -1; // mid1 earlier else return 1; } // Builds a key use to sort in TemplateCoordinate order. Returns NULL if the key could not be created (e.g. MC // tag is missing), otherwise the pointer to the provided key. static template_coordinate_key_t* template_coordinate_key(bam1_t *b, template_coordinate_key_t *key, sam_hdr_t *hdr, khash_t(const_c2c) *lib_lookup) { uint8_t *data; char *rg; khiter_t k; // defaults key->tid1 = key->tid2 = INT32_MAX; key->pos1 = key->pos2 = HTS_POS_MAX; key->neg1 = key->neg2 = false; key->mid = ""; // update values rg = (char *)bam_aux_get(b, "RG"); if (rg && rg[0] == 'Z' &&(k = kh_get(const_c2c, lib_lookup, rg + 1)) < kh_end(lib_lookup)) { key->library = kh_value(lib_lookup, k); } else { key->library = ""; } key->name = bam_get_qname(b); if (!(b->core.flag & BAM_FUNMAP)) { // read is mapped, update coordinates key->tid1 = b->core.tid; key->neg1 = bam_is_rev(b); key->pos1 = (key->neg1) ? unclipped_end(b) : unclipped_start(b); } if (b->core.flag & BAM_FPAIRED && !(b->core.flag & BAM_FMUNMAP)) { // mate is mapped, update coordinates char *cigar; if ((data = bam_aux_get(b, "MC"))) { if (!(cigar = bam_aux2Z(data))) { fprintf(stderr, "[bam_sort] error: MC tag wrong type. Please use the MC tag provided by samtools fixmate.\n"); return NULL; } } else { fprintf(stderr, "[bam_sort] error: no MC tag. Please run samtools fixmate on file first.\n"); return NULL; } key->tid2 = b->core.mtid; key->neg2 = bam_is_mrev(b); key->pos2 = (key->neg2) ? unclipped_other_end(b->core.mpos, cigar) : unclipped_other_start(b->core.mpos, cigar); } if ((data = bam_aux_get(b, "MI"))) { if (!(key->mid=bam_aux2Z(data))) { fprintf(stderr, "[bam_sort] error: MI tag wrong type (not a string).\n"); return NULL; } } // set is_upper_of_pair, and swap if we get the same key regardless of which end // of the pair it is if (key->tid1 < key->tid2 || (key->tid1 == key->tid2 && key->pos1 < key->pos2) || (key->tid1 == key->tid2 && key->pos1 == key->pos2 && !key->neg1)) { key->is_upper_of_pair = false; } else { key->is_upper_of_pair = true; // swap int tmp_tid; hts_pos_t tmp_pos; bool tmp_neg; tmp_tid = key->tid1; key->tid1 = key->tid2; key->tid2 = tmp_tid; tmp_pos = key->pos1; key->pos1 = key->pos2; key->pos2 = tmp_pos; tmp_neg = key->neg1; key->neg1 = key->neg2; key->neg2 = tmp_neg; } return key; } // Function to compare reads and determine which one is < or > the other // Handles template-coordinate, which sorts by: // 1. the earlier unclipped 5' coordinate of the read pair // 2. the higher unclipped 5' coordinate of the read pair // 3. library (from read group) // 4. the molecular identifier (if present) // 5. read name // 6. if unpaired, or if R1 has the lower coordinates of the pair // Returns a value less than, equal to or greater than zero if a is less than, // equal to or greater than b, respectively. static inline int bam1_cmp_template_coordinate(const bam1_tag a, const bam1_tag b) { if (!a.bam_record) return 1; if (!b.bam_record) return 0; const template_coordinate_key_t* key_a = a.u.key; const template_coordinate_key_t* key_b = b.u.key; int retval = 0; if (0 == retval) retval = key_a->tid1 - key_b->tid1; if (0 == retval) retval = key_a->tid2 - key_b->tid2; if (0 == retval) retval = key_a->pos1 < key_b->pos1 ? -1 : (key_a->pos1 > key_b->pos1 ? 1 : 0); if (0 == retval) retval = key_a->pos2 < key_b->pos2 ? -1 : (key_a->pos2 > key_b->pos2 ? 1 : 0); if (0 == retval) retval = key_a->neg1 == key_b->neg1 ? 0 : (key_a->neg1 ? -1 : 1); if (0 == retval) retval = key_a->neg2 == key_b->neg2 ? 0 : (key_a->neg2 ? -1 : 1); if (0 == retval) retval = strcmp(key_a->library, key_b->library); if (0 == retval) retval = template_coordinate_key_compare_mid(key_a->mid, key_b->mid); if (0 == retval) retval = strcmp(key_a->name, key_b->name); if (0 == retval) retval = key_a->is_upper_of_pair == key_b->is_upper_of_pair ? 0 : (key_a->is_upper_of_pair ? 1 : -1); return retval < 0 ? -1 : (retval > 0 ? 1 : 0); } // Function to compare reads and determine which one is < the other // Handle sort-by-pos, sort-by-name, sort-by-tag, or sort-by-template-coordinate. static inline int bam1_lt(const bam1_tag a, const bam1_tag b) { switch (g_sam_order) { case Coordinate: case QueryName: return bam1_cmp_core(a, b) < 0; case TagQueryName: case TagCoordinate: return bam1_cmp_by_tag(a, b) < 0; case MinHash: return bam1_cmp_by_minhash(a, b) < 0; case TemplateCoordinate: return bam1_cmp_template_coordinate(a, b) < 0; default: return bam1_cmp_core(a,b) < 0; } } KSORT_INIT(sort, bam1_tag, bam1_lt) typedef struct { size_t buf_len; bam1_tag *buf; const sam_hdr_t *h; int error; int large_pos; int minimiser_kmer; } worker_t; // Returns 0 for success // -1 for failure static int write_buffer(const char *fn, const char *mode, size_t l, bam1_tag *buf, const sam_hdr_t *h, int n_threads, const htsFormat *fmt, int clear_minhash, char *arg_list, int no_pg, int write_index) { size_t i; samFile* fp; char *out_idx_fn = NULL; fp = sam_open_format(fn, mode, fmt); if (fp == NULL) return -1; hts_set_opt(fp, HTS_OPT_BLOCK_SIZE, BAM_BLOCK_SIZE); if (!no_pg && sam_hdr_add_pg((sam_hdr_t *)h, "samtools", "VN", samtools_version(), arg_list ? "CL": NULL, arg_list ? arg_list : NULL, NULL)) { goto fail; } if (sam_hdr_write(fp, h) != 0) goto fail; if (write_index) if (!(out_idx_fn = auto_index(fp, fn, (sam_hdr_t *)h))) goto fail; if (n_threads > 1) hts_set_threads(fp, n_threads); for (i = 0; i < l; ++i) { bam1_t *b = buf[i].bam_record; if (clear_minhash && b->core.tid == -1) { // Remove the cached minhash value b->core.pos = -1; b->core.mpos = -1; b->core.isize = 0; } if (sam_write1(fp, h, b) < 0) goto fail; } if (write_index) { if (sam_idx_save(fp) < 0) { print_error_errno("merge", "writing index failed"); goto fail; } free(out_idx_fn); } if (sam_close(fp) < 0) return -1; return 0; fail: sam_close(fp); free(out_idx_fn); return -1; } #define NUMBASE 256 static int ks_radixsort(size_t n, bam1_tag *buf, const sam_hdr_t *h) { int curr = 0, ret = -1; ssize_t i; bam1_tag *buf_ar2[2], *bam_a, *bam_b; uint64_t max_pos = 1; uint32_t max_tid = 1, tid_bytes = 0, pos_bytes = 0, byte = 0; uint32_t tid_shift_l, tid_shift_r; int nref = sam_hdr_nref(h); // Count number of bytes needed for biggest tid and pos // Notes: Add 1 to core.pos so always positive. // Convert unmapped tid (-1) to number of references so unmapped // sort to the end. for (i = 0; i < n; i++) { bam1_t *b = buf[i].bam_record; uint32_t tid = b->core.tid == -1 ? nref : b->core.tid; uint64_t pos = ((uint64_t)(b->core.pos + 1) << 1) | bam_is_rev(b); if (max_tid < tid) max_tid = tid; if (max_pos < pos) max_pos = pos; } for (; max_pos > 0; max_pos >>= 8) pos_bytes++; for (; max_tid > 0; max_tid >>= 8) tid_bytes++; assert(pos_bytes + tid_bytes < sizeof(buf[0].u.pos_tid)); tid_shift_l = pos_bytes * 8; tid_shift_r = 64 - tid_shift_l; // Write position and tid into bam1_tag::u::pos_tid using minimum number // of bytes required. Values are stored little-endian so that we // get a least-significant digit (byte) radix sort. for (i = 0; i < n; i++) { bam1_t *b = buf[i].bam_record; uint32_t tid = b->core.tid == -1 ? nref : b->core.tid; // 'pos' here includes as many bytes of tid as will fit // in the space remaining above pos_bytes. The rest of tid // is written out separately. uint64_t pos = (bam_is_rev(b) | ((uint64_t)(b->core.pos + 1) << 1) | (tid_shift_l < 64 ? (uint64_t) tid << tid_shift_l : 0)); u64_to_le(pos, buf[i].u.pos_tid); u32_to_le(tid_shift_r < 32 ? tid >> tid_shift_r : 0, &buf[i].u.pos_tid[8]); } buf_ar2[0] = buf; buf_ar2[1] = (bam1_tag *)malloc(sizeof(bam1_tag) * n); if (buf_ar2[1] == NULL) { print_error("sort", "couldn't allocate memory for temporary buf"); goto err; } // Least-significant digit radix sort (where "digits" are bytes) for (byte = 0; byte < pos_bytes + tid_bytes; byte++) { size_t remainders[NUMBASE] = { 0 }; bam_a = buf_ar2[curr]; bam_b = buf_ar2[1-curr]; for (i = 0; i < n; ++i) remainders[bam_a[i].u.pos_tid[byte]]++; for (i = 1; i < NUMBASE; ++i) remainders[i] += remainders[i - 1]; for (i = n - 1; i >= 0; i--) { size_t j = --remainders[bam_a[i].u.pos_tid[byte]]; bam_b[j] = bam_a[i]; } curr = 1 - curr; } if (curr == 1) { bam1_tag *end = buf + n; bam_a = buf_ar2[0]; bam_b = buf_ar2[1]; while (bam_a < end) *bam_a++ = *bam_b++; } ret = 0; err: free(buf_ar2[1]); return ret; } /* * Computes the minhash of a sequence using both forward and reverse strands. * * This is used as a sort key for unmapped data, to collate like sequences * together and to improve compression ratio. * * The minhash is returned and *pos filled out with location of this hash * key in the sequence if pos != NULL. */ static uint64_t minhash(bam1_t *b, int kmer, int *pos, int *rev) { uint64_t hashf = 0, minhashf = UINT64_MAX; uint64_t hashr = 0, minhashr = UINT64_MAX; int minhashpf = 0, minhashpr = 0, i; uint64_t mask = (1L<<(2*kmer))-1; unsigned char *seq = bam_get_seq(b); int len = b->core.l_qseq; // Lookup tables for bam_seqi to 0123 fwd/rev hashes // =ACM GRSV TWYH KDBN #define X 0 unsigned char L[16] = { X,0,1,X, 2,X,X,X, 3,X,X,X, X,X,X,X, }; uint64_t R[16] = { X,3,2,X, 1,X,X,X, 0,X,X,X, X,X,X,X, }; for (i = 0; i < 16; i++) R[i] <<= 2*(kmer-1); // Punt homopolymers somewhere central in the hash space #define XOR (0xdead7878beef7878 & mask) // Initialise hash keys for (i = 0; i < kmer-1 && i < len; i++) { int base = bam_seqi(seq, i); hashf = (hashf<<2) | L[base]; hashr = (hashr>>2) | R[base]; } // Loop to find minimum for (; i < len; i++) { int base = bam_seqi(seq, i); hashf = ((hashf<<2) | L[base]) & mask; hashr = (hashr>>2) | R[base]; if (minhashf > (hashf^XOR)) minhashf = (hashf^XOR), minhashpf = i; if (minhashr > (hashr^XOR)) minhashr = (hashr^XOR), minhashpr = len-i+kmer-2; } if (minhashf <= minhashr) { if (rev) *rev = 0; if (pos) *pos = minhashpf; return minhashf; } else { if (rev) *rev = 1; if (pos) *pos = minhashpr; return minhashr; } } //--- Start of candidates to punt to htslib /*! * @abstract * Extracts the sequence (in current alignment orientation) from * a bam record and places it in buf, which is nul terminated. * * @param b The bam structure * @param buf A buffer at least b->core.l_qseq+1 bytes long */ static void bam_to_seq(bam1_t *b, char *buf) { int i; uint8_t *seq = bam_get_seq(b); for (i = 0; i < b->core.l_qseq; i++) buf[i] = seq_nt16_str[bam_seqi(seq, i)]; buf[i] = 0; } /*! * @abstract * Writes a new sequence, of length b->core.l_qseq, to a BAM record. * * If a sequence of a new length is required the caller must first make * room for it by updating the bam1_t struct. * * @param b The bam structure * @param buf A buffer at least b->core.l_qseq bytes long */ static void seq_to_bam(bam1_t *b, char *buf) { int i; uint8_t *seq = bam_get_seq(b); for (i = 0; i < b->core.l_qseq; i++) bam_set_seqi(seq, i, seq_nt16_table[(unsigned char)buf[i]]); } /*! * @abstract Reverse complements a BAM record. * * It's possible to do this inline, but complex due to the 4-bit sequence * encoding. For now I take the dumb approach. * * @param b Pointer to a BAM alignment * * @return 0 on success, -1 on failure (ENOMEM) */ static int reverse_complement(bam1_t *b) { static char comp[256] = { 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N',//00 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N',//10 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N',//20 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N',//30 // * * * * E F * * I J * L * * O '@','T','V','G', 'H','E','F','C', 'D','I','H','M', 'L','K','N','O',//40 //P Q * * * * * * X Y Z [ \ ] ^ _ 'P','Q','Y','S', 'A','A','B','W', 'X','Y','Z','[','\\','[','^','_',//50 //` * * * * E F * * I J * L * * O '`','t','v','g', 'h','e','f','c', 'd','i','j','m', 'l','k','n','o',//60 //P Q * * * * * * X Y Z { | } ~ DEL 'p','q','y','s', 'a','a','b','w', 'x','y','z','{', '|','}','~',127,//70 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N',//80 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N',//90 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N',//A0 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N',//B0 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N',//C0 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N',//D0 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N',//E0 'N','N','N','N', 'N','N','N','N', 'N','N','N','N', 'N','N','N','N',//F0 }; char seq_[10000], *seq = seq_; uint8_t *qual = bam_get_qual(b); int i, j; if (b->core.l_qseq >= 10000) if (!(seq = malloc(b->core.l_qseq+1))) return -1; bam_to_seq(b, seq); for (i = 0, j = b->core.l_qseq-1; i < j; i++, j--) { unsigned char tmp = seq[i]; seq[i] = comp[(unsigned char)seq[j]]; seq[j] = comp[tmp]; tmp = qual[i]; qual[i] = qual[j]; qual[j] = tmp; } if (i ==j) seq[i] = comp[(unsigned char)seq[i]]; seq_to_bam(b, seq); if (seq != seq_) free(seq); b->core.flag ^= 0x10; return 0; } //--- End of candidates to punt to htslib static inline void worker_minhash(worker_t *w) { int i; for (i = 0; i < w->buf_len; i++) { bam1_t *b = w->buf[i].bam_record; if (b->core.tid != -1) continue; int pos = 0, rev = 0; uint64_t mh = minhash(b, w->minimiser_kmer, &pos, &rev); if (rev) reverse_complement(b); // Store 64-bit hash in unmapped pos and mpos fields. // The position of hash is in isize, which we use for // resolving ties when sorting by hash key. // These are unused for completely unmapped data and // will be reset during final output. b->core.pos = mh>>31; b->core.mpos = mh&0x7fffffff; b->core.isize = 65535-pos >=0 ? 65535-pos : 0; } } static void *worker(void *data) { worker_t *w = (worker_t*)data; w->error = 0; switch (g_sam_order) { case Coordinate: if (ks_radixsort(w->buf_len, w->buf, w->h) < 0) { w->error = errno; return NULL; } break; case MinHash: worker_minhash(w); // no break, go to merge sort default: ks_mergesort(sort, w->buf_len, w->buf, 0); } return 0; } static int sort_blocks(size_t k, bam1_tag *buf, const sam_hdr_t *h, int n_threads, buf_region *in_mem, int large_pos, int minimiser_kmer) { int i; size_t pos, rest; pthread_t *tid; pthread_attr_t attr; worker_t *w; int n_failed = 0; if (n_threads < 1) n_threads = 1; if (k < n_threads * 64) n_threads = 1; // use a single thread if we only sort a small batch of records pthread_attr_init(&attr); pthread_attr_setdetachstate(&attr, PTHREAD_CREATE_JOINABLE); w = (worker_t*)calloc(n_threads, sizeof(worker_t)); if (!w) return -1; tid = (pthread_t*)calloc(n_threads, sizeof(pthread_t)); if (!tid) { free(w); return -1; } pos = 0; rest = k; for (i = 0; i < n_threads; ++i) { w[i].buf_len = rest / (n_threads - i); w[i].buf = &buf[pos]; w[i].h = h; w[i].large_pos = large_pos; w[i].minimiser_kmer = minimiser_kmer; in_mem[i].from = pos; in_mem[i].to = pos + w[i].buf_len; pos += w[i].buf_len; rest -= w[i].buf_len; pthread_create(&tid[i], &attr, worker, &w[i]); } for (i = 0; i < n_threads; ++i) { pthread_join(tid[i], 0); if (w[i].error != 0) { errno = w[i].error; print_error_errno("sort", "failed to sort block %d", i); n_failed++; } } free(w); free(tid); return n_failed ? -1 : n_threads; } static void lib_lookup_destroy(khash_t(const_c2c) *lib_lookup) { khiter_t k; if (lib_lookup == NULL) return; for (k = kh_begin(lib_lookup); k < kh_end(lib_lookup); k++) { if (kh_exist(lib_lookup, k)) free(kh_value(lib_lookup, k)); } kh_destroy(const_c2c, lib_lookup); } // Build an RG to LB lookup table, for the template coordinate sort. // Returns a populated hash table (which may be empty) on success; // NULL on failure. static khash_t(const_c2c) * lookup_libraries(sam_hdr_t *header) { khash_t(const_c2c) *lib_lookup = kh_init(const_c2c); kstring_t lib_name = KS_INITIALIZE; int num_rg, i, res; if (!lib_lookup) return NULL; // Iterate through any RG lines and look for library information num_rg = sam_hdr_count_lines(header, "RG"); if (num_rg < 0) goto fail; for (i = 0; i < num_rg; i++) { const char *rg_id = sam_hdr_line_name(header, "RG", i); khiter_t k; if (!rg_id) goto fail; res = sam_hdr_find_tag_pos(header, "RG", i, "LB", &lib_name); if (res < -1) // Error goto fail; if (res < 0 || !lib_name.s) // No LB tag continue; // Add to lookup table k = kh_put(const_c2c, lib_lookup, rg_id, &res); if (res < 0) // Error goto fail; if (res > 0) { // Inserted kh_value(lib_lookup, k) = ks_release(&lib_name); } } free(lib_name.s); return lib_lookup; fail: lib_lookup_destroy(lib_lookup); free(lib_name.s); return NULL; } /*! @abstract Sort an unsorted BAM file based on the provided sort order @param sam_order the order in which the sort should occur @param sort_tag the tag to use if sorting by Tag @param minimiser_kmer the kmer size when sorting by MinHash @param fn name of the file to be sorted @param prefix prefix of the temporary files (prefix.NNNN.bam are written) @param fnout name of the final output file to be written @param modeout sam_open() mode to be used to create the final output file @param max_mem approxiate maximum memory (very inaccurate) @param in_fmt input file format options @param out_fmt output file format and options @param arg_list command string for PG line @param no_pg if 1, do not add a new PG line @paran write_index create index for the output file @return 0 for successful sorting, negative on errors @discussion It may create multiple temporary subalignment files and then merge them by calling bam_merge_simple(). This function is NOT thread safe. */ int bam_sort_core_ext(SamOrder sam_order, char* sort_tag, int minimiser_kmer, const char *fn, const char *prefix, const char *fnout, const char *modeout, size_t _max_mem, int n_threads, const htsFormat *in_fmt, const htsFormat *out_fmt, char *arg_list, int no_pg, int write_index) { int ret = -1, res, i, nref, n_files = 0, n_big_files = 0, fn_counter = 0; size_t max_k, k, max_mem, bam_mem_offset; sam_hdr_t *header = NULL; samFile *fp = NULL; bam1_tag *buf = NULL; template_coordinate_keys_t *keys = NULL; bam1_t *b = bam_init1(); uint8_t *bam_mem = NULL; char **fns = NULL; size_t fns_size = 0; const char *new_so = NULL; const char *new_go = NULL; const char *new_ss = NULL; buf_region *in_mem = NULL; khash_t(const_c2c) *lib_lookup = NULL; htsThreadPool htspool = { NULL, 0 }; int num_in_mem = 0; int large_pos = 0; if (!b) { print_error("sort", "couldn't allocate memory for bam record"); return -1; } if (n_threads < 2) n_threads = 1; g_sam_order = sam_order; if (g_sam_order == TagQueryName || g_sam_order == TagCoordinate) { g_sort_tag[0] = sort_tag[0]; g_sort_tag[1] = sort_tag[0] ? sort_tag[1] : '\0'; } if (sam_order == TemplateCoordinate) { if ((keys = malloc(sizeof(template_coordinate_keys_t))) == NULL) { print_error("sort", "could not allocate memory for the top-level keys"); goto err; } keys->n = 0; keys->m = 0; keys->buffer_size = 0x10000; keys->buffers = NULL; } max_mem = _max_mem * n_threads; buf = NULL; fp = sam_open_format(fn, "r", in_fmt); if (fp == NULL) { print_error_errno("sort", "can't open \"%s\"", fn); goto err; } hts_set_opt(fp, HTS_OPT_BLOCK_SIZE, BAM_BLOCK_SIZE); header = sam_hdr_read(fp); if (header == NULL) { print_error("sort", "failed to read header from \"%s\"", fn); goto err; } // Inspect the header looking for long chromosomes // If there is one, we need to write temporary files in SAM format nref = sam_hdr_nref(header); for (i = 0; i < nref; i++) { if (sam_hdr_tid2len(header, i) > INT32_MAX) large_pos = 1; } // Also check the output format is large position compatible if (large_pos) { int compatible = (out_fmt->format == sam || (out_fmt->format == cram && out_fmt->version.major >= 4) || (out_fmt->format == unknown_format && modeout[0] == 'w' && (modeout[1] == 'z' || modeout[1] == '\0'))); if (!compatible) { print_error("sort", "output format is not compatible with very large references"); goto err; } } if (g_sam_order == TemplateCoordinate) { lib_lookup = lookup_libraries(header); if (!lib_lookup) goto err; } switch (g_sam_order) { case Coordinate: new_so = "coordinate"; break; case QueryName: new_so = "queryname"; break; case MinHash: new_so = "coordinate"; new_ss = "coordinate:minhash"; break; case TagQueryName: case TagCoordinate: new_so = "unknown"; break; case TemplateCoordinate: new_so = "unsorted"; new_go = "query"; new_ss = "unsorted:template-coordinate"; break; default: new_so = "unknown"; break; } if (new_ss == NULL && new_go == NULL) { // just SO if ((-1 == sam_hdr_update_hd(header, "SO", new_so)) && (-1 == sam_hdr_add_line(header, "HD", "VN", SAM_FORMAT_VERSION, "SO", new_so, NULL)) ) { print_error("sort", "failed to change sort order header to 'SO:%s'\n", new_so); goto err; } } else if (new_ss != NULL && new_go == NULL) { // update SO and SS, but not GO if ((-1 == sam_hdr_update_hd(header, "SO", new_so, "SS", new_ss)) && (-1 == sam_hdr_add_line(header, "HD", "VN", SAM_FORMAT_VERSION, "SO", new_so, "SS", new_ss, NULL)) ) { print_error("sort", "failed to change sort order header to 'SO:%s SS:%s'\n", new_so, new_ss); goto err; } } else if (new_ss == NULL && new_go != NULL) { // update SO and GO, but not SS if ((-1 == sam_hdr_update_hd(header, "SO", new_so, "GO", new_go)) && (-1 == sam_hdr_add_line(header, "HD", "VN", SAM_FORMAT_VERSION, "SO", new_so, "GO", new_go, NULL)) ) { print_error("sort", "failed to change sort order header to 'SO:%s GO:%s'\n", new_so, new_go); goto err; } } else { // update SO, GO, and SS if ((-1 == sam_hdr_update_hd(header, "SO", new_so, "GO", new_go, "SS", new_ss)) && (-1 == sam_hdr_add_line(header, "HD", "VN", SAM_FORMAT_VERSION, "SO", new_so, "GO", new_go, "SS", new_ss, NULL)) ) { print_error("sort", "failed to change sort order header to 'SO:%s GO:%s SS:%s'\n", new_so, new_go, new_ss); goto err; } } if (new_go == NULL) { if (-1 == sam_hdr_remove_tag_hd(header, "GO")) { print_error("sort", "failed to delete group order in header\n"); goto err; } } if (new_ss == NULL) { if (-1 == sam_hdr_remove_tag_hd(header, "SS")) { print_error("sort", "failed to delete sub sort in header\n"); goto err; } } if (n_threads > 1) { htspool.pool = hts_tpool_init(n_threads); if (!htspool.pool) { print_error_errno("sort", "failed to set up thread pool"); goto err; } hts_set_opt(fp, HTS_OPT_THREAD_POOL, &htspool); } if ((bam_mem = malloc(max_mem)) == NULL) { print_error("sort", "couldn't allocate memory for bam_mem"); goto err; } in_mem = calloc(n_threads > 0 ? n_threads : 1, sizeof(in_mem[0])); if (!in_mem) goto err; // write sub files k = max_k = bam_mem_offset = 0; size_t name_len = strlen(prefix) + 30; while ((res = sam_read1(fp, header, b)) >= 0) { int mem_full = 0; if (k == max_k) { bam1_tag *new_buf; max_k = max_k? max_k<<1 : 0x10000; if ((new_buf = realloc(buf, max_k * sizeof(bam1_tag))) == NULL) { print_error("sort", "couldn't allocate memory for buf"); goto err; } buf = new_buf; } if (sam_order == TemplateCoordinate && k >= keys->m * keys->buffer_size) { if (template_coordinate_keys_realloc(keys, k + 1) == -1) { goto err; } } // Check if the BAM record will fit in the memory limit if (bam_mem_offset + sizeof(*b) + b->l_data < max_mem) { // Copy record into the memory block buf[k].bam_record = (bam1_t *)(bam_mem + bam_mem_offset); *buf[k].bam_record = *b; buf[k].bam_record->data = (uint8_t *)((char *)buf[k].bam_record + sizeof(bam1_t)); memcpy(buf[k].bam_record->data, b->data, b->l_data); // store next BAM record in next 8-byte-aligned address after // current one bam_mem_offset = (bam_mem_offset + sizeof(*b) + b->l_data + 8 - 1) & ~((size_t)(8 - 1)); } else { // Add a pointer to the remaining record buf[k].bam_record = b; mem_full = 1; } // Set the tag if sorting by tag, or the key for template cooridinate sorting switch (g_sam_order) { case TagQueryName: case TagCoordinate: buf[k].u.tag = bam_aux_get(buf[k].bam_record, g_sort_tag); break; case TemplateCoordinate: ++keys->n; template_coordinate_key_t *key = template_coordinate_keys_get(keys, k); buf[k].u.key = template_coordinate_key(buf[k].bam_record, key, header, lib_lookup); if (buf[k].u.key == NULL) goto err; break; default: buf[k].u.tag = NULL; buf[k].u.key = NULL; } ++k; if (mem_full) { if (hts_resize(char *, n_files + 1, &fns_size, &fns, 0) < 0) goto err; int sort_res = sort_blocks(k, buf, header, n_threads, in_mem, large_pos, minimiser_kmer); if (sort_res < 0) goto err; fns[n_files] = calloc(name_len, 1); if (!fns[n_files]) goto err; const int MAX_TRIES = 1000; int tries = 0, merge_res = -1; char *sort_by_tag = (g_sam_order == TagQueryName || g_sam_order == TagCoordinate) ? sort_tag : NULL; int consolidate_from = n_files; if (n_files - n_big_files >= MAX_TMP_FILES/2) consolidate_from = n_big_files; else if (n_files >= MAX_TMP_FILES) consolidate_from = 0; for (;;) { if (tries) { snprintf(fns[n_files], name_len, "%s.%.4d-%.3d.bam", prefix, fn_counter, tries); } else { snprintf(fns[n_files], name_len, "%s.%.4d.bam", prefix, fn_counter); } if (bam_merge_simple(g_sam_order, sort_by_tag, fns[n_files], large_pos ? "wzx1" : "wbx1", header, n_files - consolidate_from, &fns[consolidate_from], n_threads, in_mem, buf, keys, lib_lookup, &htspool, "sort", NULL, NULL, NULL, 1, 0) >= 0) { merge_res = 0; break; } if (errno == EEXIST && tries < MAX_TRIES) { tries++; } else { break; } } fn_counter++; if (merge_res < 0) { if (errno != EEXIST) unlink(fns[n_files]); free(fns[n_files]); goto err; } if (consolidate_from < n_files) { for (i = consolidate_from; i < n_files; i++) { unlink(fns[i]); free(fns[i]); } fns[consolidate_from] = fns[n_files]; n_files = consolidate_from; n_big_files = consolidate_from + 1; } n_files++; k = 0; if (keys != NULL) keys->n = 0; bam_mem_offset = 0; } } if (res != -1) { print_error("sort", "truncated file. Aborting"); goto err; } // Sort last records if (k > 0) { num_in_mem = sort_blocks(k, buf, header, n_threads, in_mem, large_pos, minimiser_kmer); if (num_in_mem < 0) goto err; } else { num_in_mem = 0; } // write the final output if (n_files == 0 && num_in_mem < 2) { // a single block if (write_buffer(fnout, modeout, k, buf, header, n_threads, out_fmt, minimiser_kmer, arg_list, no_pg, write_index) != 0) { print_error_errno("sort", "failed to create \"%s\"", fnout); goto err; } } else { // then merge fprintf(stderr, "[bam_sort_core] merging from %d files and %d in-memory blocks...\n", n_files, num_in_mem); // Paranoia check - all temporary files should have a name for (i = 0; i < n_files; ++i) { if (!fns[i]) { print_error("sort", "BUG: no name stored for temporary file %d", i); abort(); } } char *sort_by_tag = (sam_order == TagQueryName || sam_order == TagCoordinate) ? sort_tag : NULL; if (bam_merge_simple(sam_order, sort_by_tag, fnout, modeout, header, n_files, fns, num_in_mem, in_mem, buf, keys, lib_lookup, &htspool, "sort", in_fmt, out_fmt, arg_list, no_pg, write_index) < 0) { // Propagate bam_merge_simple() failure; it has already emitted a // message explaining the failure, so no further message is needed. goto err; } } ret = 0; err: // free if (fns) { for (i = 0; i < n_files; ++i) { if (fns[i]) { unlink(fns[i]); free(fns[i]); } } free(fns); } bam_destroy1(b); free(buf); if (keys != NULL) { for (i = 0; i < keys->m; ++i) { free(keys->buffers[i]); } free(keys->buffers); free(keys); } free(bam_mem); free(in_mem); lib_lookup_destroy(lib_lookup); sam_hdr_destroy(header); if (fp) sam_close(fp); if (htspool.pool) hts_tpool_destroy(htspool.pool); return ret; } // Unused here but may be used by legacy samtools-using third-party code int bam_sort_core(int is_by_qname, const char *fn, const char *prefix, size_t max_mem) { int ret; char *fnout = calloc(strlen(prefix) + 4 + 1, 1); if (!fnout) return -1; sprintf(fnout, "%s.bam", prefix); SamOrder sam_order = is_by_qname ? QueryName : Coordinate; g_sam_order = sam_order; ret = bam_sort_core_ext(sam_order, NULL, 0, fn, prefix, fnout, "wb", max_mem, 0, NULL, NULL, NULL, 1, 0); free(fnout); return ret; } static void sort_usage(FILE *fp) { fprintf(fp, "Usage: samtools sort [options...] [in.bam]\n" "Options:\n" " -l INT Set compression level, from 0 (uncompressed) to 9 (best)\n" " -u Output uncompressed data (equivalent to -l 0)\n" " -m INT Set maximum memory per thread; suffix K/M/G recognized [768M]\n" " -M Use minimiser for clustering unaligned/unplaced reads\n" " -K INT Kmer size to use for minimiser [20]\n" " -n Sort by read name (not compatible with samtools index command)\n" " -t TAG Sort by value of TAG. Uses position as secondary index (or read name if -n is set)\n" " -o FILE Write final output to FILE rather than standard output\n" " -T PREFIX Write temporary files to PREFIX.nnnn.bam\n" " --no-PG\n" " Do not add a PG line\n" " --template-coordinate\n" " Sort by template-coordinate\n"); sam_global_opt_help(fp, "-.O..@.."); } static void complain_about_memory_setting(size_t max_mem) { char *suffix = ""; const size_t nine_k = 9<<10; if (max_mem > nine_k) { max_mem >>= 10; suffix = "K"; } if (max_mem > nine_k) { max_mem >>= 10; suffix = "M"; } fprintf(stderr, "[bam_sort] -m setting (%zu%s bytes) is less than the minimum required (%zuM).\n\n" "Trying to run with -m too small can lead to the creation of a very large number\n" "of temporary files. This may make sort fail due to it exceeding limits on the\n" "number of files it can have open at the same time.\n\n" "Please check your -m parameter. It should be an integer followed by one of the\n" "letters K (for kilobytes), M (megabytes) or G (gigabytes). You should ensure it\n" "is at least the minimum above, and much higher if you are sorting a large file.\n", max_mem, suffix, SORT_MIN_MEGS_PER_THREAD); } int bam_sort(int argc, char *argv[]) { size_t max_mem = SORT_DEFAULT_MEGS_PER_THREAD << 20; int c, nargs, ret, o_seen = 0, level = -1, no_pg = 0; SamOrder sam_order = Coordinate; bool by_tag = false; int minimiser_kmer = 20; char* sort_tag = NULL, *arg_list = NULL; char *fnout = "-", modeout[12]; kstring_t tmpprefix = { 0, 0, NULL }; struct stat st; sam_global_args ga = SAM_GLOBAL_ARGS_INIT; static const struct option lopts[] = { SAM_OPT_GLOBAL_OPTIONS('-', 0, 'O', 0, 0, '@'), { "threads", required_argument, NULL, '@' }, {"no-PG", no_argument, NULL, 1}, { "template-coordinate", no_argument, NULL, 2}, { NULL, 0, NULL, 0 } }; while ((c = getopt_long(argc, argv, "l:m:no:O:T:@:t:MK:u", lopts, NULL)) >= 0) { switch (c) { case 'o': fnout = optarg; o_seen = 1; break; case 'n': sam_order = QueryName; break; case 't': by_tag = true; sort_tag = optarg; break; case 'm': { char *q; max_mem = strtol(optarg, &q, 0); if (*q == 'k' || *q == 'K') max_mem <<= 10; else if (*q == 'm' || *q == 'M') max_mem <<= 20; else if (*q == 'g' || *q == 'G') max_mem <<= 30; break; } case 'T': kputs(optarg, &tmpprefix); break; case 'l': level = atoi(optarg); break; case 'u': level = 0; break; case 1: no_pg = 1; break; case 2: sam_order = TemplateCoordinate; break; case 'M': sam_order = MinHash; break; case 'K': minimiser_kmer = atoi(optarg); if (minimiser_kmer < 1) minimiser_kmer = 1; else if (minimiser_kmer > 31) minimiser_kmer = 31; break; default: if (parse_sam_global_opt(c, optarg, lopts, &ga) == 0) break; /* else fall-through */ case '?': sort_usage(stderr); ret = EXIT_FAILURE; goto sort_end; } } // Change sort order if tag sorting is requested. Must update based on secondary index if (by_tag) { sam_order = sam_order == QueryName ? TagQueryName : TagCoordinate; } nargs = argc - optind; if (nargs == 0 && isatty(STDIN_FILENO)) { sort_usage(stdout); ret = EXIT_SUCCESS; goto sort_end; } else if (nargs >= 2) { // If exactly two, user probably tried to specify legacy if (nargs == 2) fprintf(stderr, "[bam_sort] Use -T PREFIX / -o FILE to specify temporary and final output files\n"); sort_usage(stderr); ret = EXIT_FAILURE; goto sort_end; } if (ga.write_index && (sam_order == QueryName || sam_order == TagQueryName || sam_order == TagCoordinate || sam_order == TemplateCoordinate)) { fprintf(stderr, "[W::bam_sort] Ignoring --write-index as it only works for position sorted files.\n"); ga.write_index = 0; } if (!no_pg && !(arg_list = stringify_argv(argc+1, argv-1))) { print_error("sort", "failed to create arg_list"); return 1; } if (max_mem < (SORT_MIN_MEGS_PER_THREAD << 20)) { complain_about_memory_setting(max_mem); ret = EXIT_FAILURE; goto sort_end; } strcpy(modeout, "wb"); sam_open_mode(modeout+1, fnout, NULL); if (level >= 0) sprintf(strchr(modeout, '\0'), "%d", level < 9? level : 9); if (tmpprefix.l == 0) { if (strcmp(fnout, "-") != 0) { char *idx = strstr(fnout, HTS_IDX_DELIM); kputsn(fnout, idx ? idx - fnout : strlen(fnout), &tmpprefix); kputs(".tmp", &tmpprefix); } else { kputc('.', &tmpprefix); } } if (stat(tmpprefix.s, &st) == 0 && S_ISDIR(st.st_mode)) { unsigned t = ((unsigned) time(NULL)) ^ ((unsigned) clock()); if (tmpprefix.s[tmpprefix.l-1] != '/') kputc('/', &tmpprefix); ksprintf(&tmpprefix, "samtools.%d.%u.tmp", (int) getpid(), t % 10000); } ret = bam_sort_core_ext(sam_order, sort_tag, (sam_order == MinHash) ? minimiser_kmer : 0, (nargs > 0) ? argv[optind] : "-", tmpprefix.s, fnout, modeout, max_mem, ga.nthreads, &ga.in, &ga.out, arg_list, no_pg, ga.write_index); if (ret >= 0) ret = EXIT_SUCCESS; else { char dummy[4]; // If we failed on opening the input file & it has no .bam/.cram/etc // extension, the user probably tried legacy -o if (ret == -2 && o_seen && nargs > 0 && sam_open_mode(dummy, argv[optind], NULL) < 0) fprintf(stderr, "[bam_sort] Note the argument has been replaced by -T/-o options\n"); ret = EXIT_FAILURE; } sort_end: free(tmpprefix.s); free(arg_list); sam_global_args_free(&ga); return ret; }