package main import ( "bufio" "bytes" "context" "encoding/gob" "fmt" "io" "runtime" "sort" "strings" "sync" log "github.com/sirupsen/logrus" "golang.org/x/crypto/blake2b" ) type tileVariantID uint16 // 1-based type tileLibRef struct { Tag tagID Variant tileVariantID } type tileSeq map[string][]tileLibRef func (tseq tileSeq) Variants() ([]tileVariantID, int, int) { maxtag := 0 for _, refs := range tseq { for _, ref := range refs { if maxtag < int(ref.Tag) { maxtag = int(ref.Tag) } } } vars := make([]tileVariantID, maxtag+1) var kept, dropped int for _, refs := range tseq { for _, ref := range refs { if vars[int(ref.Tag)] != 0 { dropped++ } else { kept++ } vars[int(ref.Tag)] = ref.Variant } } return vars, kept, dropped } type tileLibrary struct { retainNoCalls bool skipOOO bool retainTileSequences bool taglib *tagLibrary variant [][][blake2b.Size256]byte refseqs map[string]map[string][]tileLibRef compactGenomes map[string][]tileVariantID // count [][]int seq map[[blake2b.Size256]byte][]byte variants int // if non-nil, write out any tile variants added while tiling encoder *gob.Encoder mtx sync.Mutex } func (tilelib *tileLibrary) loadTagSet(newtagset [][]byte) error { // Loading a tagset means either passing it through to the // output (if it's the first one we've seen), or just ensuring // it doesn't disagree with what we already have. if len(newtagset) == 0 { return nil } tilelib.mtx.Lock() defer tilelib.mtx.Unlock() if tilelib.taglib == nil || tilelib.taglib.Len() == 0 { tilelib.taglib = &tagLibrary{} err := tilelib.taglib.setTags(newtagset) if err != nil { return err } if tilelib.encoder != nil { err = tilelib.encoder.Encode(LibraryEntry{ TagSet: newtagset, }) if err != nil { return err } } } else if tilelib.taglib.Len() != len(newtagset) { return fmt.Errorf("cannot merge libraries with differing tagsets") } else { current := tilelib.taglib.Tags() for i := range newtagset { if !bytes.Equal(newtagset[i], current[i]) { return fmt.Errorf("cannot merge libraries with differing tagsets") } } } return nil } func (tilelib *tileLibrary) loadTileVariants(tvs []TileVariant, variantmap map[tileLibRef]tileVariantID) error { for _, tv := range tvs { // Assign a new variant ID (unique across all inputs) // for each input variant. variantmap[tileLibRef{Tag: tv.Tag, Variant: tv.Variant}] = tilelib.getRef(tv.Tag, tv.Sequence).Variant } return nil } func (tilelib *tileLibrary) loadCompactGenomes(cgs []CompactGenome, variantmap map[tileLibRef]tileVariantID, onLoadGenome func(CompactGenome)) error { log.Debugf("loadCompactGenomes: %d", len(cgs)) var wg sync.WaitGroup errs := make(chan error, 1) for _, cg := range cgs { wg.Add(1) cg := cg go func() { defer wg.Done() for i, variant := range cg.Variants { if len(errs) > 0 { return } if variant == 0 { continue } tag := tagID(i / 2) newvariant, ok := variantmap[tileLibRef{Tag: tag, Variant: variant}] if !ok { err := fmt.Errorf("oops: genome %q has variant %d for tag %d, but that variant was not in its library", cg.Name, variant, tag) select { case errs <- err: default: } return } log.Tracef("loadCompactGenomes: cg %s tag %d variant %d => %d", cg.Name, tag, variant, newvariant) cg.Variants[i] = newvariant } if onLoadGenome != nil { onLoadGenome(cg) } if tilelib.encoder != nil { err := tilelib.encoder.Encode(LibraryEntry{ CompactGenomes: []CompactGenome{cg}, }) if err != nil { select { case errs <- err: default: } return } } if tilelib.compactGenomes != nil { tilelib.mtx.Lock() defer tilelib.mtx.Unlock() tilelib.compactGenomes[cg.Name] = cg.Variants } }() } wg.Wait() go close(errs) return <-errs } func (tilelib *tileLibrary) loadCompactSequences(cseqs []CompactSequence, variantmap map[tileLibRef]tileVariantID) error { log.Debugf("loadCompactSequences: %d", len(cseqs)) for _, cseq := range cseqs { for _, tseq := range cseq.TileSequences { for i, libref := range tseq { if libref.Variant == 0 { // No variant (e.g., import // dropped tiles with // no-calls) = no translation. continue } v, ok := variantmap[libref] if !ok { return fmt.Errorf("oops: CompactSequence %q has variant %d for tag %d, but that variant was not in its library", cseq.Name, libref.Variant, libref.Tag) } tseq[i].Variant = v } } if tilelib.encoder != nil { if err := tilelib.encoder.Encode(LibraryEntry{ CompactSequences: []CompactSequence{cseq}, }); err != nil { return err } } } tilelib.mtx.Lock() defer tilelib.mtx.Unlock() if tilelib.refseqs == nil { tilelib.refseqs = map[string]map[string][]tileLibRef{} } for _, cseq := range cseqs { tilelib.refseqs[cseq.Name] = cseq.TileSequences } return nil } // Load library data from rdr. Tile variants might be renumbered in // the process; in that case, genomes variants will be renumbered to // match. // // If onLoadGenome is non-nil, call it on each CompactGenome entry. func (tilelib *tileLibrary) LoadGob(ctx context.Context, rdr io.Reader, onLoadGenome func(CompactGenome)) error { cgs := []CompactGenome{} cseqs := []CompactSequence{} variantmap := map[tileLibRef]tileVariantID{} err := DecodeLibrary(rdr, func(ent *LibraryEntry) error { if ctx.Err() != nil { return ctx.Err() } if err := tilelib.loadTagSet(ent.TagSet); err != nil { return err } if err := tilelib.loadTileVariants(ent.TileVariants, variantmap); err != nil { return err } cgs = append(cgs, ent.CompactGenomes...) cseqs = append(cseqs, ent.CompactSequences...) return nil }) if err != nil { return err } if ctx.Err() != nil { return ctx.Err() } err = tilelib.loadCompactGenomes(cgs, variantmap, onLoadGenome) if err != nil { return err } err = tilelib.loadCompactSequences(cseqs, variantmap) if err != nil { return err } return nil } type importStats struct { InputFile string InputLabel string InputLength int InputCoverage int TileCoverage int PathLength int DroppedOutOfOrderTiles int } func (tilelib *tileLibrary) TileFasta(filelabel string, rdr io.Reader) (tileSeq, []importStats, error) { ret := tileSeq{} type jobT struct { label string fasta []byte } todo := make(chan jobT) scanner := bufio.NewScanner(rdr) go func() { defer close(todo) var fasta []byte var seqlabel string for scanner.Scan() { buf := scanner.Bytes() if len(buf) > 0 && buf[0] == '>' { todo <- jobT{seqlabel, fasta} seqlabel, fasta = strings.SplitN(string(buf[1:]), " ", 2)[0], nil log.Debugf("%s %s reading fasta", filelabel, seqlabel) } else { fasta = append(fasta, bytes.ToLower(buf)...) } } todo <- jobT{seqlabel, fasta} }() type foundtag struct { pos int tagid tagID taglen int } found := make([]foundtag, 2000000) path := make([]tileLibRef, 2000000) totalFoundTags := 0 totalPathLen := 0 skippedSequences := 0 stats := make([]importStats, 0, len(todo)) for job := range todo { if len(job.fasta) == 0 { continue } else if strings.Contains(job.label, "_") { skippedSequences++ continue } log.Debugf("%s %s tiling", filelabel, job.label) found = found[:0] tilelib.taglib.FindAll(job.fasta, func(tagid tagID, pos, taglen int) { found = append(found, foundtag{pos: pos, tagid: tagid, taglen: taglen}) }) totalFoundTags += len(found) basesOut := 0 skipped := 0 path = path[:0] last := foundtag{tagid: -1} if tilelib.skipOOO { keep := longestIncreasingSubsequence(len(found), func(i int) int { return int(found[i].tagid) }) for i, x := range keep { found[i] = found[x] } skipped = len(found) - len(keep) found = found[:len(keep)] } for i, f := range found { log.Tracef("%s %s found[%d] == %#v", filelabel, job.label, i, f) if last.tagid < 0 { // first tag in sequence last = foundtag{tagid: f.tagid} continue } libref := tilelib.getRef(last.tagid, job.fasta[last.pos:f.pos+f.taglen]) path = append(path, libref) if libref.Variant > 0 { // Count output coverage from // the end of the previous tag // (if any) to the end of the // current tag, IOW don't // double-count coverage for // the previous tag. basesOut += countBases(job.fasta[last.pos+last.taglen : f.pos+f.taglen]) } else { // If we dropped this tile // (because !retainNoCalls), // set taglen=0 so the // overlapping tag is counted // toward coverage on the // following tile. f.taglen = 0 } last = f } if last.tagid < 0 { log.Warnf("%s %s no tags found", filelabel, job.label) } else { libref := tilelib.getRef(last.tagid, job.fasta[last.pos:]) path = append(path, libref) if libref.Variant > 0 { basesOut += countBases(job.fasta[last.pos+last.taglen:]) } } pathcopy := make([]tileLibRef, len(path)) copy(pathcopy, path) ret[job.label] = pathcopy basesIn := countBases(job.fasta) log.Infof("%s %s fasta in %d coverage in %d coverage out %d path len %d skipped %d", filelabel, job.label, len(job.fasta), basesIn, basesOut, len(path), skipped) stats = append(stats, importStats{ InputFile: filelabel, InputLabel: job.label, InputLength: len(job.fasta), InputCoverage: basesIn, TileCoverage: basesOut, PathLength: len(path), DroppedOutOfOrderTiles: skipped, }) totalPathLen += len(path) } log.Printf("%s tiled with total path len %d in %d sequences (skipped %d sequences with '_' in name, skipped %d out-of-order tags)", filelabel, totalPathLen, len(ret), skippedSequences, totalFoundTags-totalPathLen) return ret, stats, scanner.Err() } func (tilelib *tileLibrary) Len() int { tilelib.mtx.Lock() defer tilelib.mtx.Unlock() return tilelib.variants } // Return a tileLibRef for a tile with the given tag and sequence, // adding the sequence to the library if needed. func (tilelib *tileLibrary) getRef(tag tagID, seq []byte) tileLibRef { dropSeq := false if !tilelib.retainNoCalls { for _, b := range seq { if b != 'a' && b != 'c' && b != 'g' && b != 't' { dropSeq = true break } } } tilelib.mtx.Lock() if tilelib.variant == nil && tilelib.taglib != nil { tilelib.variant = make([][][blake2b.Size256]byte, tilelib.taglib.Len()) } if int(tag) >= len(tilelib.variant) { // If we haven't seen the tag library yet (as in a // merge), tilelib.taglib.Len() is zero. We can still // behave correctly, we just need to expand the // tilelib.variant slice as needed. if int(tag) >= cap(tilelib.variant) { // Allocate 2x capacity. newslice := make([][][blake2b.Size256]byte, int(tag)+1, (int(tag)+1)*2) copy(newslice, tilelib.variant) tilelib.variant = newslice[:int(tag)+1] } else { // Use previously allocated capacity, avoiding // copy. tilelib.variant = tilelib.variant[:int(tag)+1] } } seqhash := blake2b.Sum256(seq) for i, varhash := range tilelib.variant[tag] { if varhash == seqhash { tilelib.mtx.Unlock() return tileLibRef{Tag: tag, Variant: tileVariantID(i + 1)} } } tilelib.variants++ tilelib.variant[tag] = append(tilelib.variant[tag], seqhash) if tilelib.retainTileSequences && !dropSeq { if tilelib.seq == nil { tilelib.seq = map[[blake2b.Size256]byte][]byte{} } tilelib.seq[seqhash] = append([]byte(nil), seq...) } variant := tileVariantID(len(tilelib.variant[tag])) tilelib.mtx.Unlock() if tilelib.encoder != nil { saveSeq := seq if dropSeq { // Save the hash, but not the sequence saveSeq = nil } tilelib.encoder.Encode(LibraryEntry{ TileVariants: []TileVariant{{ Tag: tag, Variant: variant, Blake2b: seqhash, Sequence: saveSeq, }}, }) } return tileLibRef{Tag: tag, Variant: variant} } func (tilelib *tileLibrary) TileVariantSequence(libref tileLibRef) []byte { if libref.Variant == 0 || len(tilelib.variant) <= int(libref.Tag) || len(tilelib.variant[libref.Tag]) < int(libref.Variant) { return nil } return tilelib.seq[tilelib.variant[libref.Tag][libref.Variant-1]] } // Tidy deletes unreferenced tile variants and renumbers variants so // more common variants have smaller IDs. func (tilelib *tileLibrary) Tidy() { log.Print("Tidy: compute inref") inref := map[tileLibRef]bool{} for _, refseq := range tilelib.refseqs { for _, librefs := range refseq { for _, libref := range librefs { inref[libref] = true } } } log.Print("Tidy: compute remap") remap := make([][]tileVariantID, len(tilelib.variant)) throttle := throttle{Max: runtime.NumCPU() + 1} for tag, oldvariants := range tilelib.variant { tag, oldvariants := tagID(tag), oldvariants if tag%10000 == 0 { log.Printf("Tidy: tag %d", tag) } throttle.Acquire() go func() { defer throttle.Release() uses := make([]int, len(oldvariants)) for _, cg := range tilelib.compactGenomes { for phase := 0; phase < 2; phase++ { cgi := int(tag)*2 + phase if cgi < len(cg) && cg[cgi] > 0 { uses[cg[cgi]-1]++ } } } // Compute desired order of variants: // neworder[x] == index in oldvariants that // should move to position x. neworder := make([]int, len(oldvariants)) for i := range neworder { neworder[i] = i } sort.Slice(neworder, func(i, j int) bool { if cmp := uses[neworder[i]] - uses[neworder[j]]; cmp != 0 { return cmp > 0 } else { return bytes.Compare(oldvariants[neworder[i]][:], oldvariants[neworder[j]][:]) < 0 } }) // Replace tilelib.variants[tag] with a new // re-ordered slice of hashes, and make a // mapping from old to new variant IDs. remaptag := make([]tileVariantID, len(oldvariants)+1) newvariants := make([][blake2b.Size256]byte, 0, len(neworder)) for _, oldi := range neworder { if uses[oldi] > 0 || inref[tileLibRef{Tag: tag, Variant: tileVariantID(oldi + 1)}] { newvariants = append(newvariants, oldvariants[oldi]) remaptag[oldi+1] = tileVariantID(len(newvariants)) } } tilelib.variant[tag] = newvariants remap[tag] = remaptag }() } throttle.Wait() // Apply remap to genomes and reference sequences, so they // refer to the same tile variants using the changed IDs. log.Print("Tidy: apply remap") for _, cg := range tilelib.compactGenomes { for idx, variant := range cg { cg[idx] = remap[tagID(idx/2)][variant] } } for _, refcs := range tilelib.refseqs { for _, refseq := range refcs { for i, tv := range refseq { refseq[i].Variant = remap[tv.Tag][tv.Variant] } } } log.Print("Tidy: done") } func countBases(seq []byte) int { n := 0 for _, c := range seq { if isbase[c] { n++ } } return n }