/* Copyright 2011 Toshiaki Namiki (namiki@dna.bio.keio.ac.jp). This file is part of MetaVelvet. This file is originally distributed as a part of the Velvet software, and modified to solve de novo metagenome assembly problem. MetaVelvet is free software; you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation; either version 2 of the License, or (at your option) any later version. MetaVelvet is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. You should have received a copy of the GNU General Public License along with MetaVelvet; if not, write to the Free Software Foundation, Inc., 51 Franklin St, Fifth Floor, Boston, MA 02110-1301 USA */ #include #include #include #include "run.h" static void printUsage() { puts("Usage:"); puts("./velvetg directory [options]"); puts(""); puts("\tdirectory\t\t\t: working directory name"); puts(""); puts("Standard options:"); puts("\t-cov_cutoff \t: removal of low coverage nodes AFTER tour bus or allow the system to infer it"); puts("\t\t(default: no removal)"); puts("\t-ins_length \t\t: expected distance between two paired end reads (default: no read pairing)"); puts("\t-read_trkg \t\t: tracking of short read positions in assembly (default: no tracking)"); puts("\t-min_contig_lgth \t: minimum contig length exported to contigs.fa file (default: hash length * 2)"); puts("\t-amos_file \t\t: export assembly to AMOS file (default: no export)"); puts("\t-exp_cov \t: expected coverage of unique regions or allow the system to infer it"); puts("\t\t(default: no long or paired-end read resolution)"); puts(""); puts("Advanced options:"); puts("\t-ins_length2 \t\t: expected distance between two paired-end reads in the second short-read dataset (default: no read pairing)"); puts("\t-ins_length_long \t: expected distance between two long paired-end reads (default: no read pairing)"); puts("\t-ins_length*_sd \t: est. standard deviation of respective dataset (default: 10\% of corresponding length)"); puts("\t\t[replace '*' by nothing, '2' or '_long' as necessary]"); puts("\t-scaffolding \t\t: scaffolding of contigs used paired end information (default: on)"); puts("\t-max_branch_length \t: maximum length in base pair of bubble (default: 100)"); puts("\t-max_divergence : maximum divergence rate between two branches in a bubble (default: 0.2)"); puts("\t-max_gap_count \t: maximum number of gaps allowed in the alignment of the two branches of a bubble (default: 3)"); puts("\t-min_pair_count \t: minimum number of paired end connections to justify the scaffolding of two long contigs (default: 10)"); puts("\t-max_coverage \t: removal of high coverage nodes AFTER tour bus (default: no removal)"); puts("\t-long_mult_cutoff \t\t: minimum number of long reads required to merge contigs (default: 2)"); puts("\t-unused_reads \t\t: export unused reads in UnusedReads.fa file (default: no)"); puts(""); puts("Output:"); puts("\tdirectory/contigs.fa\t\t: fasta file of contigs longer than twice hash length"); puts("\tdirectory/stats.txt\t\t: stats file (tab-spaced) useful for determining appropriate coverage cutoff"); puts("\tdirectory/LastGraph\t\t: special formatted file with all the information on the final graph"); puts("\tdirectory/velvet_asm.afg\t: (if requested) AMOS compatible assembly file"); } int main(int argc, char **argv) { ReadSet *sequences = NULL; RoadMapArray *rdmaps; PreGraph *preGraph; Graph *graph; char *directory, *graphFilename, *preGraphFilename, *seqFilename, *roadmapFilename; double coverageCutoff = -1; double maxCoverageCutoff = -1; double expectedCoverage = -1; int longMultCutoff = -1; Coordinate minContigLength = -1; Coordinate minContigKmerLength; boolean *dubious = NULL; Coordinate insertLength[CATEGORIES]; Coordinate insertLengthLong = -1; Coordinate std_dev[CATEGORIES]; Coordinate std_dev_long = -1; short int accelerationBits = 24; boolean readTracking = false; boolean exportAssembly = false; boolean unusedReads = false; boolean estimateCoverage = false; boolean estimateCutoff = false; FILE *file; int arg_index, arg_int; double arg_double; char *arg; Coordinate *sequenceLengths = NULL; Category cat; boolean scaffolding = true; int pebbleRounds = 1; long long longlong_var; short int short_var; // Original double expectedCoverageMulti[100]; int numECM = 0; int ecmIndex; char *tokenPointer, *manualECM[100]; const char *splitChar = "_"; boolean flagManualECM = false; int *dummyMask; double rateChimericSubgraph = 0.0; boolean discardChimericSubgraph = false; double repeatNodeCovSD = 0.10; // Original setProgramName("velvetg"); for (cat = 0; cat < CATEGORIES; cat++) { insertLength[cat] = -1; std_dev[cat] = -1; } // Error message if (argc == 1) { puts("velvetg - de Bruijn graph construction, error removal and repeat resolution"); printf("Version %i.%i.%2.2i\n", VERSION_NUMBER, RELEASE_NUMBER, UPDATE_NUMBER); puts("\nCopyright 2007, 2008 Daniel Zerbino (zerbino@ebi.ac.uk)"); puts("This is free software; see the source for copying conditions. There is NO"); puts("warranty; not even for MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE.\n"); puts("Compilation settings:"); printf("CATEGORIES = %i\n", CATEGORIES); printf("MAXKMERLENGTH = %i\n", MAXKMERLENGTH); puts(""); printUsage(); return 1; } if (strcmp(argv[1], "--help") == 0) { printUsage(); return 0; } // Memory allocation directory = argv[1]; graphFilename = mallocOrExit(strlen(directory) + 100, char); preGraphFilename = mallocOrExit(strlen(directory) + 100, char); roadmapFilename = mallocOrExit(strlen(directory) + 100, char); seqFilename = mallocOrExit(strlen(directory) + 100, char); // Argument parsing for (arg_index = 2; arg_index < argc; arg_index++) { arg = argv[arg_index++]; if (arg_index >= argc) { puts("Unusual number of arguments!"); printUsage(); exit(1); } if (strcmp(arg, "-cov_cutoff") == 0) { if (strcmp(argv[arg_index], "auto") == 0) { estimateCutoff = true; } else { sscanf(argv[arg_index], "%lf", &coverageCutoff); } } else if (strcmp(arg, "-exp_cov") == 0) { if (strcmp(argv[arg_index], "auto") == 0) { estimateCoverage = true; readTracking = true; } else { sscanf(argv[arg_index], "%lf", &expectedCoverage); if (expectedCoverage > 0) readTracking = true; } } else if (strcmp(arg, "-ins_length") == 0) { sscanf(argv[arg_index], "%lli", &longlong_var); insertLength[0] = (Coordinate) longlong_var; if (insertLength[0] < 0) { printf("Invalid insert length: %lli\n", (long long) insertLength[0]); exit(1); } } else if (strcmp(arg, "-ins_length_sd") == 0) { sscanf(argv[arg_index], "%lli", &longlong_var); std_dev[0] = (Coordinate) longlong_var; if (std_dev[0] < 0) { printf("Invalid std deviation: %lli\n", (long long) std_dev[0]); exit(1); } } else if (strcmp(arg, "-ins_length_long") == 0) { sscanf(argv[arg_index], "%lli", &longlong_var); insertLengthLong = (Coordinate) longlong_var; } else if (strcmp(arg, "-ins_length_long_sd") == 0) { sscanf(argv[arg_index], "%lli", &longlong_var); std_dev_long = (Coordinate) longlong_var; } else if (strncmp(arg, "-ins_length", 11) == 0 && strchr(arg, 'd') == NULL) { sscanf(arg, "-ins_length%hi", &short_var); cat = (Category) short_var; if (cat < 1 || cat > CATEGORIES) { printf("Unknown option: %s\n", arg); exit(1); } sscanf(argv[arg_index], "%lli", &longlong_var); insertLength[cat - 1] = (Coordinate) longlong_var; if (insertLength[cat - 1] < 0) { printf("Invalid insert length: %lli\n", (long long) insertLength[cat - 1]); exit(1); } } else if (strncmp(arg, "-ins_length", 11) == 0) { sscanf(arg, "-ins_length%hi_sd", &short_var); cat = (Category) short_var; if (cat < 1 || cat > CATEGORIES) { printf("Unknown option: %s\n", arg); exit(1); } sscanf(argv[arg_index], "%lli", &longlong_var); std_dev[cat - 1] = (Coordinate) longlong_var; if (std_dev[cat - 1] < 0) { printf("Invalid std deviation: %lli\n", (long long) std_dev[cat - 1]); exit(1); } } else if (strcmp(arg, "-read_trkg") == 0) { readTracking = (strcmp(argv[arg_index], "yes") == 0); } else if (strcmp(arg, "-scaffolding") == 0) { scaffolding = (strcmp(argv[arg_index], "yes") == 0); } else if (strcmp(arg, "-amos_file") == 0) { exportAssembly = (strcmp(argv[arg_index], "yes") == 0); } else if (strcmp(arg, "-min_contig_lgth") == 0) { sscanf(argv[arg_index], "%lli", &longlong_var); minContigLength = (Coordinate) longlong_var; } else if (strcmp(arg, "-accel_bits") == 0) { sscanf(argv[arg_index], "%hi", &accelerationBits); if (accelerationBits < 0) { printf ("Illegal acceleration parameter: %s\n", argv[arg_index]); printUsage(); return -1; } } else if (strcmp(arg, "-max_branch_length") == 0) { sscanf(argv[arg_index], "%i", &arg_int); setMaxReadLength(arg_int); setLocalMaxReadLength(arg_int); } else if (strcmp(arg, "-max_divergence") == 0) { sscanf(argv[arg_index], "%lf", &arg_double); setMaxDivergence(arg_double); setLocalMaxDivergence(arg_double); } else if (strcmp(arg, "-max_gap_count") == 0) { sscanf(argv[arg_index], "%i", &arg_int); setMaxGaps(arg_int); setLocalMaxGaps(arg_int); } else if (strcmp(arg, "-min_pair_count") == 0) { sscanf(argv[arg_index], "%i", &arg_int); setUnreliableConnectionCutoff(arg_int); } else if (strcmp(arg, "-max_coverage") == 0) { sscanf(argv[arg_index], "%lf", &maxCoverageCutoff); } else if (strcmp(arg, "-long_mult_cutoff") == 0) { sscanf(argv[arg_index], "%i", &longMultCutoff); setMultiplicityCutoff(longMultCutoff); } else if (strcmp(arg, "-unused_reads") == 0) { unusedReads = (strcmp(argv[arg_index], "yes") == 0); if (unusedReads) readTracking = true; // Original } else if (strcmp(arg, "-rate_chimeric_sg") == 0) { sscanf(argv[arg_index], "%lf", &rateChimericSubgraph); } else if (strcmp(arg, "-discard_chimeric_sg") == 0) { discardChimericSubgraph = (strcmp(argv[arg_index], "yes") == 0); } else if (strcmp(arg, "-manual_exp_cov_multi") == 0) { flagManualECM = true; tokenPointer = argv[arg_index]; for (numECM = 0; numECM < 100; numECM++) { if ((manualECM[numECM] = strtok(tokenPointer, splitChar)) == NULL) break; tokenPointer = NULL; } for (ecmIndex = 0; ecmIndex < numECM; ecmIndex++) { sscanf(manualECM[ecmIndex], "%lf", &expectedCoverageMulti[ecmIndex]); printf("Manual Expected Coverage : %lf\n", expectedCoverageMulti[ecmIndex]); } printf("numECM : %d\n", numECM); } else if (strcmp(arg, "-repeat_cov_sd") == 0) { sscanf(argv[arg_index], "%lf", &repeatNodeCovSD); // Original } else if (strcmp(arg, "--help") == 0) { printUsage(); return 0; } else { printf("Unknown option: %s;\n", arg); printUsage(); return 1; } } // Bookkeeping logInstructions(argc, argv, directory); strcpy(seqFilename, directory); strcat(seqFilename, "/Sequences"); strcpy(roadmapFilename, directory); strcat(roadmapFilename, "/Roadmaps"); strcpy(preGraphFilename, directory); strcat(preGraphFilename, "/PreGraph"); if (!readTracking) { strcpy(graphFilename, directory); strcat(graphFilename, "/Graph"); } else { strcpy(graphFilename, directory); strcat(graphFilename, "/Graph2"); } // Graph uploading or creation if ((file = fopen(graphFilename, "r")) != NULL) { fclose(file); graph = importGraph(graphFilename); } else if ((file = fopen(preGraphFilename, "r")) != NULL) { fclose(file); sequences = importReadSet(seqFilename); convertSequences(sequences); graph = importPreGraph(preGraphFilename, sequences, readTracking, accelerationBits); sequenceLengths = getSequenceLengths(sequences, getWordLength(graph)); correctGraph(graph, sequenceLengths); exportGraph(graphFilename, graph, sequences->tSequences); } else if ((file = fopen(roadmapFilename, "r")) != NULL) { fclose(file); rdmaps = importRoadMapArray(roadmapFilename); preGraph = newPreGraph_pg(rdmaps, seqFilename); clipTips_pg(preGraph); exportPreGraph_pg(preGraphFilename, preGraph); destroyPreGraph_pg(preGraph); sequences = importReadSet(seqFilename); convertSequences(sequences); graph = importPreGraph(preGraphFilename, sequences, readTracking, accelerationBits); sequenceLengths = getSequenceLengths(sequences, getWordLength(graph)); correctGraph(graph, sequenceLengths); exportGraph(graphFilename, graph, sequences->tSequences); } else { puts("No Roadmap file to build upon! Please run velveth (see manual)"); exit(1); } // Set insert lengths and their standard deviations for (cat = 0; cat < CATEGORIES; cat++) { if (insertLength[cat] > -1 && std_dev[cat] < 0) std_dev[cat] = insertLength[cat] / 10; setInsertLengths(graph, cat, insertLength[cat], std_dev[cat]); } if (insertLengthLong > -1 && std_dev_long < 0) std_dev_long = insertLengthLong / 10; setInsertLengths(graph, CATEGORIES, insertLengthLong, std_dev_long); // Coverage cutoff if (expectedCoverage < 0 && estimateCoverage == true) { expectedCoverage = estimated_cov(graph, directory); if (coverageCutoff < 0) { coverageCutoff = expectedCoverage / 2; estimateCutoff = true; } } else { estimateCoverage = false; if (coverageCutoff < 0 && estimateCutoff) coverageCutoff = estimated_cov(graph, directory) / 2; else estimateCutoff = false; } if (coverageCutoff < 0) { puts("WARNING: NO COVERAGE CUTOFF PROVIDED"); puts("Velvet will probably leave behind many detectable errors"); puts("See manual for instructions on how to set the coverage cutoff parameter"); } // Original // Outputting Graph_EstimateCovMulti Status strcpy(graphFilename, directory); strcat(graphFilename, "/stats_EstimateCovMulti.txt"); displayGeneralStatistics(graph, graphFilename, sequences); // Estimating multiple expected coverages dummyMask = makeDummySubgraphMask(graph); if (!flagManualECM) numECM = estimated_cov_multi(graph, dummyMask, expectedCoverageMulti); free(dummyMask); // Define coverageCutoff as half of minimum exp_cov coverageCutoff = expectedCoverageMulti[numECM - 1] / (double) 2; // Original dubious = removeLowCoverageNodesAndDenounceDubiousReads(graph, coverageCutoff); removeHighCoverageNodes(graph, maxCoverageCutoff); clipTipsHard(graph); if (expectedCoverage > 0) { if (sequences == NULL) { sequences = importReadSet(seqFilename); convertSequences(sequences); } // Original // Outputting Graph Before Rock Band strcpy(graphFilename, directory); strcat(graphFilename, "/Graph_BeforeRockBand"); exportGraph(graphFilename, graph, sequences->tSequences); // Outputting G_BRB Node Sequences if (minContigLength < 2 * getWordLength(graph)) minContigKmerLength = getWordLength(graph); else minContigKmerLength = minContigLength - getWordLength(graph) + 1; strcpy(graphFilename, directory); strcat(graphFilename, "/Graph_BeforeRockBand_NODE.fasta"); exportLongNodeSequences(graphFilename, graph, minContigKmerLength); // Original // Original // Mixed length sequencing //readCoherentGraph(graph, isUniqueSolexa, expectedCoverage, sequences); // Original // Paired ends module createReadPairingArray(sequences); for (cat = 0; cat < CATEGORIES; cat++) if(pairUpReads(sequences, 2 * cat + 1)) pebbleRounds++; if (pairUpReads(sequences, 2 * CATEGORIES + 1)) pebbleRounds++; detachDubiousReads(sequences, dubious); activateGapMarkers(graph); // Original //for ( ;pebbleRounds > 0; pebbleRounds--) // exploitShortReadPairs(graph, sequences, dubious, scaffolding); // Original /* // Original // Identifying and Separating InterRepeats identifyAndSeparateInterRepeats(graph, expectedCoverageMulti); concatenateGraph(graph); // Original // Original // Outputting Graph After Separating InterRepeats strcpy(graphFilename, directory); strcat(graphFilename, "/Graph_AfterSeparation"); exportGraph(graphFilename, graph, sequences->tSequences); // Original */ // Original // Resolving Repeats for each subgraph resolveRepeatOfAllSubgraphs(graph, sequences, expectedCoverageMulti, dubious, scaffolding, pebbleRounds, rateChimericSubgraph, discardChimericSubgraph, repeatNodeCovSD); // Original } else { puts("WARNING: NO EXPECTED COVERAGE PROVIDED"); puts("Velvet will be unable to resolve any repeats"); puts("See manual for instructions on how to set the expected coverage parameter"); } free(dubious); concatenateGraph(graph); if (minContigLength < 2 * getWordLength(graph)) minContigKmerLength = getWordLength(graph); else minContigKmerLength = minContigLength - getWordLength(graph) + 1; strcpy(graphFilename, directory); strcat(graphFilename, "/contigs.fa"); exportLongNodeSequences(graphFilename, graph, minContigKmerLength); if (sequences == NULL) { sequences = importReadSet(seqFilename); convertSequences(sequences); } strcpy(graphFilename, directory); strcat(graphFilename, "/stats.txt"); displayGeneralStatistics(graph, graphFilename, sequences); strcpy(graphFilename, directory); strcat(graphFilename, "/LastGraph"); exportGraph(graphFilename, graph, sequences->tSequences); if (exportAssembly) { strcpy(graphFilename, directory); strcat(graphFilename, "/velvet_asm.afg"); exportAMOSContigs(graphFilename, graph, minContigKmerLength, sequences); } if (unusedReads) { exportUnusedReads(graph, sequences, minContigKmerLength, directory); } if (estimateCoverage) printf("Estimated Coverage = %f\n", expectedCoverage); if (estimateCutoff) printf("Estimated Coverage cutoff = %f\n", coverageCutoff); logFinalStats(graph, minContigKmerLength, directory); destroyGraph(graph); free(graphFilename); free(preGraphFilename); free(seqFilename); free(roadmapFilename); destroyReadSet(sequences); return 0; }