a6402cd35ac18b36a7d4ee1a6e41df145a519e98 hiram Tue Jul 11 16:03:50 2017 -0700 prepare to thread this process refs #18969 diff --git src/utils/crisprKmers/crisprKmers.c src/utils/crisprKmers/crisprKmers.c index 4947dc4..beed0c9 100644 --- src/utils/crisprKmers/crisprKmers.c +++ src/utils/crisprKmers/crisprKmers.c @@ -1,936 +1,954 @@ /* crisprKmers - find and annotate crispr sequences. */ + +/* Copyright (C) 2017 The Regents of the University of California + * See README in this or parent directory for licensing information. */ + #include <popcntintrin.h> #include "common.h" #include "linefile.h" #include "hash.h" #include "options.h" #include "dnautil.h" #include "dnaseq.h" #include "dnaLoad.h" #include "portable.h" #include "basicBed.h" #include "sqlNum.h" #include "binRange.h" #include "obscure.h" #include "memalloc.h" void usage() /* Explain usage and exit. */ { errAbort( - "crisprKmers - annotate crispr sequences\n" + "crisprKmers - annotate crispr guide sequences\n" "usage:\n" " crisprKmers <sequence>\n" "options:\n" " where <sequence> is a .2bit file or .fa fasta sequence\n" - " -verbose=N - control processing steps with level of verbose:\n" - " default N=1 - only find crisprs, set up listings\n" - " N=2 - empty process crispr list into new list\n" - " N=3 - only count identicals during processing\n" - " N=4 - attempt measure all off targets and\n" - " print out all crisprs as bed format\n" - " -bed=<file> - output kmers to given bed9+ file\n" - " -offTargets=<file> - output offTargets to given file\n" - " -ranges=<file> - use specified bed3 file to limit which crisprs are\n" + " -verbose=N - for debugging control processing steps with level of verbose:\n" + " -bed=<file> - output results to given bed9+ file\n" + " -offTargets=<file> - output off target data to given file\n" + " -ranges=<file> - use specified bed3 file to limit which guides are\n" " - measured, only those with any overlap to these bed items.\n" - " -dumpKmers=<file> - after scan of sequence, output kmers to file\n" + " -memLimit=N - N number of gigabytes for each thread, default: 8\n" + " when memory size goes beyond N gB program will thread\n" + " -dumpKmers=<file> - NOT VALID after scan of sequence, output kmers to file\n" " - process will exit after this, use -loadKmers to continue\n" - " -loadKmers=<file> - load kmers from previous scan of sequence from -dumpKmers\n" - " NOTE: It is faster to simply scan the sequence to get the system ready\n" - " to go. Reading in the kmer file takes longer than scanning." + " -loadKmers=<file> - NOT VALID load kmers from previous scan of sequence from -dumpKmers" ); } static char *bedFileOut = NULL; /* set with -bed=<file> argument, kmer output */ static char *offTargets = NULL; /* write offTargets to <file> */ static FILE *offFile = NULL; /* file handle to write off targets */ static char *ranges = NULL; /* use ranges <file> to limit scanning */ static struct hash *rangesHash = NULL; /* ranges into hash + binkeeper */ static char *dumpKmers = NULL; /* file name to write out kmers from scan */ static char *loadKmers = NULL; /* file name to read in kmers from previous scan */ +static int memLimit = 8; /* gB limit before going into thread mode */ +static int threadCount = 1; /* will be adjusted depending upon vmPeak */ /* Command line validation table. */ static struct optionSpec options[] = { {"bed", OPTION_STRING}, {"offTargets", OPTION_STRING}, {"ranges", OPTION_STRING}, + {"memLimit", OPTION_INT}, {"dumpKmers", OPTION_STRING}, {"loadKmers", OPTION_STRING}, {NULL, 0}, }; // sizeof(struct crispr): 32 struct crispr /* one chromosome set of crisprs */ { struct crispr *next; /* Next in list. */ long long sequence; /* sequence value in 2bit format */ long long start; /* chromosome start 0-relative */ char strand; /* strand: + or - */ }; // sizeof(struct crisprList): 72 struct crisprList /* all chromosome sets of crisprs */ { struct crisprList *next; /* Next in list. */ char *chrom; /* chrom name */ int size; /* chrom size */ struct crispr *chromCrisprs; /* all the crisprs on this chrom */ long long crisprCount; /* number of crisprs on this chrom */ long long *sequence; /* array of the sequences */ long long *start; /* array of the starts */ char *strand; /* array of the strand characters */ int **offBy; /* offBy[5][n] */ }; #define A_BASE 0 #define C_BASE 1 #define G_BASE 2 #define T_BASE 3 #define U_BASE 3 static int orderedNtVal[256]; /* values in alpha order: ACGT 00 01 10 11 */ /* for easier sorting and complementing */ static char bases[4]; /* for binary to ascii conversion */ #define fortySixBits 0x3fffffffffff #define fortyEixhtBits 0xffffffffffff #define high32bits 0xffffffff00000000 #define low32bits 0x00000000ffffffff #define high16bits 0xffff0000ffff0000 #define low16bits 0x0000ffff0000ffff #define high8bits 0xff00ff00ff00ff00 #define low8bits 0x00ff00ff00ff00ff #define high4bits 0xf0f0f0f0f0f0f0f0 #define low4bits 0x0f0f0f0f0f0f0f0f #define high2bits 0xcccccccccccccccc #define low2bits 0x3333333333333333 static void initOrderedNtVal() /* initialization of base value lookup arrays */ { int i; for (i=0; i<ArraySize(orderedNtVal); i++) orderedNtVal[i] = -1; orderedNtVal['A'] = orderedNtVal['a'] = A_BASE; orderedNtVal['C'] = orderedNtVal['c'] = C_BASE; orderedNtVal['G'] = orderedNtVal['g'] = G_BASE; orderedNtVal['T'] = orderedNtVal['t'] = T_BASE; orderedNtVal['U'] = orderedNtVal['u'] = T_BASE; bases[A_BASE] = 'A'; bases[C_BASE] = 'C'; bases[G_BASE] = 'G'; bases[T_BASE] = 'T'; } // static void initOrderedNtVal() static void timingMessage(char *prefix, long long count, char *message, long ms, char *units, char *invUnits) { double perSecond = 0.0; double inverse = 0.0; if ((ms > 0) && (count > 0)) { perSecond = 1000.0 * count / ms; inverse = 1.0 / perSecond; } verbose(1, "# %s: %lld %s @ %ld ms -> %.2f %s == %g %s\n", prefix, count, message, ms, perSecond, units, inverse, invUnits); } static struct hash *readRanges(char *bedFile) /* Read bed and return it as a hash keyed by chromName * with binKeeper values. (from: src/hg/bedIntersect/bedIntersec.c) */ { struct hash *hash = newHash(0); /* key is chromName, value is binkeeper data */ struct lineFile *lf = lineFileOpen(bedFile, TRUE); char *row[3]; +verbose(1, "# using ranges file: %s\n", bedFile); + while (lineFileNextRow(lf, row, 3)) { struct binKeeper *bk; struct bed3 *item; struct hashEl *hel = hashLookup(hash, row[0]); if (hel == NULL) { bk = binKeeperNew(0, 1024*1024*1024); hel = hashAdd(hash, row[0], bk); } bk = hel->val; AllocVar (item); item->chrom = hel->name; item->chromStart = lineFileNeedNum(lf, row, 1); item->chromEnd = lineFileNeedNum(lf, row, 2); binKeeperAdd(bk, item->chromStart, item->chromEnd, item); } lineFileClose(&lf); return hash; } // static struct hash *readRanges(char *bedFile) #ifdef NOT // Do not need this, slReverse does this job, perhaps a bit more efficiently static int slCmpStart(const void *va, const void *vb) /* Compare slPairs on start value */ { const struct crispr *a = *((struct crispr **)va); const struct crispr *b = *((struct crispr **)vb); long long aVal = a->start; long long bVal = b->start; if (aVal < bVal) return -1; else if (aVal > bVal) return 1; else return 0; } #endif /* these two kmerVal to strings routines could be reduced to one and they - * could use lookup tables to run faster + * could use lookup tables to run faster. This is *definately* not reentrant ! */ static char *kmerPAMString(long long val) /* return the ASCII string for last three bases in then binary sequence value */ { static char pamString[32]; long long twoBitMask = 0x30; int shiftCount = 4; int baseCount = 0; while (twoBitMask) { int base = (val & twoBitMask) >> shiftCount; pamString[baseCount++] = bases[base]; twoBitMask >>= 2; shiftCount -= 2; } pamString[baseCount] = 0; return pamString; } // static char *kmerPAMString(struct crispr *c, int trim) /* beware, this needs to be used immediately upon return since it is * returning its static answer */ static char *kmerValToString(long long val, int trim) /* return ASCII string for binary sequence value */ { static char kmerString[32]; long long twoBitMask = 0x300000000000; int shiftCount = 44; int baseCount = 0; while (twoBitMask && (shiftCount >= (2*trim))) { int base = (val & twoBitMask) >> shiftCount; kmerString[baseCount++] = bases[base]; twoBitMask >>= 2; shiftCount -= 2; } kmerString[baseCount] = 0; return kmerString; } // static char *kmerValToString(long long val, int trim) static long long revComp(long long val) /* reverse complement the 2-bit numerical value kmer */ { /* complement bases and add 18 0 bits * because this divide and conquer works on 64 bits, not 46, * hence the addition of the 18 zero bits which fall out * The 'val ^ fortySixBits' does the complement, the shifting and * masking does the reversing. */ register long long v = (val ^ fortySixBits) << 18; v = ((v & high32bits) >> 32) | ((v & low32bits) << 32); v = ((v & high16bits) >> 16) | ((v & low16bits) << 16); v = ((v & high8bits) >> 8) | ((v & low8bits) << 8); v = ((v & high4bits) >> 4) | ((v & low4bits) << 4); v = ((v & high2bits) >> 2) | ((v & low2bits) << 2); return v; } // static long long revComp(long long val) static void copyToArray(struct crisprList *list) /* copy the crispr list data into arrays */ { long startTime = clock1000(); struct crisprList *cl = NULL; long long itemsCopied = 0; for (cl = list; cl; cl = cl->next) { size_t memSize = cl->crisprCount * sizeof(long long); cl->sequence = (long long *)needLargeMem(memSize); cl->start = (long long *)needLargeMem(memSize); memSize = cl->crisprCount * sizeof(char); cl->strand = (char *)needLargeMem(memSize); memSize = 5 * sizeof(int *); cl->offBy = (int **)needLargeMem(memSize); memSize = cl->crisprCount * sizeof(int); int r = 0; for (r = 0; r < 5; ++r) cl->offBy[r] = (int *)needLargeZeroedMem(memSize); long long i = 0; struct crispr *c = NULL; for (c = cl->chromCrisprs; c; c = c->next) { ++itemsCopied; cl->sequence[i] = c->sequence; cl->start[i] = c->start; cl->strand[i++] = c->strand; } } long elapsedMs = clock1000() - startTime; timingMessage("copyToArray:", itemsCopied, "items copied", elapsedMs, "items/sec", "seconds/item"); } // static void copyToArray(struct crisprList *list) */ static struct crisprList *generateKmers(struct dnaSeq *seq) { struct crispr *crisprSet = NULL; struct crisprList *returnList = NULL; AllocVar(returnList); returnList->chrom = cloneString(seq->name); returnList->size = seq->size; int i; DNA *dna; long long chromPosition = 0; long long startGap = 0; long long gapCount = 0; int kmerLength = 0; long long kmerVal = 0; long long endsAG = (A_BASE << 2) | G_BASE; long long endsGG = (G_BASE << 2) | G_BASE; long long beginsCT = (long long)((C_BASE << 2) | T_BASE) << 42; long long beginsCC = (long long)((C_BASE << 2) | C_BASE) << 42; long long reverseMask = (long long)0xf << 42; verbose(4, "# endsAG: %032llx\n", endsAG); verbose(4, "# endsGG: %032llx\n", endsGG); verbose(4, "# beginsCT: %032llx\n", beginsCT); verbose(4, "# beginsCC: %032llx\n", beginsCC); verbose(4, "# 46 bits: %032llx\n", (long long) fortySixBits); dna=seq->dna; for (i=0; i < seq->size; ++i) { int val = orderedNtVal[(int)dna[i]]; if (val >= 0) { kmerVal = fortySixBits & ((kmerVal << 2) | val); ++kmerLength; if (kmerLength > 22) { if ((endsAG == (kmerVal & 0xf)) || (endsGG == (kmerVal & 0xf))) { /* have match for positive strand */ struct crispr *oneCrispr = NULL; AllocVar(oneCrispr); oneCrispr->start = chromPosition - 22; oneCrispr->strand = '+'; oneCrispr->sequence = kmerVal; slAddHead(&crisprSet, oneCrispr); } if ((beginsCT == (kmerVal & reverseMask)) || (beginsCC == (kmerVal & reverseMask))) { /* have match for negative strand */ struct crispr *oneCrispr = NULL; AllocVar(oneCrispr); oneCrispr->start = chromPosition - 22; oneCrispr->strand = '-'; oneCrispr->sequence = revComp(kmerVal); slAddHead(&crisprSet, oneCrispr); } } } // if (val >= 0) else { ++gapCount; startGap = chromPosition; /* skip all N's == any value not = 0,1,2,3 */ while ( ((i+1) < seq->size) && (orderedNtVal[(int)dna[i+1]] < 0)) { ++chromPosition; ++i; } if (startGap != chromPosition) verbose(4, "#GAP %s\t%lld\t%lld\t%lld\t%lld\t%s\n", seq->name, startGap, chromPosition, gapCount, chromPosition-startGap, "+"); else verbose(4, "#GAP %s\t%lld\t%lld\t%lld\t%lld\t%s\n", seq->name, startGap, chromPosition+1, gapCount, 1+chromPosition-startGap, "+"); kmerLength = 0; /* reset, start over */ kmerVal = 0; } // else if (val >= 0) ++chromPosition; } // for (i=0; i < seq->size; ++i) // slReverse(&crisprSet); // save time, order not important at this time returnList->chromCrisprs = crisprSet; returnList->crisprCount = slCount(crisprSet); return returnList; } // static struct crisprList *generateKmers(struct dnaSeq *seq) static char *itemColor(int **offBy, long long index) { static char *notUnique = "150,150,150"; // static char *notUniqueAlt = "150,150,150"; static char *twoMany = "120,120,120"; // static char *twoManyAlt = "120,120,120"; static char *highScore = "0,255,0"; // > 70 static char *mediumScore = "128,128,0"; // > 50 static char *lowScore = "255,0,0"; // >= 50 // static char *lowestScore = "80,80,80"; // < 50 // static char *lowestScoreAlt = "80,80,80"; // < 50 if (offBy[0][index]) return notUnique; else { int offSum = offBy[1][index] + offBy[2][index] + offBy[3][index] + offBy[4][index]; if (offSum < 100) return highScore; else if (offSum < 150) return mediumScore; else if (offSum < 250) return lowScore; else return twoMany; } } static void countsOutput(struct crisprList *all) /* everything has been scanned and counted, print out all the data from arrays*/ { long startTime = clock1000(); long long itemsOutput = 0; FILE *bedFH = NULL; if (bedFileOut) bedFH = mustOpen(bedFileOut, "w"); struct crisprList *list; long long totalOut = 0; for (list = all; list; list = list->next) { long long c = 0; for (c = 0; c < list->crisprCount; ++c) { ++itemsOutput; int negativeOffset = 0; if (list->strand[c] == '-') negativeOffset = 3; long long txStart = list->start[c] + negativeOffset; long long txEnd = txStart + 20; int totalOffs = list->offBy[0][c] + list->offBy[1][c] + list->offBy[2][c] + list->offBy[3][c] + list->offBy[4][c]; char *color = itemColor(list->offBy, c); if (0 == totalOffs) verbose(1, "# PERFECT score %s:%lld %c\t%s\n", list->chrom, list->start[c], list->strand[c], kmerValToString(list->sequence[c], 3)); if (bedFH) fprintf(bedFH, "%s\t%lld\t%lld\t%d,%d,%d,%d,%d\t%d\t%c\t%lld\t%lld\t%s\t%s\t%s\t%s\t%s\t%d\t%d\t%d\t%d\n", list->chrom, list->start[c], list->start[c]+23, list->offBy[0][c], list->offBy[1][c], list->offBy[2][c], list->offBy[3][c], list->offBy[4][c], list->offBy[0][c], list->strand[c], txStart, txEnd, color, color, color, kmerValToString(list->sequence[c], 3), kmerPAMString(list->sequence[c]), list->offBy[1][c], list->offBy[2][c], list->offBy[3][c], list->offBy[4][c]); if (list->offBy[0][c]) verbose(3, "# array identical: %d %s:%lld %c\t%s\n", list->offBy[0][c], list->chrom, list->start[c], list->strand[c], kmerValToString(list->sequence[c], 3)); ++totalOut; } } if (bedFH) carefulClose(&bedFH); long elapsedMs = clock1000() - startTime; timingMessage("copyToArray:", itemsOutput, "items output", elapsedMs, "items/sec", "seconds/item"); } // static void countsOutput(struct crisprList *all) static struct crisprList *scanSequence(char *inFile) /* scan the given file, return list of crisprs */ { verbose(1, "#\tscanning sequence file: %s\n", inFile); dnaUtilOpen(); struct dnaLoad *dl = dnaLoadOpen(inFile); struct dnaSeq *seq; struct crisprList *listReturn = NULL; long elapsedMs = 0; long scanStart = clock1000(); long long totalCrisprs = 0; /* scanning all sequences, setting up crisprs on the listReturn */ while ((seq = dnaLoadNext(dl)) != NULL) { if (startsWithNoCase("chrUn", seq->name) || rStringIn("hap", seq->name) || rStringIn("_alt", seq->name) ) { verbose(1, "# skip chrom: %s\n", seq->name); continue; } long startTime = clock1000(); struct crisprList *oneList = generateKmers(seq); slAddHead(&listReturn, oneList); totalCrisprs += oneList->crisprCount; elapsedMs = clock1000() - startTime; timingMessage(seq->name, oneList->crisprCount, "crisprs", elapsedMs, "crisprs/sec", "seconds/crispr"); } elapsedMs = clock1000() - scanStart; timingMessage("scanSequence", totalCrisprs, "total crisprs", elapsedMs, "crisprs/sec", "seconds/crispr"); return listReturn; } /* static crisprList *scanSequence(char *inFile) */ static struct crisprList *rangeExtraction(struct crisprList **allReference) /* given ranges in global rangesHash, construct new list of crisprs that * have any type of overlap with the ranges, also extract those items from * the all list. Returns new list. */ { struct crisprList *all = *allReference; struct crisprList *listReturn = NULL; struct crisprList *list = NULL; int inputChromCount = slCount(all); long long returnListCrisprCount = 0; long long examinedCrisprCount = 0; struct crisprList *prevChromList = NULL; long elapsedMs = 0; long scanStart = clock1000(); struct crisprList *nextList = NULL; for (list = all; list; list = nextList) { nextList = list->next; // remember before perhaps lost long long beforeCrisprCount = list->crisprCount; examinedCrisprCount += list->crisprCount; struct crispr *c = NULL; struct binKeeper *bk = hashFindVal(rangesHash, list->chrom); struct crispr *newCrispr = NULL; if (bk != NULL) { struct crispr *prevCrispr = NULL; struct crispr *next = NULL; for (c = list->chromCrisprs; c; c = next) { struct binElement *hitList = NULL; next = c->next; // remember before perhaps lost int start = c->start; if (c->strand == '-') start += 2; int end = start + 20; hitList = binKeeperFind(bk, start, end); if (hitList) { if (prevCrispr) // remove this one from the all list prevCrispr->next = next; else list->chromCrisprs = next; // removing the first one c->next = NULL; slAddHead(&newCrispr, c); // constructing new list } else prevCrispr = c; // remains on all list slFreeList(&hitList); } } if (newCrispr) { struct crisprList *newItem = NULL; AllocVar(newItem); newItem->chrom = cloneString(list->chrom); newItem->size = list->size; newItem->chromCrisprs = newCrispr; newItem->crisprCount = slCount(newCrispr); returnListCrisprCount += newItem->crisprCount; slAddHead(&listReturn, newItem); if (NULL == list->chromCrisprs) // all have been removed for this chrom { if (prevChromList) // remove it from the chrom list prevChromList->next = nextList; else all = nextList; // removing the first one } else { list->crisprCount = slCount(list->chromCrisprs); long long removedCrisprs = beforeCrisprCount - list->crisprCount; verbose(1, "# range scan chrom %s had %lld crisprs, removed %lld leaving %lld target\n", list->chrom, beforeCrisprCount, removedCrisprs, list->crisprCount); } } prevChromList = list; } // for (list = all; list; list = list->next) elapsedMs = clock1000() - scanStart; verbose(1, "# range scanning %d chroms, return %d selected chroms, leaving %d chroms\n", inputChromCount, slCount(listReturn), slCount(all)); long long targetCrisprCount = examinedCrisprCount - returnListCrisprCount; timingMessage("range scan", examinedCrisprCount, "examined crisprs", elapsedMs, "crisprs/sec", "seconds/crispr"); timingMessage("range scan", targetCrisprCount, "remaining target crisprs", elapsedMs, "crisprs/sec", "seconds/crispr"); timingMessage("range scan", returnListCrisprCount, "returned query crisprs", elapsedMs, "crisprs/sec", "seconds/crispr"); if (NULL == all) { allReference = NULL; // they have all been removed verbose(1, "# range scan, every single chrom has been removed\n"); } else if (*allReference != all) { verbose(1, "# range scan, first chrom list has been moved from %p to %p\n", (void *)*allReference, (void *)all); *allReference = all; } return listReturn; } // static crisprList *rangeExtraction(crisprList *all) static int hitScoreM[20] = {0,0,0.014,0,0,0.395,0.317,0,0.389,0.079,0.445,0.508,0.613,0.851,0.732,0.828,0.615,0.804,0.685,0.583}; static int calcHitScore(long long sequence1, long long sequence2) { double score1 = 1.0; int mmCount = 0; int lastMmPos = -1; /* the XOR will determine differences in two sequences, the shift # right 6 removes the PAM sequence */ long long misMatch = (sequence1 ^ sequence2) >> 6; int distCount = 0; int distSum = 0; int pos = 0; for (pos = 0; pos < 20; ++pos) { int diff = misMatch & 0x3; if (diff) { ++mmCount; if (lastMmPos != -1) { distSum += pos-lastMmPos; ++distCount; } score1 *= 1 - hitScoreM[pos]; lastMmPos = pos; } misMatch >>= 2; } double score2 = 1.0; if (distCount > 1) { double avgDist = (double)distSum / distCount; score2 = 1.0 / (((19-avgDist)/19.0) * 4 + 1); } double score3 = 1.0; if (mmCount > 0) score3 = 1.0 / (mmCount * mmCount); return (int)(score1 * score2 * score3 * 100); } static void recordOffTargets(struct crisprList *query, struct crisprList *target, int bitsOn, long long qIndex, long long tIndex) /* bitsOn is from 1 to 4, record this match when less than 1000 total */ { if (query->offBy[0][qIndex] ) // no need to accumulate if 0-mismatch > 0 return; if (offFile) { int i = 0; int bitsOnSum = 0; for (i = 1; i < 5; ++i) bitsOnSum += query->offBy[i][qIndex]; if (bitsOnSum < 1000) // could be command line option limit { /* needs to be two fprintfs, as the kmer*String() function * returns are confused due to static returns */ int hitScore = calcHitScore(query->sequence[qIndex], target->sequence[tIndex]); fprintf(offFile, "%s:%lld %c %s %s %d\t", query->chrom, query->start[qIndex], query->strand[qIndex], kmerValToString(query->sequence[qIndex], 3), kmerPAMString(query->sequence[qIndex]), hitScore); fprintf(offFile, "%s:%lld %c %s %s\t%d\n", target->chrom, target->start[tIndex], target->strand[tIndex], kmerValToString(target->sequence[tIndex], 3), kmerPAMString(target->sequence[tIndex]), bitsOn); } } } // static void recordOffTargets(struct crisprList *query, // struct crisprList *target, int bitsOn, long long qIndex, // long long tIndex) static void queryVsAll(struct crisprList *query, struct crisprList *target) /* run the query crisprs list against the target list in the array structures */ { struct crisprList *qList = NULL; long long totalCrisprsQuery = 0; long long totalCrisprsTarget = 0; long long totalCompares = 0; long processStart = clock1000(); long elapsedMs = 0; for (qList = query; qList; qList = qList->next) { long long qCount = 0; totalCrisprsQuery += qList->crisprCount; verbose(1, "# queryVsAll %lld query crisprs on chrom %s\n", qList->crisprCount, qList->chrom); for (qCount = 0; qCount < qList->crisprCount; ++qCount) { struct crisprList *tList = NULL; for (tList = target; tList; tList = tList->next) { long long tCount = 0; totalCompares += tList->crisprCount; // if (0 == qCount) // { // totalCrisprsTarget += tList->crisprCount; // verbose(1, "# queryVsAll %lld target crisprs on chrom %s %lld targets %lld compares\n", tList->crisprCount, tList->chrom, totalCrisprsTarget, totalCompares); // } for (tCount = 0; tCount < tList->crisprCount; ++tCount) { /* the XOR will determine differences in two sequences * the shift right 6 removes the PAM sequence */ long long misMatch = (qList->sequence[qCount] ^ tList->sequence[tCount]) >> 6; if (misMatch) { /* possible misMatch bit values: 01 10 11 * turn those three values into just: 01 */ misMatch = (misMatch | (misMatch >> 1)) & 0x5555555555; int bitsOn = _mm_popcnt_u64(misMatch); if (bitsOn < 5) { recordOffTargets(qList, tList, bitsOn, qCount, tCount); qList->offBy[bitsOn][qCount] += 1; // tList->offBy[bitsOn][tCount] += 1; not needed } } else { /* no misMatch, identical crisprs */ qList->offBy[0][qCount] += 1; // tList->offBy[0][tCount] += 1; not needed } } // for (tCount = 0; tCount < tList->crisprCount; ++tCount) } // for (tList = target; tList; tList = tList->next) } // for (qCount = 0; qCount < qList->crisprCount; ++qCount) } // for (qList = query; qList; qList = qList->next) verbose(1, "# done with scanning, check timing\n"); elapsedMs = clock1000() - processStart; timingMessage("queryVsAll", totalCrisprsQuery, "query crisprs processed", elapsedMs, "crisprs/sec", "seconds/crispr"); timingMessage("queryVsAll", totalCrisprsTarget, "vs target crisprs", elapsedMs, "crisprs/sec", "seconds/crispr"); timingMessage("queryVsAll", totalCompares, "total comparisons", elapsedMs, "compares/sec", "seconds/compare"); } /* static struct crisprList *queryVsAll(struct crisprList *query, struct crisprList *target) */ static void allVsAll(struct crisprList *all) /* run this 'all' list vs. itself avoiding self to self comparisons */ { struct crisprList *qList = NULL; long long totalCrisprsQuery = 0; long long totalCrisprsCompare = 0; long processStart = clock1000(); long elapsedMs = 0; /* query runs through all chroms */ for (qList = all; qList; qList = qList->next) { long long qCount = 0; totalCrisprsQuery += qList->crisprCount; verbose(1, "# allVsAll %lld query crisprs on chrom %s\n", qList->crisprCount, qList->chrom); /* query runs through all kmers on this chrom */ for (qCount = 0; qCount < qList->crisprCount; ++qCount) { /* target starts on same chrom as query, and at next kmer after query for this first chrom */ long long tStart = qCount+1; struct crisprList *tList = NULL; for (tList = qList; tList; tList = tList->next) { long long tCount = tStart; totalCrisprsCompare += tList->crisprCount - tStart; for (tCount = tStart; tCount < tList->crisprCount; ++tCount) { /* the XOR will determine differences in two sequences * the shift right 6 removes the PAM sequence */ long long misMatch = (qList->sequence[qCount] ^ tList->sequence[tCount]) >> 6; if (misMatch) { /* possible misMatch bit values: 01 10 11 * turn those three values into just: 01 */ misMatch = (misMatch | (misMatch >> 1)) & 0x5555555555; int bitsOn = _mm_popcnt_u64(misMatch); if (bitsOn < 5) { recordOffTargets(qList, tList, bitsOn, qCount, tCount); qList->offBy[bitsOn][qCount] += 1; tList->offBy[bitsOn][tCount] += 1; } } else { /* no misMatch, identical crisprs */ qList->offBy[0][qCount] += 1; tList->offBy[0][tCount] += 1; } } // for (tCount = 0; tCount < tList->crisprCount; ++tCount) tStart = 0; /* following chroms run through all */ } // for (tList = target; tList; tList = tList->next) } // for (qCount = 0; qCount < qList->crisprCount; ++qCount) } // for (qList = query; qList; qList = qList->next) elapsedMs = clock1000() - processStart; timingMessage("allVsAll", totalCrisprsQuery, "crisprs processed", elapsedMs, "crisprs/sec", "seconds/crispr"); timingMessage("allVsAll", totalCrisprsCompare, "total comparisons", elapsedMs, "compares/sec", "seconds/compare"); } /* static struct crisprList *allVsAll(struct crisprList *query, struct crisprList *target) */ static struct crisprList *readKmers(char *fileIn) /* read in kmer list from 'fileIn', return list structure */ { verbose(1, "# reading crisprs from: %s\n", fileIn); struct crisprList *listReturn = NULL; struct lineFile *lf = lineFileOpen(fileIn, TRUE); char *row[10]; int wordCount = 0; long long crisprsInput = 0; long startMs = clock1000(); while (0 < (wordCount = lineFileChopNextTab(lf, row, ArraySize(row))) ) { if (3 != wordCount) errAbort("expecing three words at line %d in file %s, found: %d", lf->lineIx, fileIn, wordCount); struct crisprList *newItem = NULL; AllocVar(newItem); newItem->chrom = cloneString(row[0]); newItem->crisprCount = sqlLongLong(row[1]); newItem->size = sqlLongLong(row[2]); newItem->chromCrisprs = NULL; slAddHead(&listReturn, newItem); long long verifyCount = 0; while ( (verifyCount < newItem->crisprCount) && (0 < (wordCount = lineFileChopNextTab(lf, row, ArraySize(row)))) ) { if (3 != wordCount) errAbort("expecing three words at line %d in file %s, found: %d", lf->lineIx, fileIn, wordCount); ++verifyCount; struct crispr *oneCrispr = NULL; AllocVar(oneCrispr); oneCrispr->sequence = sqlLongLong(row[0]); oneCrispr->start = sqlLongLong(row[1]); oneCrispr->strand = row[2][0]; slAddHead(&newItem->chromCrisprs, oneCrispr); } if (verifyCount != newItem->crisprCount) errAbort("expecting %lld kmer items at line %d in file %s, found: %lld", newItem->crisprCount, lf->lineIx, fileIn, verifyCount); crisprsInput += verifyCount; } lineFileClose(&lf); long elapsedMs = clock1000() - startMs; timingMessage("readKmers", crisprsInput, "crisprs read in", elapsedMs, "crisprs/sec", "seconds/crispr"); return listReturn; } // static struct crisprList *readKmers(char *fileIn) static void writeKmers(struct crisprList *all, char *fileOut) /* write kmer list 'all' to 'fileOut' */ { FILE *fh = mustOpen(fileOut, "w"); struct crisprList *list = NULL; long long crisprsWritten = 0; long startMs = clock1000(); slReverse(&all); for (list = all; list; list = list->next) { fprintf(fh, "%s\t%lld\t%d\n", list->chrom, list->crisprCount, list->size); struct crispr *c = NULL; slReverse(&list->chromCrisprs); for (c = list->chromCrisprs; c; c = c->next) { fprintf(fh, "%lld\t%lld\t%c\n", c->sequence, c->start, c->strand); ++crisprsWritten; } } carefulClose(&fh); long elapsedMs = clock1000() - startMs; timingMessage("writeKmers", crisprsWritten, "crisprs written", elapsedMs, "crisprs/sec", "seconds/crispr"); } // static void writeKmers(struct crisprList *all, char *fileOut) static void crisprKmers(char *sequence) /* crisprKmers - find and annotate crispr sequences. */ { -struct crisprList *queryCrisprs = NULL; +struct crisprList *queryGuides = NULL; // struct crisprList *countedCrisprs = NULL; -struct crisprList *allCrisprs = NULL; +struct crisprList *allGuides = NULL; if (loadKmers) - allCrisprs = readKmers(loadKmers); + allGuides = readKmers(loadKmers); else - allCrisprs = scanSequence(sequence); + allGuides = scanSequence(sequence); if (dumpKmers) { - writeKmers(allCrisprs, dumpKmers); + writeKmers(allGuides, dumpKmers); return; } +long long vmPeak = currentVmPeak(); +verbose(1, "# vmPeak after scanSequence: %lld kB\n", vmPeak); + /* processing those crisprs */ if (verboseLevel() > 1) { if (offTargets) offFile = mustOpen(offTargets, "w"); /* if ranges have been specified, construct queryList of kmers to measure */ if (rangesHash) { - /* result here is two exclusive sets: query, and allCrisprs - * the query crisprs have been extracted from the allCrisprs */ - queryCrisprs = rangeExtraction(& allCrisprs); - copyToArray(queryCrisprs); - if (allCrisprs) // if there are any left on the all list + /* result here is two exclusive sets: query, and allGuides + * the query crisprs have been extracted from the allGuides */ + queryGuides = rangeExtraction(& allGuides); + } + if (queryGuides) + copyToArray(queryGuides); + if (allGuides) + copyToArray(allGuides); + vmPeak = currentVmPeak(); + verbose(1, "# vmPeak after copyToArray: %lld kB\n", vmPeak); + /* larger example: 62646196 kB */ + if ((vmPeak >> 20) > 8) // the >> 20 converts kB to gB { - copyToArray(allCrisprs); - /* first run up query vs. all */ - queryVsAll(queryCrisprs, allCrisprs); + threadCount = 1 + ((vmPeak >> 20) / 8); + verbose(1, "# over 8 Gb at %lld kB, threadCount: %d\n", vmPeak, threadCount); } + if (queryGuides) // when range selected some query sequences + { + if (allGuides) // if there are any left on the all list + queryVsAll(queryGuides, allGuides); /* then run up the query vs. itself avoiding self vs. self */ - allVsAll(queryCrisprs); - countsOutput(queryCrisprs); + allVsAll(queryGuides); + countsOutput(queryGuides); } else { - copyToArray(allCrisprs); - /* run up all vs. all avoiding self vs. self */ - allVsAll(allCrisprs); - countsOutput(allCrisprs); + allVsAll(allGuides); /* run up all vs. all avoiding self vs. self */ + countsOutput(allGuides); } + carefulClose(&offFile); } } // static void crisprKmers(char *sequence) int main(int argc, char *argv[]) /* Process command line, initialize translation arrays and call the process */ { optionInit(&argc, argv, options); if (argc != 2) usage(); verbose(0, "# running verboseLevel: %d\n", verboseLevel()); bedFileOut = optionVal("bed", bedFileOut); dumpKmers = optionVal("dumpKmers", dumpKmers); loadKmers = optionVal("loadKmers", loadKmers); offTargets = optionVal("offTargets", offTargets); +memLimit = optionInt("memLimit", memLimit); ranges = optionVal("ranges", ranges); if (ranges) rangesHash = readRanges(ranges); initOrderedNtVal(); /* set up orderedNtVal[] */ crisprKmers(argv[1]); + if (verboseLevel() > 1) printVmPeak(); return 0; }