#include #include #include #include #include #include #include #include #include #include #include #include "kmer_utils.h" #include "nnls.h" #include "quikr.h" #include "quikr_functions.h" #ifdef Linux #include #endif void usage() { printf("Usage: multifasta_to_otu [OPTION...] - create a QIIME OTU table based on Quikr results. \n\n" "Options:\n\n" "-i, --input-directory\n" " the directory containing the samples' fasta files of reads (note each file should correspond to a separate sample)\n\n" "-f,--input-filelist\n" " a file containing list of fasta files to process seperated by newline (same rules apply as input-directory)\n\n" "-s, --sensing-matrix\n" " location of the sensing matrix. (sensing from quikr_train)\n\n" "-k,--kmer\n" " specify what size of kmer to use. (default value is 6)\n\n" "-l,--lambda\n" " lambda value to use. (default value is 10000)\n\n" "-r,--rare-percent\n" " remove mers from classification if their values are less than the x percentile of values in the sample (default value is 10000)\n\n" "-j, --jobs\n" " specifies how many jobs to run at once. (default value is the number of CPUs)\n\n" "-o, --output\n" " the OTU table, with NUM_READS_PRESENT for each sample which is compatible with QIIME's convert_biom.py (or a sequence table if not OTU's)\n\n" "-v, --verbose\n" " verbose mode.\n\n" "-V, --version\n" " print version.\n"); } char **get_fasta_files_from_file(char *fn, int file_override) { char **files; int files_count = 0; // getline stuff ssize_t read; size_t len = 0; char *line = NULL; FILE *fh = fopen(fn, "r"); if(fh == NULL) { fprintf(stderr, "Error opening %s - %s\n", fn, strerror(errno)); exit(EXIT_FAILURE); } files = malloc(sizeof(char **)); while ((read = getline(&line, &len, fh)) != -1) { char *file = malloc(sizeof(char) * (strlen(line) + 1)); check_malloc(file, NULL); strncpy(file, line, strlen(line) + 1 ); file[strlen(file)- 1] = '\0'; if(access(file, F_OK) == 0 || file_override) { files[files_count] = file; files_count++; files = realloc(files, sizeof(char *) * (files_count + 1)); if(files == NULL) { fprintf(stderr, "could not realloc keys\n"); exit(EXIT_FAILURE); } } else { fprintf(stderr, "Warning: ignoring %s (%s)\n", file, strerror(errno)); errno = 0; free(file); } } files_count++; files = realloc(files, sizeof(char *) * files_count); if(files == NULL) { fprintf(stderr, "could not realloc keys\n"); exit(EXIT_FAILURE); } files[files_count - 1] = NULL; fclose(fh); return files; } char **get_fasta_files_from_directory(char *directory) { DIR *dh; struct dirent *e; char **headers; long long count = -2; // -2 for ../ and ./ long long i = 0; // open our directory dh = opendir(directory); if(dh == NULL) { fprintf(stderr, "could not open %s\n", directory); exit(EXIT_FAILURE); } while((e = readdir(dh))) count++; e = NULL; rewinddir(dh); if(count == 0) { fprintf(stderr, "%s is empty\n", directory); exit(EXIT_FAILURE); } headers = malloc(count * sizeof(char *)); check_malloc(headers, NULL); int array_pos = 0; for(i = 0; i < count; i++) { char *ext = NULL; e = readdir(dh); if(strcmp(e->d_name, "..") == 0 || strcmp(e->d_name, ".") == 0) { i--; continue; } ext = strrchr(e->d_name, '.'); if(str_eq(ext, ".fasta") || str_eq(ext, ".fa") || str_eq(ext, ".fna")) { char *header = malloc(strlen(directory) + strlen(e->d_name) + 1); check_malloc(header, NULL); sprintf(header, "%s/%s", directory, e->d_name); headers[array_pos] = header; } else { continue; } array_pos++; } headers[array_pos] = NULL; closedir(dh); return headers; } int main(int argc, char **argv) { int c; char *input_fasta_directory = NULL; char *input_fasta_filelist = NULL; char *input_fasta_filenamelist = NULL; char *sensing_matrix_filename = NULL; char *output_filename = NULL; unsigned long long i = 0; unsigned long long j = 0; unsigned long long width = 0; unsigned int kmer = 6; unsigned long long lambda = 10000; double rare_percent = 1.0; unsigned int jobs = 1; long done = 0; unsigned long long dir_count = 0; #ifdef Linux jobs = get_nprocs(); #endif #ifdef Darwin jobs = sysconf (_SC_NPROCESSORS_ONLN); #endif int verbose = 0; static struct option long_options[] = { {"input-directory", required_argument, 0, 'i'}, {"input-filelist", required_argument, 0, 'f'}, {"input-filename-list", required_argument, 0, 'n'}, {"kmer", required_argument, 0, 'k'}, {"lambda", required_argument, 0, 'l'}, {"jobs", required_argument, 0, 'j'}, {"output", required_argument, 0, 'o'}, {"sensing-matrix", required_argument, 0, 's'}, {"rare-percent", required_argument, 0, 'r'}, {"verbose", no_argument, 0, 'v'}, {"help", no_argument, 0, 'h'}, {"version", no_argument, 0, 'V'}, {0, 0, 0, 0} }; while (1) { int option_index = 0; c = getopt_long (argc, argv, "f:k:l:s:i:o:n:j:r:hvV", long_options, &option_index); if (c == -1) break; switch (c) { case 'i': input_fasta_directory = optarg; break; case 'f': input_fasta_filelist = optarg; break; case 'n': printf("using -n\n"); input_fasta_filenamelist = optarg; break; case 'j': jobs = atoi(optarg); break; case 'k': kmer = atoi(optarg); break; case 'l': lambda = atoi(optarg); break; case 'o': output_filename = optarg; break; case 'r': rare_percent = atof(optarg); break; case 's': sensing_matrix_filename = optarg; break; case 'v': verbose = 1; break; case 'V': printf("%s\n", VERSION); exit(EXIT_SUCCESS); break; case 'h': usage(); exit(EXIT_SUCCESS); break; default: break; } } if(sensing_matrix_filename == NULL) { fprintf(stderr, "Error: sensing matrix filename (-s) must be specified\n\n"); usage(); exit(EXIT_FAILURE); } if(output_filename == NULL) { fprintf(stderr, "Error: output filename (-o) must be specified\n\n"); usage(); exit(EXIT_FAILURE); } // input fasta parsing if(input_fasta_directory == NULL && input_fasta_filelist == NULL) { fprintf(stderr, "Error: input fasta directory (-i) or input fasta filelist (-f) must be specified\n\n"); usage(); exit(EXIT_FAILURE); } if(input_fasta_directory != NULL && input_fasta_filelist != NULL) { fprintf(stderr, "Error: input fasta directory (-i) and input fasta filelist (-f) cannot be used concurrently\n\n"); usage(); exit(EXIT_FAILURE); } if(rare_percent <= 0 || rare_percent > 1.0) { fprintf(stderr, "Error: rare percent must be between 0 and 1\n"); exit(EXIT_FAILURE); } if(verbose) { printf("kmer: %u\n", kmer); printf("rare: %lf\n", rare_percent); printf("lambda: %llu\n", lambda); printf("input directory: %s\n", input_fasta_directory); printf("input filelist: %s\n", input_fasta_filelist); printf("sensing database: %s\n", sensing_matrix_filename); printf("output: %s\n", output_filename); printf("number of jobs to run at once: %d\n", jobs); } if(access (sensing_matrix_filename, F_OK) == -1) { fprintf(stderr, "Error: could not find %s\n", sensing_matrix_filename); exit(EXIT_FAILURE); } struct matrix *sensing_matrix = load_sensing_matrix(sensing_matrix_filename, kmer); if(kmer == 0) { fprintf(stdout, "Warning: zero is not a valid kmer, inferring kmer from sensing matrix (%d)\n", sensing_matrix->kmer); kmer = sensing_matrix->kmer; } // load filenames char **filenames = NULL; char **filenames_alternative = NULL; if(input_fasta_directory != NULL) { filenames = get_fasta_files_from_directory(input_fasta_directory); } else { filenames = get_fasta_files_from_file(input_fasta_filelist, 0); if(input_fasta_filenamelist != NULL) { filenames_alternative = get_fasta_files_from_file(input_fasta_filenamelist, 1); } } while(filenames[dir_count] != NULL) dir_count++; if(dir_count == 0) { fprintf(stderr, "Error: No files loaded from input\n"); exit(EXIT_FAILURE); } // 4 "ACGT" ^ Kmer gives us the size of output rows width = pow(4, kmer); double *sensing_matrix_ptr = sensing_matrix->matrix; unsigned long long sequences = sensing_matrix->sequences; if(verbose) { printf("directory count: %llu\n", dir_count); printf("width: %llu\n", width); printf("sequences: %llu\n", sequences); } unsigned long long *solutions = malloc(dir_count * sequences * sizeof(unsigned long long)); check_malloc(solutions, NULL); long long *file_sequence_count = calloc(dir_count, sizeof(long long)); check_malloc(file_sequence_count, NULL); #ifdef OMP omp_set_num_threads(jobs); #endif printf("Beginning to process samples\n"); //#pragma omp parallel for shared(solutions, sensing_matrix_ptr, done) for(size_t i = 0; i < dir_count; i++ ) { size_t x = 0; size_t y = 0; size_t z = 0; unsigned long long file_sequence_count = 0; unsigned long long rare_value = 0; unsigned long long rare_width = 0; printf("processing %s\n", filenames[i]); file_sequence_count = count_sequences(filenames[i]); printf("%s has %llu sequences\n", filenames[i], file_sequence_count); // load counts matrix double *count_matrix = malloc(width * sizeof(double)); check_malloc(count_matrix, NULL); // convert our matrix into doubles { unsigned long long *integer_counts = get_kmer_counts_from_file(filenames[i], kmer); for(x = 0; x < width; x++) { count_matrix[x] = (double)integer_counts[x]; } free(integer_counts); } // get_rare_value get_rare_value(count_matrix, width, rare_percent, &rare_value, &rare_width); if(verbose) printf("there are %llu values less than %llu\n", rare_width, rare_value); // add a extra space for our zero's array, so we can set the first column to 1's rare_width++; // store our count matrix double *count_matrix_rare = calloc(rare_width, sizeof(double)); check_malloc(count_matrix_rare, NULL); double *sensing_matrix_rare = calloc(rare_width * sequences, sizeof(double)); check_malloc(sensing_matrix_rare, NULL); // copy only kmers from our original counts that match our rareness percentage // in both our count matrix and our sensing matrix // // y = 1 because we are offsetting the array by 1, so we can set the first row to all 1's for(x = 0, y = 1; x < width; x++) { if(count_matrix[x] <= rare_value) { count_matrix_rare[y] = count_matrix[x]; for(z = 0; z < sequences; z++) sensing_matrix_rare[z*rare_width + y] = sensing_matrix_ptr[z*width + x]; y++; } } // normalize our kmer counts and our sensing_matrix normalize_matrix(count_matrix_rare, 1, rare_width); normalize_matrix(sensing_matrix_rare, sequences, rare_width); // multiply our kmer counts and sensing matrix by lambda for(x = 1; x < rare_width; x++) count_matrix_rare[x] *= lambda; //TODO use one loop for(x = 0; x < sequences; x++) { for(y = 1; y < rare_width; y++) { sensing_matrix_rare[rare_width*x + y] *= lambda; } } // count_matrix's first element should be zero count_matrix_rare[0] = 0; // stack one's on our first row of our sensing matrix for(x = 0; x < sequences; x++) { sensing_matrix_rare[x*rare_width] = 1.0; } double *solution = nnls(sensing_matrix_rare, count_matrix_rare, sequences, rare_width); // normalize our solution normalize_matrix(solution, 1, sequences); // add the current solution to the solutions array for(unsigned long long z = 0; z < sequences; z++ ) { solutions[sensing_matrix->sequences*i + z] = (unsigned long long)round(solution[z] * file_sequence_count); } done++; printf("%ld/%llu samples processed\n", done, dir_count); free(solution); free(count_matrix_rare); free(count_matrix); free(sensing_matrix_rare); } // output our matrix FILE *output_fh = fopen(output_filename, "w"); if(output_fh == NULL) { fprintf(stderr, "Could not open %s for writing\n", output_filename); exit(EXIT_FAILURE); } fprintf(output_fh, "# QIIME vQuikr OTU table\n"); fprintf(output_fh, "#OTU_ID\t"); // print our filename headers char **filename_array_to_use = filenames; if(filenames_alternative != NULL) { printf("using alternative: %s %s\n", filenames_alternative[0], filenames[0]); filename_array_to_use = filenames_alternative; printf("using alternative: %s %s\n", filenames_alternative[0], filenames[0]); } for(i = 0; i < dir_count - 1; i++) { fprintf(output_fh, "%s\t", basename(filename_array_to_use[i])); } fprintf(output_fh, "%s\n", basename(filename_array_to_use[dir_count - 1])); for(j = 0; j < sequences; j++) { double column_sum = 0.; for(i = 0; i < dir_count; i++) { column_sum += solutions[sensing_matrix->sequences*i + j]; } // if our column is zero, don't bother printing the row if(column_sum != 0) { fprintf(output_fh, "%s\t", sensing_matrix->headers[j]); for(i = 0; i < dir_count - 1; i++) { fprintf(output_fh, "%llu\t", solutions[sensing_matrix->sequences*i + j]); } fprintf(output_fh, "%llu\n", solutions[sensing_matrix->sequences*(dir_count - 1) + j]); } } fclose(output_fh); return EXIT_SUCCESS; }