slicenumpy.go

   1 // Copyright (C) The Lightning Authors. All rights reserved.
   2 //
   3 // SPDX-License-Identifier: AGPL-3.0
   4
   5 package lightning
   6
   7 import (
   8         "bufio"
   9         "bytes"
  10         "encoding/gob"
  11         "encoding/json"
  12         "errors"
  13         "flag"
  14         "fmt"
  15         "io"
  16         "io/ioutil"
  17         "math"
  18         "net/http"
  19         _ "net/http/pprof"
  20         "os"
  21         "regexp"
  22         "runtime"
  23         "runtime/debug"
  24         "sort"
  25         "strconv"
  26         "strings"
  27         "sync/atomic"
  28         "unsafe"
  29
  30         "git.arvados.org/arvados.git/sdk/go/arvados"
  31         "github.com/arvados/lightning/hgvs"
  32         "github.com/james-bowman/nlp"
  33         "github.com/kshedden/gonpy"
  34         "github.com/sirupsen/logrus"
  35         log "github.com/sirupsen/logrus"
  36         "golang.org/x/crypto/blake2b"
  37         "gonum.org/v1/gonum/mat"
  38 )
  39
  40 const annotationMaxTileSpan = 100
  41
  42 type sliceNumpy struct {
  43         filter          filter
  44         threads         int
  45         chi2Cases       []bool
  46         chi2PValue      float64
  47         pcaComponents   int
  48         minCoverage     int
  49         includeVariant1 bool
  50         debugTag        tagID
  51
  52         cgnames         []string
  53         samples         []sampleInfo
  54         trainingSet     []int // samples index => training set index, or -1 if not in training set
  55         trainingSetSize int
  56         pvalue          func(onehot []bool) float64
  57         pvalueCallCount int64
  58 }
  59
  60 func (cmd *sliceNumpy) RunCommand(prog string, args []string, stdin io.Reader, stdout, stderr io.Writer) int {
  61         err := cmd.run(prog, args, stdin, stdout, stderr)
  62         if err != nil {
  63                 fmt.Fprintf(stderr, "%s\n", err)
  64                 return 1
  65         }
  66         return 0
  67 }
  68
  69 func (cmd *sliceNumpy) run(prog string, args []string, stdin io.Reader, stdout, stderr io.Writer) error {
  70         flags := flag.NewFlagSet("", flag.ContinueOnError)
  71         flags.SetOutput(stderr)
  72         pprof := flags.String("pprof", "", "serve Go profile data at http://`[addr]:port`")
  73         runlocal := flags.Bool("local", false, "run on local host (default: run in an arvados container)")
  74         arvadosRAM := flags.Int("arvados-ram", 750000000000, "amount of memory to request for arvados container (`bytes`)")
  75         arvadosVCPUs := flags.Int("arvados-vcpus", 96, "number of VCPUs to request for arvados container")
  76         projectUUID := flags.String("project", "", "project `UUID` for output data")
  77         priority := flags.Int("priority", 500, "container request priority")
  78         preemptible := flags.Bool("preemptible", true, "request preemptible instance")
  79         inputDir := flags.String("input-dir", "./in", "input `directory`")
  80         outputDir := flags.String("output-dir", "./out", "output `directory`")
  81         ref := flags.String("ref", "", "reference name (if blank, choose last one that appears in input)")
  82         regionsFilename := flags.String("regions", "", "only output columns/annotations that intersect regions in specified bed `file`")
  83         expandRegions := flags.Int("expand-regions", 0, "expand specified regions by `N` base pairs on each side`")
  84         mergeOutput := flags.Bool("merge-output", false, "merge output into one matrix.npy and one matrix.annotations.csv")
  85         hgvsSingle := flags.Bool("single-hgvs-matrix", false, "also generate hgvs-based matrix")
  86         hgvsChunked := flags.Bool("chunked-hgvs-matrix", false, "also generate hgvs-based matrix per chromosome")
  87         onehotSingle := flags.Bool("single-onehot", false, "generate one-hot tile-based matrix")
  88         onehotChunked := flags.Bool("chunked-onehot", false, "generate one-hot tile-based matrix per input chunk")
  89         samplesFilename := flags.String("samples", "", "`samples.csv` file with training/validation and case/control groups (see 'lightning choose-samples')")
  90         caseControlOnly := flags.Bool("case-control-only", false, "drop samples that are not in case/control groups")
  91         onlyPCA := flags.Bool("pca", false, "run principal component analysis, write components to pca.npy and samples.csv")
  92         flags.IntVar(&cmd.pcaComponents, "pca-components", 4, "number of PCA components to compute / use in logistic regression")
  93         maxPCATiles := flags.Int("max-pca-tiles", 0, "maximum tiles to use as PCA input (filter, then drop every 2nd colum pair until below max)")
  94         debugTag := flags.Int("debug-tag", -1, "log debugging details about specified tag")
  95         flags.IntVar(&cmd.threads, "threads", 16, "number of memory-hungry assembly threads, and number of VCPUs to request for arvados container")
  96         flags.Float64Var(&cmd.chi2PValue, "chi2-p-value", 1, "do Χ² test (or logistic regression if -samples file has PCA components) and omit columns with p-value above this threshold")
  97         flags.BoolVar(&cmd.includeVariant1, "include-variant-1", false, "include most common variant when building one-hot matrix")
  98         cmd.filter.Flags(flags)
  99         err := flags.Parse(args)
 100         if err == flag.ErrHelp {
 101                 return nil
 102         } else if err != nil {
 103                 return err
 104         } else if flags.NArg() > 0 {
 105                 return fmt.Errorf("errant command line arguments after parsed flags: %v", flags.Args())
 106         }
 107
 108         if *pprof != "" {
 109                 go func() {
 110                         log.Println(http.ListenAndServe(*pprof, nil))
 111                 }()
 112         }
 113
 114         if cmd.chi2PValue != 1 && *samplesFilename == "" {
 115                 return fmt.Errorf("cannot use provided -chi2-p-value=%f because -samples= value is empty", cmd.chi2PValue)
 116         }
 117
 118         cmd.debugTag = tagID(*debugTag)
 119
 120         if !*runlocal {
 121                 runner := arvadosContainerRunner{
 122                         Name:        "lightning slice-numpy",
 123                         Client:      arvados.NewClientFromEnv(),
 124                         ProjectUUID: *projectUUID,
 125                         RAM:         int64(*arvadosRAM),
 126                         VCPUs:       *arvadosVCPUs,
 127                         Priority:    *priority,
 128                         KeepCache:   2,
 129                         APIAccess:   true,
 130                         Preemptible: *preemptible,
 131                 }
 132                 err = runner.TranslatePaths(inputDir, regionsFilename, samplesFilename)
 133                 if err != nil {
 134                         return err
 135                 }
 136                 runner.Args = []string{"slice-numpy", "-local=true",
 137                         "-pprof=:6060",
 138                         "-input-dir=" + *inputDir,
 139                         "-output-dir=/mnt/output",
 140                         "-threads=" + fmt.Sprintf("%d", cmd.threads),
 141                         "-regions=" + *regionsFilename,
 142                         "-expand-regions=" + fmt.Sprintf("%d", *expandRegions),
 143                         "-merge-output=" + fmt.Sprintf("%v", *mergeOutput),
 144                         "-single-hgvs-matrix=" + fmt.Sprintf("%v", *hgvsSingle),
 145                         "-chunked-hgvs-matrix=" + fmt.Sprintf("%v", *hgvsChunked),
 146                         "-single-onehot=" + fmt.Sprintf("%v", *onehotSingle),
 147                         "-chunked-onehot=" + fmt.Sprintf("%v", *onehotChunked),
 148                         "-samples=" + *samplesFilename,
 149                         "-case-control-only=" + fmt.Sprintf("%v", *caseControlOnly),
 150                         "-pca=" + fmt.Sprintf("%v", *onlyPCA),
 151                         "-pca-components=" + fmt.Sprintf("%d", cmd.pcaComponents),
 152                         "-max-pca-tiles=" + fmt.Sprintf("%d", *maxPCATiles),
 153                         "-chi2-p-value=" + fmt.Sprintf("%f", cmd.chi2PValue),
 154                         "-include-variant-1=" + fmt.Sprintf("%v", cmd.includeVariant1),
 155                         "-debug-tag=" + fmt.Sprintf("%d", cmd.debugTag),
 156                 }
 157                 runner.Args = append(runner.Args, cmd.filter.Args()...)
 158                 var output string
 159                 output, err = runner.Run()
 160                 if err != nil {
 161                         return err
 162                 }
 163                 fmt.Fprintln(stdout, output)
 164                 return nil
 165         }
 166
 167         infiles, err := allFiles(*inputDir, matchGobFile)
 168         if err != nil {
 169                 return err
 170         }
 171         if len(infiles) == 0 {
 172                 err = fmt.Errorf("no input files found in %s", *inputDir)
 173                 return err
 174         }
 175         sort.Strings(infiles)
 176
 177         var refseq map[string][]tileLibRef
 178         var reftiledata = make(map[tileLibRef][]byte, 11000000)
 179         in0, err := open(infiles[0])
 180         if err != nil {
 181                 return err
 182         }
 183
 184         matchGenome, err := regexp.Compile(cmd.filter.MatchGenome)
 185         if err != nil {
 186                 err = fmt.Errorf("-match-genome: invalid regexp: %q", cmd.filter.MatchGenome)
 187                 return err
 188         }
 189
 190         if *samplesFilename != "" {
 191                 cmd.samples, err = loadSampleInfo(*samplesFilename)
 192                 if err != nil {
 193                         return err
 194                 }
 195                 if len(cmd.samples[0].pcaComponents) > 0 {
 196                         cmd.pvalue = glmPvalueFunc(cmd.samples, cmd.pcaComponents)
 197                         // Unfortunately, statsmodel/glm lib logs
 198                         // stuff to os.Stdout when it panics on an
 199                         // unsolvable problem. We recover() from the
 200                         // panic in glm.go, but we also need to
 201                         // commandeer os.Stdout to avoid producing
 202                         // large quantities of logs.
 203                         stdoutWas := os.Stdout
 204                         defer func() { os.Stdout = stdoutWas }()
 205                         os.Stdout, err = os.Open(os.DevNull)
 206                         if err != nil {
 207                                 return err
 208                         }
 209                 }
 210         } else if *caseControlOnly {
 211                 return fmt.Errorf("-case-control-only does not make sense without -samples")
 212         }
 213
 214         cmd.cgnames = nil
 215         var tagset [][]byte
 216         err = DecodeLibrary(in0, strings.HasSuffix(infiles[0], ".gz"), func(ent *LibraryEntry) error {
 217                 if len(ent.TagSet) > 0 {
 218                         tagset = ent.TagSet
 219                 }
 220                 for _, cseq := range ent.CompactSequences {
 221                         if cseq.Name == *ref || *ref == "" {
 222                                 refseq = cseq.TileSequences
 223                         }
 224                 }
 225                 for _, cg := range ent.CompactGenomes {
 226                         if matchGenome.MatchString(cg.Name) {
 227                                 cmd.cgnames = append(cmd.cgnames, cg.Name)
 228                         }
 229                 }
 230                 for _, tv := range ent.TileVariants {
 231                         if tv.Ref {
 232                                 reftiledata[tileLibRef{tv.Tag, tv.Variant}] = tv.Sequence
 233                         }
 234                 }
 235                 return nil
 236         })
 237         if err != nil {
 238                 return err
 239         }
 240         in0.Close()
 241         if refseq == nil {
 242                 err = fmt.Errorf("%s: reference sequence not found", infiles[0])
 243                 return err
 244         }
 245         if len(tagset) == 0 {
 246                 err = fmt.Errorf("tagset not found")
 247                 return err
 248         }
 249
 250         taglib := &tagLibrary{}
 251         err = taglib.setTags(tagset)
 252         if err != nil {
 253                 return err
 254         }
 255         taglen := taglib.TagLen()
 256         sort.Strings(cmd.cgnames)
 257
 258         if len(cmd.cgnames) == 0 {
 259                 return fmt.Errorf("fatal: 0 matching samples in library, nothing to do")
 260         }
 261         cmd.trainingSet = make([]int, len(cmd.cgnames))
 262         if *samplesFilename == "" {
 263                 cmd.trainingSetSize = len(cmd.cgnames)
 264                 for i, name := range cmd.cgnames {
 265                         cmd.samples = append(cmd.samples, sampleInfo{
 266                                 id:         trimFilenameForLabel(name),
 267                                 isTraining: true,
 268                         })
 269                         cmd.trainingSet[i] = i
 270                 }
 271         } else if len(cmd.cgnames) != len(cmd.samples) {
 272                 return fmt.Errorf("mismatched sample list: %d samples in library, %d in %s", len(cmd.cgnames), len(cmd.samples), *samplesFilename)
 273         } else {
 274                 for i, name := range cmd.cgnames {
 275                         if s := trimFilenameForLabel(name); s != cmd.samples[i].id {
 276                                 return fmt.Errorf("mismatched sample list: sample %d is %q in library, %q in %s", i, s, cmd.samples[i].id, *samplesFilename)
 277                         }
 278                 }
 279                 if *caseControlOnly {
 280                         for i := 0; i < len(cmd.samples); i++ {
 281                                 if !cmd.samples[i].isTraining && !cmd.samples[i].isValidation {
 282                                         if i+1 < len(cmd.samples) {
 283                                                 copy(cmd.samples[i:], cmd.samples[i+1:])
 284                                                 copy(cmd.cgnames[i:], cmd.cgnames[i+1:])
 285                                         }
 286                                         cmd.samples = cmd.samples[:len(cmd.samples)-1]
 287                                         cmd.cgnames = cmd.cgnames[:len(cmd.cgnames)-1]
 288                                         i--
 289                                 }
 290                         }
 291                 }
 292                 cmd.chi2Cases = nil
 293                 cmd.trainingSetSize = 0
 294                 for i := range cmd.cgnames {
 295                         if cmd.samples[i].isTraining {
 296                                 cmd.trainingSet[i] = cmd.trainingSetSize
 297                                 cmd.trainingSetSize++
 298                                 cmd.chi2Cases = append(cmd.chi2Cases, cmd.samples[i].isCase)
 299                         } else {
 300                                 cmd.trainingSet[i] = -1
 301                         }
 302                 }
 303                 if cmd.pvalue == nil {
 304                         cmd.pvalue = func(onehot []bool) float64 {
 305                                 return pvalue(onehot, cmd.chi2Cases)
 306                         }
 307                 }
 308         }
 309         if cmd.filter.MinCoverage == 1 {
 310                 // In the generic formula below, floating point
 311                 // arithmetic can effectively push the coverage
 312                 // threshold above 1.0, which is impossible/useless.
 313                 // 1.0 needs to mean exactly 100% coverage.
 314                 cmd.minCoverage = len(cmd.cgnames)
 315         } else {
 316                 cmd.minCoverage = int(math.Ceil(cmd.filter.MinCoverage * float64(len(cmd.cgnames))))
 317         }
 318
 319         {
 320                 samplesOutFilename := *outputDir + "/samples.csv"
 321                 log.Infof("writing sample metadata to %s", samplesOutFilename)
 322                 var f *os.File
 323                 f, err = os.Create(samplesOutFilename)
 324                 if err != nil {
 325                         return err
 326                 }
 327                 defer f.Close()
 328                 for i, si := range cmd.samples {
 329                         var cc, tv string
 330                         if si.isCase {
 331                                 cc = "1"
 332                         } else if si.isControl {
 333                                 cc = "0"
 334                         }
 335                         if si.isTraining {
 336                                 tv = "1"
 337                         } else {
 338                                 tv = "0"
 339                         }
 340                         _, err = fmt.Fprintf(f, "%d,%s,%s,%s\n", i, si.id, cc, tv)
 341                         if err != nil {
 342                                 err = fmt.Errorf("write %s: %w", samplesOutFilename, err)
 343                                 return err
 344                         }
 345                 }
 346                 err = f.Close()
 347                 if err != nil {
 348                         err = fmt.Errorf("close %s: %w", samplesOutFilename, err)
 349                         return err
 350                 }
 351                 log.Print("done")
 352         }
 353
 354         log.Info("indexing reference tiles")
 355         type reftileinfo struct {
 356                 variant  tileVariantID
 357                 seqname  string // chr1
 358                 pos      int    // distance from start of chromosome to starttag
 359                 tiledata []byte // acgtggcaa...
 360                 excluded bool   // true if excluded by regions file
 361                 nexttag  tagID  // tagID of following tile (-1 for last tag of chromosome)
 362         }
 363         isdup := map[tagID]bool{}
 364         reftile := map[tagID]*reftileinfo{}
 365         for seqname, cseq := range refseq {
 366                 pos := 0
 367                 lastreftag := tagID(-1)
 368                 for _, libref := range cseq {
 369                         if cmd.filter.MaxTag >= 0 && libref.Tag > tagID(cmd.filter.MaxTag) {
 370                                 continue
 371                         }
 372                         tiledata := reftiledata[libref]
 373                         if len(tiledata) == 0 {
 374                                 err = fmt.Errorf("missing tiledata for tag %d variant %d in %s in ref", libref.Tag, libref.Variant, seqname)
 375                                 return err
 376                         }
 377                         foundthistag := false
 378                         taglib.FindAll(tiledata[:len(tiledata)-1], func(tagid tagID, offset, _ int) {
 379                                 if !foundthistag && tagid == libref.Tag {
 380                                         foundthistag = true
 381                                         return
 382                                 }
 383                                 if dupref, ok := reftile[tagid]; ok {
 384                                         log.Printf("dropping reference tile %+v from %s @ %d, tag not unique, also found inside %+v from %s @ %d", tileLibRef{Tag: tagid, Variant: dupref.variant}, dupref.seqname, dupref.pos, libref, seqname, pos+offset+1)
 385                                         delete(reftile, tagid)
 386                                 } else {
 387                                         log.Printf("found tag %d at offset %d inside tile variant %+v on %s @ %d", tagid, offset, libref, seqname, pos+offset+1)
 388                                 }
 389                                 isdup[tagid] = true
 390                         })
 391                         if isdup[libref.Tag] {
 392                                 log.Printf("dropping reference tile %+v from %s @ %d, tag not unique", libref, seqname, pos)
 393                         } else if reftile[libref.Tag] != nil {
 394                                 log.Printf("dropping reference tile %+v from %s @ %d, tag not unique", tileLibRef{Tag: libref.Tag, Variant: reftile[libref.Tag].variant}, reftile[libref.Tag].seqname, reftile[libref.Tag].pos)
 395                                 delete(reftile, libref.Tag)
 396                                 log.Printf("dropping reference tile %+v from %s @ %d, tag not unique", libref, seqname, pos)
 397                                 isdup[libref.Tag] = true
 398                         } else {
 399                                 reftile[libref.Tag] = &reftileinfo{
 400                                         seqname:  seqname,
 401                                         variant:  libref.Variant,
 402                                         tiledata: tiledata,
 403                                         pos:      pos,
 404                                         nexttag:  -1,
 405                                 }
 406                                 if lastreftag >= 0 {
 407                                         reftile[lastreftag].nexttag = libref.Tag
 408                                 }
 409                                 lastreftag = libref.Tag
 410                         }
 411                         pos += len(tiledata) - taglen
 412                 }
 413                 log.Printf("... %s done, len %d", seqname, pos+taglen)
 414         }
 415
 416         var mask *mask
 417         if *regionsFilename != "" {
 418                 log.Printf("loading regions from %s", *regionsFilename)
 419                 mask, err = makeMask(*regionsFilename, *expandRegions)
 420                 if err != nil {
 421                         return err
 422                 }
 423                 log.Printf("before applying mask, len(reftile) == %d", len(reftile))
 424                 log.Printf("deleting reftile entries for regions outside %d intervals", mask.Len())
 425                 for _, rt := range reftile {
 426                         if !mask.Check(strings.TrimPrefix(rt.seqname, "chr"), rt.pos, rt.pos+len(rt.tiledata)) {
 427                                 rt.excluded = true
 428                         }
 429                 }
 430                 log.Printf("after applying mask, len(reftile) == %d", len(reftile))
 431         }
 432
 433         type hgvsColSet map[hgvs.Variant][2][]int8
 434         encodeHGVS := throttle{Max: len(refseq)}
 435         encodeHGVSTodo := map[string]chan hgvsColSet{}
 436         tmpHGVSCols := map[string]*os.File{}
 437         if *hgvsChunked {
 438                 for seqname := range refseq {
 439                         var f *os.File
 440                         f, err = os.Create(*outputDir + "/tmp." + seqname + ".gob")
 441                         if err != nil {
 442                                 return err
 443                         }
 444                         defer os.Remove(f.Name())
 445                         bufw := bufio.NewWriterSize(f, 1<<24)
 446                         enc := gob.NewEncoder(bufw)
 447                         tmpHGVSCols[seqname] = f
 448                         todo := make(chan hgvsColSet, 128)
 449                         encodeHGVSTodo[seqname] = todo
 450                         encodeHGVS.Go(func() error {
 451                                 for colset := range todo {
 452                                         err := enc.Encode(colset)
 453                                         if err != nil {
 454                                                 encodeHGVS.Report(err)
 455                                                 for range todo {
 456                                                 }
 457                                                 return err
 458                                         }
 459                                 }
 460                                 return bufw.Flush()
 461                         })
 462                 }
 463         }
 464
 465         var toMerge [][]int16
 466         if *mergeOutput || *hgvsSingle {
 467                 toMerge = make([][]int16, len(infiles))
 468         }
 469         var onehotIndirect [][2][]uint32 // [chunkIndex][axis][index]
 470         var onehotChunkSize []uint32
 471         var onehotXrefs [][]onehotXref
 472         if *onehotSingle || *onlyPCA {
 473                 onehotIndirect = make([][2][]uint32, len(infiles))
 474                 onehotChunkSize = make([]uint32, len(infiles))
 475                 onehotXrefs = make([][]onehotXref, len(infiles))
 476         }
 477         chunkStartTag := make([]tagID, len(infiles))
 478
 479         throttleMem := throttle{Max: cmd.threads} // TODO: estimate using mem and data size
 480         throttleNumpyMem := throttle{Max: cmd.threads/2 + 1}
 481         log.Info("generating annotations and numpy matrix for each slice")
 482         var errSkip = errors.New("skip infile")
 483         var done int64
 484         for infileIdx, infile := range infiles {
 485                 infileIdx, infile := infileIdx, infile
 486                 throttleMem.Go(func() error {
 487                         seq := make(map[tagID][]TileVariant, 50000)
 488                         cgs := make(map[string]CompactGenome, len(cmd.cgnames))
 489                         f, err := open(infile)
 490                         if err != nil {
 491                                 return err
 492                         }
 493                         defer f.Close()
 494                         log.Infof("%04d: reading %s", infileIdx, infile)
 495                         err = DecodeLibrary(f, strings.HasSuffix(infile, ".gz"), func(ent *LibraryEntry) error {
 496                                 for _, tv := range ent.TileVariants {
 497                                         if tv.Ref {
 498                                                 continue
 499                                         }
 500                                         // Skip tile with no
 501                                         // corresponding ref tile, if
 502                                         // mask is in play (we can't
 503                                         // determine coordinates for
 504                                         // these)
 505                                         if mask != nil && reftile[tv.Tag] == nil {
 506                                                 continue
 507                                         }
 508                                         // Skip tile whose
 509                                         // corresponding ref tile is
 510                                         // outside target regions --
 511                                         // unless it's a potential
 512                                         // spanning tile.
 513                                         if mask != nil && reftile[tv.Tag].excluded &&
 514                                                 (int(tv.Tag+1) >= len(tagset) ||
 515                                                         (bytes.HasSuffix(tv.Sequence, tagset[tv.Tag+1]) && reftile[tv.Tag+1] != nil && !reftile[tv.Tag+1].excluded)) {
 516                                                 continue
 517                                         }
 518                                         if tv.Tag == cmd.debugTag {
 519                                                 log.Printf("infile %d %s tag %d variant %d hash %x", infileIdx, infile, tv.Tag, tv.Variant, tv.Blake2b[:3])
 520                                         }
 521                                         variants := seq[tv.Tag]
 522                                         if len(variants) == 0 {
 523                                                 variants = make([]TileVariant, 100)
 524                                         }
 525                                         for len(variants) <= int(tv.Variant) {
 526                                                 variants = append(variants, TileVariant{})
 527                                         }
 528                                         variants[int(tv.Variant)] = tv
 529                                         seq[tv.Tag] = variants
 530                                 }
 531                                 for _, cg := range ent.CompactGenomes {
 532                                         if cmd.filter.MaxTag >= 0 && cg.StartTag > tagID(cmd.filter.MaxTag) {
 533                                                 return errSkip
 534                                         }
 535                                         if !matchGenome.MatchString(cg.Name) {
 536                                                 continue
 537                                         }
 538                                         // pad to full slice size
 539                                         // to avoid out-of-bounds
 540                                         // checks later
 541                                         if sliceSize := 2 * int(cg.EndTag-cg.StartTag); len(cg.Variants) < sliceSize {
 542                                                 cg.Variants = append(cg.Variants, make([]tileVariantID, sliceSize-len(cg.Variants))...)
 543                                         }
 544                                         cgs[cg.Name] = cg
 545                                 }
 546                                 return nil
 547                         })
 548                         if err == errSkip {
 549                                 return nil
 550                         } else if err != nil {
 551                                 return fmt.Errorf("%04d: DecodeLibrary(%s): err", infileIdx, infile)
 552                         }
 553                         tagstart := cgs[cmd.cgnames[0]].StartTag
 554                         tagend := cgs[cmd.cgnames[0]].EndTag
 555                         chunkStartTag[infileIdx] = tagstart
 556
 557                         // TODO: filters
 558
 559                         log.Infof("%04d: renumber/dedup variants for tags %d-%d", infileIdx, tagstart, tagend)
 560                         variantRemap := make([][]tileVariantID, tagend-tagstart)
 561                         throttleCPU := throttle{Max: runtime.GOMAXPROCS(0)}
 562                         for tag, variants := range seq {
 563                                 tag, variants := tag, variants
 564                                 throttleCPU.Go(func() error {
 565                                         alleleCoverage := 0
 566                                         count := make(map[[blake2b.Size256]byte]int, len(variants))
 567
 568                                         rt := reftile[tag]
 569                                         if rt != nil {
 570                                                 count[blake2b.Sum256(rt.tiledata)] = 0
 571                                         }
 572
 573                                         for cgname, cg := range cgs {
 574                                                 idx := int(tag-tagstart) * 2
 575                                                 for allele := 0; allele < 2; allele++ {
 576                                                         v := cg.Variants[idx+allele]
 577                                                         if v > 0 && len(variants[v].Sequence) > 0 {
 578                                                                 count[variants[v].Blake2b]++
 579                                                                 alleleCoverage++
 580                                                         }
 581                                                         if v > 0 && tag == cmd.debugTag {
 582                                                                 log.Printf("tag %d cg %s allele %d tv %d hash %x count is now %d", tag, cgname, allele, v, variants[v].Blake2b[:3], count[variants[v].Blake2b])
 583                                                         }
 584                                                 }
 585                                         }
 586                                         if alleleCoverage < cmd.minCoverage*2 {
 587                                                 idx := int(tag-tagstart) * 2
 588                                                 for _, cg := range cgs {
 589                                                         cg.Variants[idx] = 0
 590                                                         cg.Variants[idx+1] = 0
 591                                                 }
 592                                                 if tag == cmd.debugTag {
 593                                                         log.Printf("tag %d alleleCoverage %d < min %d, sample data wiped", tag, alleleCoverage, cmd.minCoverage*2)
 594                                                 }
 595                                                 return nil
 596                                         }
 597
 598                                         // hash[i] will be the hash of
 599                                         // the variant(s) that should
 600                                         // be at rank i (0-based).
 601                                         hash := make([][blake2b.Size256]byte, 0, len(count))
 602                                         for b := range count {
 603                                                 hash = append(hash, b)
 604                                         }
 605                                         sort.Slice(hash, func(i, j int) bool {
 606                                                 bi, bj := &hash[i], &hash[j]
 607                                                 if ci, cj := count[*bi], count[*bj]; ci != cj {
 608                                                         return ci > cj
 609                                                 } else {
 610                                                         return bytes.Compare((*bi)[:], (*bj)[:]) < 0
 611                                                 }
 612                                         })
 613                                         // rank[b] will be the 1-based
 614                                         // new variant number for
 615                                         // variants whose hash is b.
 616                                         rank := make(map[[blake2b.Size256]byte]tileVariantID, len(hash))
 617                                         for i, h := range hash {
 618                                                 rank[h] = tileVariantID(i + 1)
 619                                         }
 620                                         if tag == cmd.debugTag {
 621                                                 for h, r := range rank {
 622                                                         log.Printf("tag %d rank(%x) = %v", tag, h[:3], r)
 623                                                 }
 624                                         }
 625                                         // remap[v] will be the new
 626                                         // variant number for original
 627                                         // variant number v.
 628                                         remap := make([]tileVariantID, len(variants))
 629                                         for i, tv := range variants {
 630                                                 remap[i] = rank[tv.Blake2b]
 631                                         }
 632                                         if tag == cmd.debugTag {
 633                                                 for in, out := range remap {
 634                                                         if out > 0 {
 635                                                                 log.Printf("tag %d remap %d => %d", tag, in, out)
 636                                                         }
 637                                                 }
 638                                         }
 639                                         variantRemap[tag-tagstart] = remap
 640                                         if rt != nil {
 641                                                 refrank := rank[blake2b.Sum256(rt.tiledata)]
 642                                                 if tag == cmd.debugTag {
 643                                                         log.Printf("tag %d reftile variant %d => %d", tag, rt.variant, refrank)
 644                                                 }
 645                                                 rt.variant = refrank
 646                                         }
 647                                         return nil
 648                                 })
 649                         }
 650                         throttleCPU.Wait()
 651
 652                         var onehotChunk [][]int8
 653                         var onehotXref []onehotXref
 654
 655                         var annotationsFilename string
 656                         if *onlyPCA {
 657                                 annotationsFilename = "/dev/null"
 658                         } else {
 659                                 annotationsFilename = fmt.Sprintf("%s/matrix.%04d.annotations.csv", *outputDir, infileIdx)
 660                                 log.Infof("%04d: writing %s", infileIdx, annotationsFilename)
 661                         }
 662                         annof, err := os.Create(annotationsFilename)
 663                         if err != nil {
 664                                 return err
 665                         }
 666                         annow := bufio.NewWriterSize(annof, 1<<20)
 667                         outcol := 0
 668                         for tag := tagstart; tag < tagend; tag++ {
 669                                 rt := reftile[tag]
 670                                 if rt == nil && mask != nil {
 671                                         // With no ref tile, we don't
 672                                         // have coordinates to say
 673                                         // this is in the desired
 674                                         // regions -- so it's not.
 675                                         // TODO: handle ref spanning
 676                                         // tile case.
 677                                         continue
 678                                 }
 679                                 if rt != nil && rt.excluded {
 680                                         // TODO: don't skip yet --
 681                                         // first check for spanning
 682                                         // tile variants that
 683                                         // intersect non-excluded ref
 684                                         // tiles.
 685                                         continue
 686                                 }
 687                                 if cmd.filter.MaxTag >= 0 && tag > tagID(cmd.filter.MaxTag) {
 688                                         break
 689                                 }
 690                                 remap := variantRemap[tag-tagstart]
 691                                 if remap == nil {
 692                                         // was not assigned above,
 693                                         // because minCoverage
 694                                         outcol++
 695                                         continue
 696                                 }
 697                                 maxv := tileVariantID(0)
 698                                 for _, v := range remap {
 699                                         if maxv < v {
 700                                                 maxv = v
 701                                         }
 702                                 }
 703                                 if *onehotChunked || *onehotSingle || *onlyPCA {
 704                                         onehot, xrefs := cmd.tv2homhet(cgs, maxv, remap, tag, tagstart, seq)
 705                                         if tag == cmd.debugTag {
 706                                                 log.WithFields(logrus.Fields{
 707                                                         "onehot": onehot,
 708                                                         "xrefs":  xrefs,
 709                                                 }).Info("tv2homhet()")
 710                                         }
 711                                         onehotChunk = append(onehotChunk, onehot...)
 712                                         onehotXref = append(onehotXref, xrefs...)
 713                                 }
 714                                 if *onlyPCA {
 715                                         outcol++
 716                                         continue
 717                                 }
 718                                 if rt == nil {
 719                                         // Reference does not use any
 720                                         // variant of this tile
 721                                         //
 722                                         // TODO: diff against the
 723                                         // relevant portion of the
 724                                         // ref's spanning tile
 725                                         outcol++
 726                                         continue
 727                                 }
 728                                 fmt.Fprintf(annow, "%d,%d,%d,=,%s,%d,,,\n", tag, outcol, rt.variant, rt.seqname, rt.pos)
 729                                 variants := seq[tag]
 730                                 reftilestr := strings.ToUpper(string(rt.tiledata))
 731
 732                                 done := make([]bool, maxv+1)
 733                                 variantDiffs := make([][]hgvs.Variant, maxv+1)
 734                                 for v, tv := range variants {
 735                                         v := remap[v]
 736                                         if v == 0 || v == rt.variant || done[v] {
 737                                                 continue
 738                                         } else {
 739                                                 done[v] = true
 740                                         }
 741                                         if len(tv.Sequence) < taglen {
 742                                                 continue
 743                                         }
 744                                         // if reftilestr doesn't end
 745                                         // in the same tag as tv,
 746                                         // extend reftilestr with
 747                                         // following ref tiles until
 748                                         // it does (up to an arbitrary
 749                                         // sanity-check limit)
 750                                         reftilestr := reftilestr
 751                                         endtagstr := strings.ToUpper(string(tv.Sequence[len(tv.Sequence)-taglen:]))
 752                                         for i, rt := 0, rt; i < annotationMaxTileSpan && !strings.HasSuffix(reftilestr, endtagstr) && rt.nexttag >= 0; i++ {
 753                                                 rt = reftile[rt.nexttag]
 754                                                 if rt == nil {
 755                                                         break
 756                                                 }
 757                                                 reftilestr += strings.ToUpper(string(rt.tiledata[taglen:]))
 758                                         }
 759                                         if mask != nil && !mask.Check(strings.TrimPrefix(rt.seqname, "chr"), rt.pos, rt.pos+len(reftilestr)) {
 760                                                 continue
 761                                         }
 762                                         if !strings.HasSuffix(reftilestr, endtagstr) {
 763                                                 fmt.Fprintf(annow, "%d,%d,%d,,%s,%d,,,\n", tag, outcol, v, rt.seqname, rt.pos)
 764                                                 continue
 765                                         }
 766                                         if lendiff := len(reftilestr) - len(tv.Sequence); lendiff < -1000 || lendiff > 1000 {
 767                                                 fmt.Fprintf(annow, "%d,%d,%d,,%s,%d,,,\n", tag, outcol, v, rt.seqname, rt.pos)
 768                                                 continue
 769                                         }
 770                                         diffs, _ := hgvs.Diff(reftilestr, strings.ToUpper(string(tv.Sequence)), 0)
 771                                         for i := range diffs {
 772                                                 diffs[i].Position += rt.pos
 773                                         }
 774                                         for _, diff := range diffs {
 775                                                 fmt.Fprintf(annow, "%d,%d,%d,%s:g.%s,%s,%d,%s,%s,%s\n", tag, outcol, v, rt.seqname, diff.String(), rt.seqname, diff.Position, diff.Ref, diff.New, diff.Left)
 776                                         }
 777                                         if *hgvsChunked {
 778                                                 variantDiffs[v] = diffs
 779                                         }
 780                                 }
 781                                 if *hgvsChunked {
 782                                         // We can now determine, for each HGVS
 783                                         // variant (diff) in this reftile
 784                                         // region, whether a given genome
 785                                         // phase/allele (1) has the variant, (0) has
 786                                         // =ref or a different variant in that
 787                                         // position, or (-1) is lacking
 788                                         // coverage / couldn't be diffed.
 789                                         hgvsCol := hgvsColSet{}
 790                                         for _, diffs := range variantDiffs {
 791                                                 for _, diff := range diffs {
 792                                                         if _, ok := hgvsCol[diff]; ok {
 793                                                                 continue
 794                                                         }
 795                                                         hgvsCol[diff] = [2][]int8{
 796                                                                 make([]int8, len(cmd.cgnames)),
 797                                                                 make([]int8, len(cmd.cgnames)),
 798                                                         }
 799                                                 }
 800                                         }
 801                                         for row, name := range cmd.cgnames {
 802                                                 variants := cgs[name].Variants[(tag-tagstart)*2:]
 803                                                 for ph := 0; ph < 2; ph++ {
 804                                                         v := variants[ph]
 805                                                         if int(v) >= len(remap) {
 806                                                                 v = 0
 807                                                         } else {
 808                                                                 v = remap[v]
 809                                                         }
 810                                                         if v == rt.variant {
 811                                                                 // hgvsCol[*][ph][row] is already 0
 812                                                         } else if len(variantDiffs[v]) == 0 {
 813                                                                 // lacking coverage / couldn't be diffed
 814                                                                 for _, col := range hgvsCol {
 815                                                                         col[ph][row] = -1
 816                                                                 }
 817                                                         } else {
 818                                                                 for _, diff := range variantDiffs[v] {
 819                                                                         hgvsCol[diff][ph][row] = 1
 820                                                                 }
 821                                                         }
 822                                                 }
 823                                         }
 824                                         for diff, colpair := range hgvsCol {
 825                                                 allele2homhet(colpair)
 826                                                 if !cmd.filterHGVScolpair(colpair) {
 827                                                         delete(hgvsCol, diff)
 828                                                 }
 829                                         }
 830                                         if len(hgvsCol) > 0 {
 831                                                 encodeHGVSTodo[rt.seqname] <- hgvsCol
 832                                         }
 833                                 }
 834                                 outcol++
 835                         }
 836                         err = annow.Flush()
 837                         if err != nil {
 838                                 return err
 839                         }
 840                         err = annof.Close()
 841                         if err != nil {
 842                                 return err
 843                         }
 844
 845                         if *onehotChunked {
 846                                 // transpose onehotChunk[col][row] to numpy[row*ncols+col]
 847                                 rows := len(cmd.cgnames)
 848                                 cols := len(onehotChunk)
 849                                 log.Infof("%04d: preparing onehot numpy (rows=%d, cols=%d, mem=%d)", infileIdx, rows, cols, rows*cols)
 850                                 throttleNumpyMem.Acquire()
 851                                 out := onehotcols2int8(onehotChunk)
 852                                 fnm := fmt.Sprintf("%s/onehot.%04d.npy", *outputDir, infileIdx)
 853                                 err = writeNumpyInt8(fnm, out, rows, cols)
 854                                 if err != nil {
 855                                         return err
 856                                 }
 857                                 fnm = fmt.Sprintf("%s/onehot-columns.%04d.npy", *outputDir, infileIdx)
 858                                 err = writeNumpyInt32(fnm, onehotXref2int32(onehotXref), 4, len(onehotXref))
 859                                 if err != nil {
 860                                         return err
 861                                 }
 862                                 debug.FreeOSMemory()
 863                                 throttleNumpyMem.Release()
 864                         }
 865                         if *onehotSingle || *onlyPCA {
 866                                 onehotIndirect[infileIdx] = onehotChunk2Indirect(onehotChunk)
 867                                 onehotChunkSize[infileIdx] = uint32(len(onehotChunk))
 868                                 onehotXrefs[infileIdx] = onehotXref
 869                                 n := len(onehotIndirect[infileIdx][0])
 870                                 log.Infof("%04d: keeping onehot coordinates in memory (n=%d, mem=%d)", infileIdx, n, n*8*2)
 871                         }
 872                         if !(*onehotSingle || *onehotChunked || *onlyPCA) || *mergeOutput || *hgvsSingle {
 873                                 log.Infof("%04d: preparing numpy (rows=%d, cols=%d)", infileIdx, len(cmd.cgnames), 2*outcol)
 874                                 throttleNumpyMem.Acquire()
 875                                 rows := len(cmd.cgnames)
 876                                 cols := 2 * outcol
 877                                 out := make([]int16, rows*cols)
 878                                 for row, name := range cmd.cgnames {
 879                                         outidx := row * cols
 880                                         for col, v := range cgs[name].Variants {
 881                                                 tag := tagstart + tagID(col/2)
 882                                                 if cmd.filter.MaxTag >= 0 && tag > tagID(cmd.filter.MaxTag) {
 883                                                         break
 884                                                 }
 885                                                 if rt := reftile[tag]; rt == nil || rt.excluded {
 886                                                         continue
 887                                                 }
 888                                                 if v == 0 {
 889                                                         out[outidx] = 0 // tag not found / spanning tile
 890                                                 } else if variants, ok := seq[tag]; ok && int(v) < len(variants) && len(variants[v].Sequence) > 0 {
 891                                                         out[outidx] = int16(variantRemap[tag-tagstart][v])
 892                                                 } else {
 893                                                         out[outidx] = -1 // low quality tile variant
 894                                                 }
 895                                                 if tag == cmd.debugTag {
 896                                                         log.Printf("tag %d row %d col %d outidx %d v %d out %d", tag, row, col, outidx, v, out[outidx])
 897                                                 }
 898                                                 outidx++
 899                                         }
 900                                 }
 901                                 seq = nil
 902                                 cgs = nil
 903                                 debug.FreeOSMemory()
 904                                 throttleNumpyMem.Release()
 905                                 if *mergeOutput || *hgvsSingle {
 906                                         log.Infof("%04d: matrix fragment %d rows x %d cols", infileIdx, rows, cols)
 907                                         toMerge[infileIdx] = out
 908                                 }
 909                                 if !*mergeOutput && !*onehotChunked && !*onehotSingle {
 910                                         fnm := fmt.Sprintf("%s/matrix.%04d.npy", *outputDir, infileIdx)
 911                                         err = writeNumpyInt16(fnm, out, rows, cols)
 912                                         if err != nil {
 913                                                 return err
 914                                         }
 915                                 }
 916                         }
 917                         debug.FreeOSMemory()
 918                         log.Infof("%s: done (%d/%d)", infile, int(atomic.AddInt64(&done, 1)), len(infiles))
 919                         return nil
 920                 })
 921         }
 922         if err = throttleMem.Wait(); err != nil {
 923                 return err
 924         }
 925
 926         if *hgvsChunked {
 927                 log.Info("flushing hgvsCols temp files")
 928                 for seqname := range refseq {
 929                         close(encodeHGVSTodo[seqname])
 930                 }
 931                 err = encodeHGVS.Wait()
 932                 if err != nil {
 933                         return err
 934                 }
 935                 for seqname := range refseq {
 936                         log.Infof("%s: reading hgvsCols from temp file", seqname)
 937                         f := tmpHGVSCols[seqname]
 938                         _, err = f.Seek(0, io.SeekStart)
 939                         if err != nil {
 940                                 return err
 941                         }
 942                         var hgvsCols hgvsColSet
 943                         dec := gob.NewDecoder(bufio.NewReaderSize(f, 1<<24))
 944                         for err == nil {
 945                                 err = dec.Decode(&hgvsCols)
 946                         }
 947                         if err != io.EOF {
 948                                 return err
 949                         }
 950                         log.Infof("%s: sorting %d hgvs variants", seqname, len(hgvsCols))
 951                         variants := make([]hgvs.Variant, 0, len(hgvsCols))
 952                         for v := range hgvsCols {
 953                                 variants = append(variants, v)
 954                         }
 955                         sort.Slice(variants, func(i, j int) bool {
 956                                 vi, vj := &variants[i], &variants[j]
 957                                 if vi.Position != vj.Position {
 958                                         return vi.Position < vj.Position
 959                                 } else if vi.Ref != vj.Ref {
 960                                         return vi.Ref < vj.Ref
 961                                 } else {
 962                                         return vi.New < vj.New
 963                                 }
 964                         })
 965                         rows := len(cmd.cgnames)
 966                         cols := len(variants) * 2
 967                         log.Infof("%s: building hgvs matrix (rows=%d, cols=%d, mem=%d)", seqname, rows, cols, rows*cols)
 968                         out := make([]int8, rows*cols)
 969                         for varIdx, variant := range variants {
 970                                 hgvsCols := hgvsCols[variant]
 971                                 for row := range cmd.cgnames {
 972                                         for ph := 0; ph < 2; ph++ {
 973                                                 out[row*cols+varIdx+ph] = hgvsCols[ph][row]
 974                                         }
 975                                 }
 976                         }
 977                         err = writeNumpyInt8(fmt.Sprintf("%s/hgvs.%s.npy", *outputDir, seqname), out, rows, cols)
 978                         if err != nil {
 979                                 return err
 980                         }
 981                         out = nil
 982
 983                         fnm := fmt.Sprintf("%s/hgvs.%s.annotations.csv", *outputDir, seqname)
 984                         log.Infof("%s: writing hgvs column labels to %s", seqname, fnm)
 985                         var hgvsLabels bytes.Buffer
 986                         for varIdx, variant := range variants {
 987                                 fmt.Fprintf(&hgvsLabels, "%d,%s:g.%s\n", varIdx, seqname, variant.String())
 988                         }
 989                         err = ioutil.WriteFile(fnm, hgvsLabels.Bytes(), 0666)
 990                         if err != nil {
 991                                 return err
 992                         }
 993                 }
 994         }
 995
 996         if *mergeOutput || *hgvsSingle {
 997                 var annow *bufio.Writer
 998                 var annof *os.File
 999                 if *mergeOutput {
1000                         annoFilename := fmt.Sprintf("%s/matrix.annotations.csv", *outputDir)
1001                         annof, err = os.Create(annoFilename)
1002                         if err != nil {
1003                                 return err
1004                         }
1005                         annow = bufio.NewWriterSize(annof, 1<<20)
1006                 }
1007
1008                 rows := len(cmd.cgnames)
1009                 cols := 0
1010                 for _, chunk := range toMerge {
1011                         cols += len(chunk) / rows
1012                 }
1013                 log.Infof("merging output matrix (rows=%d, cols=%d, mem=%d) and annotations", rows, cols, rows*cols*2)
1014                 var out []int16
1015                 if *mergeOutput {
1016                         out = make([]int16, rows*cols)
1017                 }
1018                 hgvsCols := map[string][2][]int16{} // hgvs -> [[g0,g1,g2,...], [g0,g1,g2,...]] (slice of genomes for each phase)
1019                 startcol := 0
1020                 for outIdx, chunk := range toMerge {
1021                         chunkcols := len(chunk) / rows
1022                         if *mergeOutput {
1023                                 for row := 0; row < rows; row++ {
1024                                         copy(out[row*cols+startcol:], chunk[row*chunkcols:(row+1)*chunkcols])
1025                                 }
1026                         }
1027                         toMerge[outIdx] = nil
1028
1029                         annotationsFilename := fmt.Sprintf("%s/matrix.%04d.annotations.csv", *outputDir, outIdx)
1030                         log.Infof("reading %s", annotationsFilename)
1031                         buf, err := os.ReadFile(annotationsFilename)
1032                         if err != nil {
1033                                 return err
1034                         }
1035                         if *mergeOutput {
1036                                 err = os.Remove(annotationsFilename)
1037                                 if err != nil {
1038                                         return err
1039                                 }
1040                         }
1041                         for _, line := range bytes.Split(buf, []byte{'\n'}) {
1042                                 if len(line) == 0 {
1043                                         continue
1044                                 }
1045                                 fields := bytes.SplitN(line, []byte{','}, 9)
1046                                 tag, _ := strconv.Atoi(string(fields[0]))
1047                                 incol, _ := strconv.Atoi(string(fields[1]))
1048                                 tileVariant, _ := strconv.Atoi(string(fields[2]))
1049                                 hgvsID := string(fields[3])
1050                                 seqname := string(fields[4])
1051                                 pos, _ := strconv.Atoi(string(fields[5]))
1052                                 refseq := fields[6]
1053                                 if hgvsID == "" {
1054                                         // Null entry for un-diffable
1055                                         // tile variant
1056                                         continue
1057                                 }
1058                                 if hgvsID == "=" {
1059                                         // Null entry for ref tile
1060                                         continue
1061                                 }
1062                                 if mask != nil && !mask.Check(strings.TrimPrefix(seqname, "chr"), pos, pos+len(refseq)) {
1063                                         // The tile intersects one of
1064                                         // the selected regions, but
1065                                         // this particular HGVS
1066                                         // variant does not.
1067                                         continue
1068                                 }
1069                                 hgvsColPair := hgvsCols[hgvsID]
1070                                 if hgvsColPair[0] == nil {
1071                                         // values in new columns start
1072                                         // out as -1 ("no data yet")
1073                                         // or 0 ("=ref") here, may
1074                                         // change to 1 ("hgvs variant
1075                                         // present") below, either on
1076                                         // this line or a future line.
1077                                         hgvsColPair = [2][]int16{make([]int16, len(cmd.cgnames)), make([]int16, len(cmd.cgnames))}
1078                                         rt, ok := reftile[tagID(tag)]
1079                                         if !ok {
1080                                                 err = fmt.Errorf("bug: seeing annotations for tag %d, but it has no reftile entry", tag)
1081                                                 return err
1082                                         }
1083                                         for ph := 0; ph < 2; ph++ {
1084                                                 for row := 0; row < rows; row++ {
1085                                                         v := chunk[row*chunkcols+incol*2+ph]
1086                                                         if tileVariantID(v) == rt.variant {
1087                                                                 hgvsColPair[ph][row] = 0
1088                                                         } else {
1089                                                                 hgvsColPair[ph][row] = -1
1090                                                         }
1091                                                 }
1092                                         }
1093                                         hgvsCols[hgvsID] = hgvsColPair
1094                                         if annow != nil {
1095                                                 hgvsref := hgvs.Variant{
1096                                                         Position: pos,
1097                                                         Ref:      string(refseq),
1098                                                         New:      string(refseq),
1099                                                 }
1100                                                 fmt.Fprintf(annow, "%d,%d,%d,%s:g.%s,%s,%d,%s,%s,%s\n", tag, incol+startcol/2, rt.variant, seqname, hgvsref.String(), seqname, pos, refseq, refseq, fields[8])
1101                                         }
1102                                 }
1103                                 if annow != nil {
1104                                         fmt.Fprintf(annow, "%d,%d,%d,%s,%s,%d,%s,%s,%s\n", tag, incol+startcol/2, tileVariant, hgvsID, seqname, pos, refseq, fields[7], fields[8])
1105                                 }
1106                                 for ph := 0; ph < 2; ph++ {
1107                                         for row := 0; row < rows; row++ {
1108                                                 v := chunk[row*chunkcols+incol*2+ph]
1109                                                 if int(v) == tileVariant {
1110                                                         hgvsColPair[ph][row] = 1
1111                                                 }
1112                                         }
1113                                 }
1114                         }
1115
1116                         startcol += chunkcols
1117                 }
1118                 if *mergeOutput {
1119                         err = annow.Flush()
1120                         if err != nil {
1121                                 return err
1122                         }
1123                         err = annof.Close()
1124                         if err != nil {
1125                                 return err
1126                         }
1127                         err = writeNumpyInt16(fmt.Sprintf("%s/matrix.npy", *outputDir), out, rows, cols)
1128                         if err != nil {
1129                                 return err
1130                         }
1131                 }
1132                 out = nil
1133
1134                 if *hgvsSingle {
1135                         cols = len(hgvsCols) * 2
1136                         log.Printf("building hgvs-based matrix: %d rows x %d cols", rows, cols)
1137                         out = make([]int16, rows*cols)
1138                         hgvsIDs := make([]string, 0, cols/2)
1139                         for hgvsID := range hgvsCols {
1140                                 hgvsIDs = append(hgvsIDs, hgvsID)
1141                         }
1142                         sort.Strings(hgvsIDs)
1143                         var hgvsLabels bytes.Buffer
1144                         for idx, hgvsID := range hgvsIDs {
1145                                 fmt.Fprintf(&hgvsLabels, "%d,%s\n", idx, hgvsID)
1146                                 for ph := 0; ph < 2; ph++ {
1147                                         hgvscol := hgvsCols[hgvsID][ph]
1148                                         for row, val := range hgvscol {
1149                                                 out[row*cols+idx*2+ph] = val
1150                                         }
1151                                 }
1152                         }
1153                         err = writeNumpyInt16(fmt.Sprintf("%s/hgvs.npy", *outputDir), out, rows, cols)
1154                         if err != nil {
1155                                 return err
1156                         }
1157
1158                         fnm := fmt.Sprintf("%s/hgvs.annotations.csv", *outputDir)
1159                         log.Printf("writing hgvs labels: %s", fnm)
1160                         err = ioutil.WriteFile(fnm, hgvsLabels.Bytes(), 0777)
1161                         if err != nil {
1162                                 return err
1163                         }
1164                 }
1165         }
1166         if *onehotSingle || *onlyPCA {
1167                 nzCount := 0
1168                 for _, part := range onehotIndirect {
1169                         nzCount += len(part[0])
1170                 }
1171                 onehot := make([]uint32, nzCount*2) // [r,r,r,...,c,c,c,...]
1172                 var xrefs []onehotXref
1173                 chunkOffset := uint32(0)
1174                 outcol := 0
1175                 for i, part := range onehotIndirect {
1176                         for i := range part[1] {
1177                                 part[1][i] += chunkOffset
1178                         }
1179                         copy(onehot[outcol:], part[0])
1180                         copy(onehot[outcol+nzCount:], part[1])
1181                         xrefs = append(xrefs, onehotXrefs[i]...)
1182
1183                         outcol += len(part[0])
1184                         chunkOffset += onehotChunkSize[i]
1185
1186                         part[0] = nil
1187                         part[1] = nil
1188                         onehotXrefs[i] = nil
1189                         debug.FreeOSMemory()
1190                 }
1191                 if *onehotSingle {
1192                         fnm := fmt.Sprintf("%s/onehot.npy", *outputDir)
1193                         err = writeNumpyUint32(fnm, onehot, 2, nzCount)
1194                         if err != nil {
1195                                 return err
1196                         }
1197                         fnm = fmt.Sprintf("%s/onehot-columns.npy", *outputDir)
1198                         err = writeNumpyInt32(fnm, onehotXref2int32(xrefs), 5, len(xrefs))
1199                         if err != nil {
1200                                 return err
1201                         }
1202                         fnm = fmt.Sprintf("%s/stats.json", *outputDir)
1203                         j, err := json.Marshal(map[string]interface{}{
1204                                 "pvalueCallCount": cmd.pvalueCallCount,
1205                         })
1206                         if err != nil {
1207                                 return err
1208                         }
1209                         err = os.WriteFile(fnm, j, 0777)
1210                         if err != nil {
1211                                 return err
1212                         }
1213                 }
1214                 if *onlyPCA {
1215                         cols := 0
1216                         for _, c := range onehot[nzCount:] {
1217                                 if int(c) >= cols {
1218                                         cols = int(c) + 1
1219                                 }
1220                         }
1221                         if cols == 0 {
1222                                 return fmt.Errorf("cannot do PCA: one-hot matrix is empty")
1223                         }
1224                         log.Printf("have %d one-hot cols", cols)
1225                         stride := 1
1226                         for *maxPCATiles > 0 && cols > *maxPCATiles*2 {
1227                                 cols = (cols + 1) / 2
1228                                 stride = stride * 2
1229                         }
1230                         if cols%2 == 1 {
1231                                 // we work with pairs of columns
1232                                 cols++
1233                         }
1234                         log.Printf("creating full matrix (%d rows) and training matrix (%d rows) with %d cols, stride %d", len(cmd.cgnames), cmd.trainingSetSize, cols, stride)
1235                         mtxFull := mat.NewDense(len(cmd.cgnames), cols, nil)
1236                         mtxTrain := mat.NewDense(cmd.trainingSetSize, cols, nil)
1237                         for i, c := range onehot[nzCount:] {
1238                                 if int(c/2)%stride == 0 {
1239                                         outcol := int(c/2)/stride*2 + int(c)%2
1240                                         mtxFull.Set(int(onehot[i]), outcol, 1)
1241                                         if trainRow := cmd.trainingSet[int(onehot[i])]; trainRow >= 0 {
1242                                                 mtxTrain.Set(trainRow, outcol, 1)
1243                                         }
1244                                 }
1245                         }
1246                         log.Print("fitting")
1247                         transformer := nlp.NewPCA(cmd.pcaComponents)
1248                         transformer.Fit(mtxTrain.T())
1249                         log.Printf("transforming")
1250                         pca, err := transformer.Transform(mtxFull.T())
1251                         if err != nil {
1252                                 return err
1253                         }
1254                         pca = pca.T()
1255                         outrows, outcols := pca.Dims()
1256                         log.Printf("copying result to numpy output array: %d rows, %d cols", outrows, outcols)
1257                         out := make([]float64, outrows*outcols)
1258                         for i := 0; i < outrows; i++ {
1259                                 for j := 0; j < outcols; j++ {
1260                                         out[i*outcols+j] = pca.At(i, j)
1261                                 }
1262                         }
1263                         fnm := fmt.Sprintf("%s/pca.npy", *outputDir)
1264                         log.Printf("writing numpy: %s", fnm)
1265                         output, err := os.OpenFile(fnm, os.O_CREATE|os.O_TRUNC|os.O_WRONLY, 0777)
1266                         if err != nil {
1267                                 return err
1268                         }
1269                         npw, err := gonpy.NewWriter(nopCloser{output})
1270                         if err != nil {
1271                                 return fmt.Errorf("gonpy.NewWriter: %w", err)
1272                         }
1273                         npw.Shape = []int{outrows, outcols}
1274                         err = npw.WriteFloat64(out)
1275                         if err != nil {
1276                                 return fmt.Errorf("WriteFloat64: %w", err)
1277                         }
1278                         err = output.Close()
1279                         if err != nil {
1280                                 return err
1281                         }
1282                         log.Print("done")
1283
1284                         samplesOutFilename := *outputDir + "/samples.csv"
1285                         log.Infof("writing sample metadata to %s", samplesOutFilename)
1286                         var f *os.File
1287                         f, err = os.Create(samplesOutFilename)
1288                         if err != nil {
1289                                 return err
1290                         }
1291                         defer f.Close()
1292                         pcaLabels := ""
1293                         for i := 0; i < outcols; i++ {
1294                                 pcaLabels += fmt.Sprintf(",PCA%d", i)
1295                         }
1296                         _, err = fmt.Fprintf(f, "Index,SampleID,CaseControl,TrainingValidation%s\n", pcaLabels)
1297                         if err != nil {
1298                                 return err
1299                         }
1300                         for i, si := range cmd.samples {
1301                                 var cc, tv string
1302                                 if si.isCase {
1303                                         cc = "1"
1304                                 } else if si.isControl {
1305                                         cc = "0"
1306                                 }
1307                                 if si.isTraining {
1308                                         tv = "1"
1309                                 } else if si.isValidation {
1310                                         tv = "0"
1311                                 }
1312                                 var pcavals string
1313                                 for c := 0; c < outcols; c++ {
1314                                         pcavals += fmt.Sprintf(",%f", pca.At(i, c))
1315                                 }
1316                                 _, err = fmt.Fprintf(f, "%d,%s,%s,%s%s\n", i, si.id, cc, tv, pcavals)
1317                                 if err != nil {
1318                                         err = fmt.Errorf("write %s: %w", samplesOutFilename, err)
1319                                         return err
1320                                 }
1321                         }
1322                         err = f.Close()
1323                         if err != nil {
1324                                 err = fmt.Errorf("close %s: %w", samplesOutFilename, err)
1325                                 return err
1326                         }
1327                         log.Print("done")
1328                 }
1329         }
1330         if !*mergeOutput && !*onehotChunked && !*onehotSingle && !*onlyPCA {
1331                 tagoffsetFilename := *outputDir + "/chunk-tag-offset.csv"
1332                 log.Infof("writing tag offsets to %s", tagoffsetFilename)
1333                 var f *os.File
1334                 f, err = os.Create(tagoffsetFilename)
1335                 if err != nil {
1336                         return err
1337                 }
1338                 defer f.Close()
1339                 for idx, offset := range chunkStartTag {
1340                         _, err = fmt.Fprintf(f, "%q,%d\n", fmt.Sprintf("matrix.%04d.npy", idx), offset)
1341                         if err != nil {
1342                                 err = fmt.Errorf("write %s: %w", tagoffsetFilename, err)
1343                                 return err
1344                         }
1345                 }
1346                 err = f.Close()
1347                 if err != nil {
1348                         err = fmt.Errorf("close %s: %w", tagoffsetFilename, err)
1349                         return err
1350                 }
1351         }
1352
1353         return nil
1354 }
1355
1356 type sampleInfo struct {
1357         id            string
1358         isCase        bool
1359         isControl     bool
1360         isTraining    bool
1361         isValidation  bool
1362         pcaComponents []float64
1363 }
1364
1365 // Read samples.csv file with case/control and training/validation
1366 // flags.
1367 func loadSampleInfo(samplesFilename string) ([]sampleInfo, error) {
1368         var si []sampleInfo
1369         f, err := open(samplesFilename)
1370         if err != nil {
1371                 return nil, err
1372         }
1373         buf, err := io.ReadAll(f)
1374         f.Close()
1375         if err != nil {
1376                 return nil, err
1377         }
1378         lineNum := 0
1379         for _, csv := range bytes.Split(buf, []byte{'\n'}) {
1380                 lineNum++
1381                 if len(csv) == 0 {
1382                         continue
1383                 }
1384                 split := strings.Split(string(csv), ",")
1385                 if len(split) < 4 {
1386                         return nil, fmt.Errorf("%d fields < 4 in %s line %d: %q", len(split), samplesFilename, lineNum, csv)
1387                 }
1388                 if split[0] == "Index" && split[1] == "SampleID" && split[2] == "CaseControl" && split[3] == "TrainingValidation" {
1389                         continue
1390                 }
1391                 idx, err := strconv.Atoi(split[0])
1392                 if err != nil {
1393                         if lineNum == 1 {
1394                                 return nil, fmt.Errorf("header does not look right: %q", csv)
1395                         }
1396                         return nil, fmt.Errorf("%s line %d: index: %s", samplesFilename, lineNum, err)
1397                 }
1398                 if idx != len(si) {
1399                         return nil, fmt.Errorf("%s line %d: index %d out of order", samplesFilename, lineNum, idx)
1400                 }
1401                 var pcaComponents []float64
1402                 if len(split) > 4 {
1403                         for _, s := range split[4:] {
1404                                 f, err := strconv.ParseFloat(s, 64)
1405                                 if err != nil {
1406                                         return nil, fmt.Errorf("%s line %d: cannot parse float %q: %s", samplesFilename, lineNum, s, err)
1407                                 }
1408                                 pcaComponents = append(pcaComponents, f)
1409                         }
1410                 }
1411                 si = append(si, sampleInfo{
1412                         id:            split[1],
1413                         isCase:        split[2] == "1",
1414                         isControl:     split[2] == "0",
1415                         isTraining:    split[3] == "1",
1416                         isValidation:  split[3] == "0",
1417                         pcaComponents: pcaComponents,
1418                 })
1419         }
1420         return si, nil
1421 }
1422
1423 func (cmd *sliceNumpy) filterHGVScolpair(colpair [2][]int8) bool {
1424         if cmd.chi2PValue >= 1 {
1425                 return true
1426         }
1427         col0 := make([]bool, 0, len(cmd.chi2Cases))
1428         col1 := make([]bool, 0, len(cmd.chi2Cases))
1429         cases := make([]bool, 0, len(cmd.chi2Cases))
1430         for i, c := range cmd.chi2Cases {
1431                 if colpair[0][i] < 0 {
1432                         continue
1433                 }
1434                 col0 = append(col0, colpair[0][i] != 0)
1435                 col1 = append(col1, colpair[1][i] != 0)
1436                 cases = append(cases, c)
1437         }
1438         return len(cases) >= cmd.minCoverage &&
1439                 (pvalue(col0, cases) <= cmd.chi2PValue || pvalue(col1, cases) <= cmd.chi2PValue)
1440 }
1441
1442 func writeNumpyUint32(fnm string, out []uint32, rows, cols int) error {
1443         output, err := os.Create(fnm)
1444         if err != nil {
1445                 return err
1446         }
1447         defer output.Close()
1448         bufw := bufio.NewWriterSize(output, 1<<26)
1449         npw, err := gonpy.NewWriter(nopCloser{bufw})
1450         if err != nil {
1451                 return err
1452         }
1453         log.WithFields(log.Fields{
1454                 "filename": fnm,
1455                 "rows":     rows,
1456                 "cols":     cols,
1457                 "bytes":    rows * cols * 4,
1458         }).Infof("writing numpy: %s", fnm)
1459         npw.Shape = []int{rows, cols}
1460         npw.WriteUint32(out)
1461         err = bufw.Flush()
1462         if err != nil {
1463                 return err
1464         }
1465         return output.Close()
1466 }
1467
1468 func writeNumpyInt32(fnm string, out []int32, rows, cols int) error {
1469         output, err := os.Create(fnm)
1470         if err != nil {
1471                 return err
1472         }
1473         defer output.Close()
1474         bufw := bufio.NewWriterSize(output, 1<<26)
1475         npw, err := gonpy.NewWriter(nopCloser{bufw})
1476         if err != nil {
1477                 return err
1478         }
1479         log.WithFields(log.Fields{
1480                 "filename": fnm,
1481                 "rows":     rows,
1482                 "cols":     cols,
1483                 "bytes":    rows * cols * 4,
1484         }).Infof("writing numpy: %s", fnm)
1485         npw.Shape = []int{rows, cols}
1486         npw.WriteInt32(out)
1487         err = bufw.Flush()
1488         if err != nil {
1489                 return err
1490         }
1491         return output.Close()
1492 }
1493
1494 func writeNumpyInt16(fnm string, out []int16, rows, cols int) error {
1495         output, err := os.Create(fnm)
1496         if err != nil {
1497                 return err
1498         }
1499         defer output.Close()
1500         bufw := bufio.NewWriterSize(output, 1<<26)
1501         npw, err := gonpy.NewWriter(nopCloser{bufw})
1502         if err != nil {
1503                 return err
1504         }
1505         log.WithFields(log.Fields{
1506                 "filename": fnm,
1507                 "rows":     rows,
1508                 "cols":     cols,
1509                 "bytes":    rows * cols * 2,
1510         }).Infof("writing numpy: %s", fnm)
1511         npw.Shape = []int{rows, cols}
1512         npw.WriteInt16(out)
1513         err = bufw.Flush()
1514         if err != nil {
1515                 return err
1516         }
1517         return output.Close()
1518 }
1519
1520 func writeNumpyInt8(fnm string, out []int8, rows, cols int) error {
1521         output, err := os.Create(fnm)
1522         if err != nil {
1523                 return err
1524         }
1525         defer output.Close()
1526         bufw := bufio.NewWriterSize(output, 1<<26)
1527         npw, err := gonpy.NewWriter(nopCloser{bufw})
1528         if err != nil {
1529                 return err
1530         }
1531         log.WithFields(log.Fields{
1532                 "filename": fnm,
1533                 "rows":     rows,
1534                 "cols":     cols,
1535                 "bytes":    rows * cols,
1536         }).Infof("writing numpy: %s", fnm)
1537         npw.Shape = []int{rows, cols}
1538         npw.WriteInt8(out)
1539         err = bufw.Flush()
1540         if err != nil {
1541                 return err
1542         }
1543         return output.Close()
1544 }
1545
1546 func allele2homhet(colpair [2][]int8) {
1547         a, b := colpair[0], colpair[1]
1548         for i, av := range a {
1549                 bv := b[i]
1550                 if av < 0 || bv < 0 {
1551                         // no-call
1552                         a[i], b[i] = -1, -1
1553                 } else if av > 0 && bv > 0 {
1554                         // hom
1555                         a[i], b[i] = 1, 0
1556                 } else if av > 0 || bv > 0 {
1557                         // het
1558                         a[i], b[i] = 0, 1
1559                 } else {
1560                         // ref (or a different variant in same position)
1561                         // (this is a no-op) a[i], b[i] = 0, 0
1562                 }
1563         }
1564 }
1565
1566 type onehotXref struct {
1567         tag     tagID
1568         variant tileVariantID
1569         hom     bool
1570         pvalue  float64
1571 }
1572
1573 const onehotXrefSize = unsafe.Sizeof(onehotXref{})
1574
1575 // Build onehot matrix (m[tileVariantIndex][genome] == 0 or 1) for all
1576 // variants of a single tile/tag#.
1577 //
1578 // Return nil if no tile variant passes Χ² filter.
1579 func (cmd *sliceNumpy) tv2homhet(cgs map[string]CompactGenome, maxv tileVariantID, remap []tileVariantID, tag, chunkstarttag tagID, seq map[tagID][]TileVariant) ([][]int8, []onehotXref) {
1580         if tag == cmd.debugTag {
1581                 tv := make([]tileVariantID, len(cmd.cgnames)*2)
1582                 for i, name := range cmd.cgnames {
1583                         copy(tv[i*2:(i+1)*2], cgs[name].Variants[(tag-chunkstarttag)*2:])
1584                 }
1585                 log.WithFields(logrus.Fields{
1586                         "cgs[i].Variants[tag*2+j]": tv,
1587                         "maxv":                     maxv,
1588                         "remap":                    remap,
1589                         "tag":                      tag,
1590                         "chunkstarttag":            chunkstarttag,
1591                 }).Info("tv2homhet()")
1592         }
1593         if maxv < 1 || (maxv < 2 && !cmd.includeVariant1) {
1594                 // everyone has the most common variant (of the variants we don't drop)
1595                 return nil, nil
1596         }
1597         tagoffset := tag - chunkstarttag
1598         coverage := 0
1599         for _, cg := range cgs {
1600                 alleles := 0
1601                 for _, v := range cg.Variants[tagoffset*2 : tagoffset*2+2] {
1602                         if v > 0 && int(v) < len(seq[tag]) && len(seq[tag][v].Sequence) > 0 {
1603                                 alleles++
1604                         }
1605                 }
1606                 if alleles == 2 {
1607                         coverage++
1608                 }
1609         }
1610         if coverage < cmd.minCoverage {
1611                 return nil, nil
1612         }
1613         // "observed" array for p-value calculation (training set
1614         // only)
1615         obs := make([][]bool, (maxv+1)*2) // 2 slices (hom + het) for each variant#
1616         // one-hot output (all samples)
1617         outcols := make([][]int8, (maxv+1)*2)
1618         for i := range obs {
1619                 obs[i] = make([]bool, cmd.trainingSetSize)
1620                 outcols[i] = make([]int8, len(cmd.cgnames))
1621         }
1622         for cgid, name := range cmd.cgnames {
1623                 tsid := cmd.trainingSet[cgid]
1624                 cgvars := cgs[name].Variants[tagoffset*2:]
1625                 tv0, tv1 := remap[cgvars[0]], remap[cgvars[1]]
1626                 for v := tileVariantID(1); v <= maxv; v++ {
1627                         if tv0 == v && tv1 == v {
1628                                 if tsid >= 0 {
1629                                         obs[v*2][tsid] = true
1630                                 }
1631                                 outcols[v*2][cgid] = 1
1632                         } else if tv0 == v || tv1 == v {
1633                                 if tsid >= 0 {
1634                                         obs[v*2+1][tsid] = true
1635                                 }
1636                                 outcols[v*2+1][cgid] = 1
1637                         }
1638                 }
1639         }
1640         var onehot [][]int8
1641         var xref []onehotXref
1642         for col := 2; col < len(obs); col++ {
1643                 // col 0,1 correspond to tile variant 0, i.e.,
1644                 // no-call; col 2,3 correspond to the most common
1645                 // variant; so we (normally) start at col 4.
1646                 if col < 4 && !cmd.includeVariant1 {
1647                         continue
1648                 }
1649                 atomic.AddInt64(&cmd.pvalueCallCount, 1)
1650                 p := cmd.pvalue(obs[col])
1651                 if cmd.chi2PValue < 1 && !(p < cmd.chi2PValue) {
1652                         continue
1653                 }
1654                 onehot = append(onehot, outcols[col])
1655                 xref = append(xref, onehotXref{
1656                         tag:     tag,
1657                         variant: tileVariantID(col >> 1),
1658                         hom:     col&1 == 0,
1659                         pvalue:  p,
1660                 })
1661         }
1662         return onehot, xref
1663 }
1664
1665 // convert a []onehotXref with length N to a numpy-style []int32
1666 // matrix with N columns, one row per field of onehotXref struct.
1667 //
1668 // Hom/het row contains hom=0, het=1.
1669 //
1670 // P-value row contains 1000000x actual p-value.
1671 func onehotXref2int32(xrefs []onehotXref) []int32 {
1672         xcols := len(xrefs)
1673         xdata := make([]int32, 5*xcols)
1674         for i, xref := range xrefs {
1675                 xdata[i] = int32(xref.tag)
1676                 xdata[xcols+i] = int32(xref.variant)
1677                 if xref.hom {
1678                         xdata[xcols*2+i] = 1
1679                 }
1680                 xdata[xcols*3+i] = int32(xref.pvalue * 1000000)
1681                 xdata[xcols*4+i] = int32(-math.Log10(xref.pvalue) * 1000000)
1682         }
1683         return xdata
1684 }
1685
1686 // transpose onehot data from in[col][row] to numpy-style
1687 // out[row*cols+col].
1688 func onehotcols2int8(in [][]int8) []int8 {
1689         if len(in) == 0 {
1690                 return nil
1691         }
1692         cols := len(in)
1693         rows := len(in[0])
1694         out := make([]int8, rows*cols)
1695         for row := 0; row < rows; row++ {
1696                 outrow := out[row*cols:]
1697                 for col, incol := range in {
1698                         outrow[col] = incol[row]
1699                 }
1700         }
1701         return out
1702 }
1703
1704 // Return [2][]uint32{rowIndices, colIndices} indicating which
1705 // elements of matrixT[c][r] have non-zero values.
1706 func onehotChunk2Indirect(matrixT [][]int8) [2][]uint32 {
1707         var nz [2][]uint32
1708         for c, col := range matrixT {
1709                 for r, val := range col {
1710                         if val != 0 {
1711                                 nz[0] = append(nz[0], uint32(r))
1712                                 nz[1] = append(nz[1], uint32(c))
1713                         }
1714                 }
1715         }
1716         return nz
1717 }