10x-vdj-bcr-full-length.yaml

flags:
  - species
pipeline:
  - mitool-parse
  - mitool-refine-tags
  - mitool-consensus
  - align
  - refineTagsAndSort
  - extend
  - assemble
  - assembleContigs
  - assembleCells
  - exportClones
  - exportCloneGroups
mitool:
  parse:
    pattern: "^(CELL:N{16})(UMI:N{10})\\^(R2:*)"
    unstranded: false
    maxErrorBudget: 10.0
    trimmingQualityThreshold: null
    trimmingWindowSize: null
    replaceWildcards: true
    splitBySample: true
  refineTags:
    tagTypes:
      - Cell
      - Molecule
    whitelists:
      CELL: builtin:737K-august-2016
    parameters:
      correctionPower: 0.001
      backgroundSubstitutionRate: 0.001
      backgroundIndelRate: 1.0E-5
      minQuality: 12
      maxSubstitutions: 2
      maxIndels: 2
      maxTotalErrors: 3
      postFilter: null
  consensus:
    - parameters:
        aAssemblerParameters:
          bandWidth: 4
          scoring:
            type: linear
            alphabet: nucleotide
            subsMatrix: "simple(match = 5, mismatch = -4)"
            gapPenalty: -19
          minAlignmentScore: 499
          maxNormalizedAlignmentPenalty: 0.46
          trimMinimalSumQuality: 0
          trimReferenceRegion: true
          maxQuality: 45
        kMapParameters:
          k: 9
          useQ1Score: true
          coreSequenceKMers: 20
          coreSequenceRecordScore: 20
          neighbourhoodSize: 2
        positiveMinQualityShare: 0.29
        positiveMinIntersectionQuality: 379
        minAlignmentScoreForDiscardRecord: 0
        maxNormalizedAlignmentPenaltyForDiscardRecord: 0.0
        minRecordSharePerConsensus: 0.001
        minRecordsPerConsensus: 3
        useWeightsForRecordsPerConsensus: true
        minRecordWeightForUse: 0
        maxIterationsPerConsensus: 8
        maxConsensuses: 0
      groupingTagTypes:
        - Cell
        - Molecule
      payloadTagTypes:
        - Targets
      saveUnusedReads: true
      saveDiscardedReads: true
      inferMinRecordsPerConsensus: true
    - parameters:
        aAssemblerParameters:
          bandWidth: 4
          scoring:
            type: linear
            alphabet: nucleotide
            subsMatrix: "simple(match = 5, mismatch = -4)"
            gapPenalty: -19
          minAlignmentScore: 51
          maxNormalizedAlignmentPenalty: 0.42
          trimMinimalSumQuality: 0
          trimReferenceRegion: true
          maxQuality: 45
        kMapParameters:
          k: 9
          useQ1Score: true
          coreSequenceKMers: 20
          coreSequenceRecordScore: 20
          neighbourhoodSize: 2
        positiveMinQualityShare: 0.11
        positiveMinIntersectionQuality: 101
        minAlignmentScoreForDiscardRecord: 0
        maxNormalizedAlignmentPenaltyForDiscardRecord: 0.0
        minRecordSharePerConsensus: 0.3
        minRecordsPerConsensus: 3
        useWeightsForRecordsPerConsensus: true
        minRecordWeightForUse: 0
        maxIterationsPerConsensus: 8
        maxConsensuses: 0
      groupingTagTypes:
        - Cell
        - Molecule
      payloadTagTypes:
        - Targets
      saveUnusedReads: true
      saveDiscardedReads: true
      inferMinRecordsPerConsensus: true
    - parameters:
        aAssemblerParameters:
          bandWidth: 4
          scoring:
            type: linear
            alphabet: nucleotide
            subsMatrix: "simple(match = 5, mismatch = -4)"
            gapPenalty: -19
          minAlignmentScore: 41
          maxNormalizedAlignmentPenalty: 0.35
          trimMinimalSumQuality: 0
          trimReferenceRegion: true
          maxQuality: 45
        kMapParameters:
          k: 9
          useQ1Score: true
          coreSequenceKMers: 20
          coreSequenceRecordScore: 20
          neighbourhoodSize: 2
        positiveMinQualityShare: 0.77
        positiveMinIntersectionQuality: 130
        minAlignmentScoreForDiscardRecord: 0
        maxNormalizedAlignmentPenaltyForDiscardRecord: 0.0
        minRecordSharePerConsensus: 0.001
        minRecordsPerConsensus: 3
        useWeightsForRecordsPerConsensus: false
        minRecordWeightForUse: 0
        maxIterationsPerConsensus: 8
        maxConsensuses: 0
      groupingTagTypes:
        - Cell
      payloadTagTypes:
        - Targets
      saveUnusedReads: true
      saveDiscardedReads: true
      inferMinRecordsPerConsensus: true
    - parameters:
        aAssemblerParameters:
          bandWidth: 4
          scoring:
            type: linear
            alphabet: nucleotide
            subsMatrix: "simple(match = 5, mismatch = -4)"
            gapPenalty: -19
          minAlignmentScore: 181
          maxNormalizedAlignmentPenalty: 0.59
          trimMinimalSumQuality: 0
          trimReferenceRegion: true
          maxQuality: 45
        kMapParameters:
          k: 9
          useQ1Score: true
          coreSequenceKMers: 20
          coreSequenceRecordScore: 20
          neighbourhoodSize: 2
        positiveMinQualityShare: 0.51
        positiveMinIntersectionQuality: 51
        minAlignmentScoreForDiscardRecord: 0
        maxNormalizedAlignmentPenaltyForDiscardRecord: 0.0
        minRecordSharePerConsensus: 0.001
        minRecordsPerConsensus: 3
        useWeightsForRecordsPerConsensus: false
        minRecordWeightForUse: 2
        maxIterationsPerConsensus: 8
        maxConsensuses: 0
      groupingTagTypes:
        - Cell
      payloadTagTypes:
        - Targets
      saveUnusedReads: true
      saveDiscardedReads: true
      inferMinRecordsPerConsensus: true
align:
  species: ""
  libraryName: default
  trimmingQualityThreshold: 0
  trimmingWindowSize: 6
  chains: ALL
  replaceWildcards: true
  overlapPairedReads: true
  bamDropNonVDJ: false
  writeFailedAlignments: false
  tagPattern: "^(CELL:N{16})(UMI:N{10})\\^(R2:*)"
  tagUnstranded: false
  tagMaxBudget: 10.0
  headerExtractors: []
  readIdAsCellTag: false
  sampleTable: null
  tagsValidations:
    - type: MustContainTagType
      tagType: Cell
    - type: MustContainTagType
      tagType: Molecule
  splitBySample: true
  limit: null
  parameters:
    vParameters:
      geneFeatureToAlign: VTranscriptWithP
      minSumScore: 60
      relativeMinScore: 0.7
      maxHits: 5
      edgeRealignmentMinScoreOverride: 35
      parameters:
        type: kaligner2
        mapperNValue: 9
        mapperKValue: 1
        floatingLeftBound: false
        floatingRightBound: true
        mapperAbsoluteMinClusterScore: 250
        mapperExtraClusterScore: -38
        mapperMatchScore: 127
        mapperMismatchScore: -14
        mapperOffsetShiftScore: -82
        mapperSlotCount: 6
        mapperMaxClusters: 15
        mapperMaxClusterIndels: 4
        mapperKMersPerPosition: 4
        mapperAbsoluteMinScore: 250
        mapperRelativeMinScore: 0.7
        mapperMinSeedsDistance: 6
        mapperMaxSeedsDistance: 6
        alignmentStopPenalty: 0
        absoluteMinScore: 150
        relativeMinScore: 0.7
        maxHits: 3
        scoring:
          type: affine
          alphabet: nucleotide
          subsMatrix: "simple(match = 10, mismatch = -19)"
          gapOpenPenalty: -62
          gapExtensionPenalty: -11
      rightForceRealignmentTrigger: CDR3Begin(-6)
    dParameters:
      geneFeatureToAlign: DRegionWithP
      relativeMinScore: 0.85
      absoluteMinScore: 25.0
      maxHits: 3
      scoring:
        type: linear
        alphabet: nucleotide
        subsMatrix: "simple(match = 5, mismatch = -9)"
        gapPenalty: -12
    jParameters:
      geneFeatureToAlign: JRegionWithP
      minSumScore: 150
      relativeMinScore: 0.97
      maxHits: 5
      parameters:
        type: kaligner2
        mapperNValue: 8
        mapperKValue: 1
        floatingLeftBound: true
        floatingRightBound: false
        mapperAbsoluteMinClusterScore: 102
        mapperExtraClusterScore: -38
        mapperMatchScore: 95
        mapperMismatchScore: -14
        mapperOffsetShiftScore: -82
        mapperSlotCount: 6
        mapperMaxClusters: 4
        mapperMaxClusterIndels: 4
        mapperKMersPerPosition: 4
        mapperAbsoluteMinScore: 100
        mapperRelativeMinScore: 0.8
        mapperMinSeedsDistance: 5
        mapperMaxSeedsDistance: 5
        alignmentStopPenalty: 0
        absoluteMinScore: 150
        relativeMinScore: 0.8
        maxHits: 3
        scoring:
          type: affine
          alphabet: nucleotide
          subsMatrix: "simple(match = 10, mismatch = -19)"
          gapOpenPenalty: -62
          gapExtensionPenalty: -11
    cParameters:
      geneFeatureToAlign: CExon1
      minSumScore: 40
      relativeMinScore: 0.97
      maxHits: 5
      parameters:
        type: kaligner
        mapperKValue: 5
        floatingLeftBound: false
        floatingRightBound: false
        mapperAbsoluteMinScore: 95.0
        mapperRelativeMinScore: 0.63
        mapperMatchScore: 128.0
        mapperMismatchPenalty: -0.1
        mapperOffsetShiftPenalty: -0.3
        mapperMinSeedsDistance: 8
        mapperMaxSeedsDistance: 18
        minAlignmentLength: 15
        maxAdjacentIndels: 2
        alignmentStopPenalty: -1000
        absoluteMinScore: 40.0
        relativeMinScore: 0.87
        maxHits: 4
        scoring:
          type: linear
          alphabet: nucleotide
          subsMatrix: "simple(match = 5, mismatch = -9)"
          gapPenalty: -12
    vjAlignmentOrder: VThenJ
    libraryStructure: Unknown
    includeDScore: false
    includeCScore: false
    minSumScore: 120.0
    relativeMinVFR3CDR3Score: 0.7
    allowPartialAlignments: true
    allowNoCDR3PartAlignments: true
    allowChimeras: false
    readsLayout: ReverseOnly
    mergerParameters:
      qualityMergingAlgorithm: MaxSubtraction
      partsLayout: null
      minimalOverlap: 13
      minimalMatchQualitySum: 364
      maxQuality: 50
      minimalIdentity: 0.7
      identityType: MinimalQualityWeighted
    fixSeed: true
    alignmentBoundaryTolerance: 5
    minChimeraDetectionScore: 120
    vjOverlapWindow: 3
    saveOriginalSequence: false
    saveOriginalReads: false
    smartForceEdgeAlignments: true
    retriesForPartialAlignments: 3
    retriesAlignmentOfNotCoveredPart: 2
refineTagsAndSort:
  whitelists: {}
  runCorrection: true
  dontCorrectTagsTypes:
    - Cell
    - Molecule
  parameters:
    correctionPower: 0.001
    backgroundSubstitutionRate: 0.001
    backgroundIndelRate: 1.0E-5
    minQuality: 12
    maxSubstitutions: 2
    maxIndels: 2
    maxTotalErrors: 3
    postFilter:
      type: filter_and
      operands:
        - type: filter_groups
          groupingKeys:
            - allTags:Cell
          predicates:
            - metrics:
                - type: group_metric_nunique
                  reportHist:
                    log: true
                    binNumber: 0
                    minBinWidth: 0.2
                    multiplyWeightByKey: false
                  keys:
                    - allTags:Molecule
              operator:
                type: group_operator_advanced_thresholding
                algo:
                  type: otsu
                histTransformers:
                  - type: inflate
                    fraction: 0.15
                    value: 1.0
                logX: true
                minimalSample: 20
                fallbackThreshold: 1.0
                accept: High
              expectedSorting:
                - allTags:Molecule
              requiredSequences: []
          expectedSorting:
            - allTags:Cell
            - allTags:Molecule
          requiredSequences: []
        - type: filter_groups
          groupingKeys:
            - allTags:Cell
            - allTags:Molecule
          predicates:
            - metrics:
                - type: group_metric_kmer_diversity
                  sequences:
                    - targets
                  k: 12
                  minimalQuality: 20
                  topShare: 0.5
                  minSequenceCount: 4
                  reportHist:
                    log: false
                    binNumber: 0
                    minBinWidth: 6.0
                    multiplyWeightByKey: false
              operator:
                type: group_operator_range
                lower: 150.0
                acceptNaN: true
              expectedSorting: []
              requiredSequences:
                - targets
          expectedSorting:
            - allTags:Cell
            - allTags:Molecule
          requiredSequences:
            - targets
      expectedSorting:
        - allTags:Cell
        - allTags:Molecule
      requiredSequences:
        - targets
assemblePartial:
  overlappedOnly: false
  dropPartial: false
  cellLevel: false
  parameters:
    kValue: 12
    kOffset: -7
    minimalAssembleOverlap: 12
    minimalNOverlap: 7
    minimalNOverlapShare: 0.65
    minimalAlignmentMergeIdentity: 0.85
    mergerParameters:
      qualityMergingAlgorithm: SumSubtraction
      partsLayout: CollinearDirect
      minimalOverlap: 20
      minimalMatchQualitySum: 0
      maxQuality: 45
      minimalIdentity: 0.95
      identityType: Unweighted
    maxLeftParts: 256000
    maxLeftMatches: 6144
extend:
  vAnchor: CDR3Begin
  jAnchor: FR4Begin
  minimalVScore: 50
  minimalJScore: 50
assemble:
  sortBySequence: false
  clnaOutput: true
  cellLevel: true
  consensusAssemblerParameters:
    assembler:
      aAssemblerParameters:
        bandWidth: 4
        scoring:
          type: linear
          alphabet: nucleotide
          subsMatrix: "simple(match = 5, mismatch = -4)"
          gapPenalty: -14
        minAlignmentScore: 40
        maxNormalizedAlignmentPenalty: 0.2
        trimMinimalSumQuality: 0
        trimReferenceRegion: false
        maxQuality: 45
      maxIterations: 6
      minAltSeedQualityScore: 11
      minAltSeedNormalizedPenalty: 0.35
      altSeedPenaltyTolerance: 0.5
      minRecordSharePerConsensus: 0.02
      minRecordsPerConsensus: 0
      minRecursiveRecordShare: 0.1
      minQualityScore: 0
      maxConsensuses: 2
    minTagSuffixShare: 0.0
    isolateChains: true
    empiricalAlignmentAssignment:
      uniqGeneOccurrenceWithinTag:
        - V
        - J
      uniqGeneOccurrenceWithinCluster:
        - V
        - J
      matchAlignmentsWithoutVJHitsByTags: true
  cloneAssemblerParameters:
    assemblingFeatures:
      - CDR3
    minimalClonalSequenceLength: 12
    qualityAggregationType: BetaScore
    cloneClusteringParameters:
      searchDepth: 2
      allowedMutationsInNRegions: 1
      searchParameters: twoMismatchesOrIndels
      clusteringFilter:
        type: advanced
        correctionPower: 0.001
        backgroundSubstitutionRate: 5.0E-4
        backgroundIndelRate: 2.0E-4
    cloneFactoryParameters:
      vParameters:
        relativeMinScore: 0.8
        maxAlignmentWidthLinear: 5
        maxAlignmentWidthAffine: 500
      jParameters:
        maxAlignmentWidthLinear: 5
        maxAlignmentWidthAffine: 500
      cParameters:
        maxAlignmentWidthLinear: 5
        maxAlignmentWidthAffine: 500
      dParameters:
        relativeMinScore: null
        absoluteMinScore: null
        maxHits: null
        scoring: null
    separateByV: false
    separateByJ: false
    separateByC: false
    maximalPreClusteringRatio: 1.0
    preClusteringScoreFilteringRatio: 2.0
    preClusteringCountFilteringRatio: 2.0
    addReadsCountOnClustering: false
    badQualityThreshold: 0
    maxBadPointsPercent: 0.7
    mappingThreshold: 2of5
    minimalQuality: 0
    postFilters:
      - type: filter_in_groups
        isolationKeys:
          - clone
        nestedFilter:
          type: filter_groups
          groupingKeys:
            - allTags:Cell
          predicates:
            - metrics:
                - type: group_metric_sum_weight
                  reportHist:
                    log: true
                    binNumber: 0
                    minBinWidth: 0.1
                    multiplyWeightByKey: false
              operator:
                type: group_operator_cumtop
                share: 0.99
                accept: High
              expectedSorting: []
              requiredSequences: []
          expectedSorting:
            - allTags:Cell
          requiredSequences: []
        aggregateReport: true
        expectedSorting:
          - clone
          - allTags:Cell
  inferMinRecordsPerConsensus: false
assembleContigs:
  ignoreTags: false
  parameters:
    branchingMinimalQualityShare: 0.1
    branchingMinimalSumQuality: 60
    decisiveBranchingSumQualityThreshold: 120
    alignedSequenceEdgeDelta: 3
    alignmentEdgeRegionSize: 7
    minimalNonEdgePointsFraction: 0.25
    minimalMeanNormalizedQuality: 5.0
    outputMinimalQualityShare: 0.75
    outputMinimalSumQuality: 0
    subCloningRegions:
      - VDJRegion
    assemblingRegions:
      - VDJRegion
    postFiltering:
      type: OnlyUnambiguouslyCovering
      geneFeatures:
        - VDJRegion
    trimmingParameters:
      averageQualityThreshold: 10.0
      windowSize: 8
    minimalContigLength: 20
    alignedRegionsOnly: true
    discardAmbiguousNucleotideCalls: false
    useOnlyFullAlignments:
      cumtop: 0.95
      fallbackToTopAlignment: true
  cloneFactoryParameters:
    vParameters:
      relativeMinScore: 0.97
      maxAlignmentWidthLinear: 5
      maxAlignmentWidthAffine: 500
    jParameters:
      maxAlignmentWidthLinear: 5
      maxAlignmentWidthAffine: 500
    cParameters:
      maxAlignmentWidthLinear: 5
      maxAlignmentWidthAffine: 500
    dParameters:
      relativeMinScore: null
      absoluteMinScore: null
      maxHits: null
      scoring: null
assembleCells:
  algorithm:
    type: SingleCellGroupingByOverlappingCellIds
    minOverlapForSmaller:
      percentage: 0.8
      roundingMode: UP
    minOverlapForBigger:
      percentage: 0.2
      roundingMode: UP
    countCellBarcodesWithOneClone: true
    thresholdForAssigningLeftoverCells:
      percentage: 0.6
      roundingMode: DOWN
    nonFunctional:
      type: OverlapWithFoundGroups
      minOverlapForClone:
        percentage: 0.9
        roundingMode: UP
      minOverlapForGroup:
        percentage: 0.5
        roundingMode: UP
exportAlignments:
  chains: ALL
  noHeader: false
  fields:
    - field: -readIds
    - field: -tags
      args:
        - Cell
    - field: -tags
      args:
        - Molecule
    - field: -targetSequences
    - field: -targetQualities
    - field: -vHitsWithScore
    - field: -dHitsWithScore
    - field: -jHitsWithScore
    - field: -cHitsWithScore
    - field: -vAlignments
    - field: -dAlignments
    - field: -jAlignments
    - field: -cAlignments
    - field: -allNFeaturesWithMinQuality
    - field: -allAAFeatures
    - field: -defaultAnchorPoints
    - field: -topChains
exportClones:
  splitByTagType: null
  filterOutOfFrames: false
  filterStops: false
  chains: ALL
  noHeader: false
  fields:
    - field: -cloneId
    - field: -cellGroup
    - field: -tags
      args:
        - Cell
    - field: -readCount
    - field: -readFraction
    - field: -uniqueTagCount
      args:
        - Molecule
    - field: -uniqueTagFraction
      args:
        - Molecule
    - field: -targetSequences
    - field: -targetQualities
    - field: -vHit
    - field: -dHit
    - field: -jHit
    - field: -cHit
    - field: -vHitsWithScore
    - field: -dHitsWithScore
    - field: -jHitsWithScore
    - field: -cHitsWithScore
    - field: -vAlignments
    - field: -dAlignments
    - field: -jAlignments
    - field: -cAlignments
    - field: -allNFeaturesWithMinQuality
    - field: -allAAFeatures
    - field: -defaultAnchorPoints
    - field: -topChains
  splitFilesBy: []
  groupClonesBy: []
exportCloneGroups:
  filterOutOfFrames: false
  filterStops: false
  filterOutGroupsWithOneClone: false
  showSecondaryChains: true
  types: []
  noHeader: false
  fields:
    - field: -cellGroup
    - field: -groupReadCount
    - field: -groupUniqueTagCount
      args:
        - Cell
    - field: -groupUniqueTagCount
      args:
        - Molecule
    - field: -uniqClonesPerChainCount
    - field: -readCount
    - field: -uniqueTagCount
      args:
        - Molecule
    - field: -vHit
    - field: -dHit
    - field: -jHit
    - field: -cHit
    - field: -nFeature
      args:
        - CDR3
    - field: -aaFeature
      args:
        - CDR3
  sortChainsBy: Auto
qc:
  checks:
    - type: SuccessfullyAlignedReads
      upper: 0.6
      middle: 0.4
      label: Successfully aligned reads
    - type: UnassignedAlignments
      upper: 0.4
      middle: 0.2
      label: Unassigned alignments in clonotype assembly
    - type: AlignmentsDroppedLowQuality
      upper: 0.4
      middle: 0.2
      label: Alignments dropped due to low sequence quality
    - type: ReadsDroppedInTagRefinement
      upper: 0.5
      middle: 0.3
      label: Reads dropped in tags error correction and filtering
    - type: TagArtificialDiversityEliminated
      tag: CELL
      upper: 0.9
      middle: 0.8
      label: CELLs artificial diversity eliminated
    - type: ReadsDroppedInTagFiltering
      upper: 0.4
      middle: 0.2
      label: Reads dropped in tags filtering
    - type: CellBarcodesWithFoundGroups
      upper: 0.75
      middle: 0.6
      label: Cell barcodes used in result groups