Workflow: BD Rhapsody™ WTA Analysis Pipeline

Fetched 2024-10-17 18:30:29 GMT

The BD Rhapsody™ WTA Analysis Pipeline is used to create sequencing libraries from single cell transcriptomes without having to specify a targeted panel. After sequencing, the analysis pipeline takes the FASTQ files, a reference genome file and a transcriptome annotation file for gene alignment. The pipeline generates molecular counts per cell, read counts per cell, metrics, and an alignment file.

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Inputs

ID Type Title Doc
Reads File[] Reads
Run_Name String (Optional) Run Name

This is a name for output files, for example Experiment1_Metrics_Summary.csv. Default if left empty is to name run based on a library. Any non-alpha numeric characters will be changed to a hyphen.

Subsample Float (Optional) Subsample Reads

Any number of reads >1 or a fraction between 0 < n < 1 to indicate the percentage of reads to subsample.

Tag_Names String[] (Optional) Sample Tag Names

Specify the Sample Tag number followed by - (hyphen) and a sample name to appear in the output files. For example: 4-Ramos. Should be alpha numeric, with + - and _ allowed. Any special characters: &, (), [], {}, <>, ?, | will be corrected to underscores.

VDJ_Version https://w3id.org/cwl/view/git/46d4472af75afacfb13ed92d149a4decd015cf43/workflow1_11.cwl#main/VDJ_Version/VDJ_Version (Optional) VDJ Species Version

The VDJ species and chain types. This option should only be set for VDJ experiment.

Subsample_seed Integer (Optional) Subsample Seed

For use when replicating a previous subsampling run only. Obtain the seed generated from the log file for the SplitFastQ node.

AbSeq_Reference File[] (Optional) AbSeq Reference
Basic_Algo_Only Boolean (Optional) Disable Refined Putative Cell Calling

Determine putative cells using only the basic algorithm (minimum second derivative along the cumulative reads curve). The refined algorithm attempts to remove false positives and recover false negatives, but may not be ideal for certain complex mixtures of cell types. Does not apply if Exact Cell Count is set.

Exact_Cell_Count Integer (Optional) Exact Cell Count

Set a specific number (>=1) of cells as putative, based on those with the highest error-corrected read count

Reference_Genome File Reference Genome
Putative_Cell_Call https://w3id.org/cwl/view/git/46d4472af75afacfb13ed92d149a4decd015cf43/workflow1_11.cwl#main/Putative_Cell_Call/Putative_Cell_Call (Optional) Putative Cell Calling

Specify the data to be used for putative cell calling. mRNA is the default selected option.

Sample_Tags_Version https://w3id.org/cwl/view/git/46d4472af75afacfb13ed92d149a4decd015cf43/workflow1_11.cwl#main/Sample_Tags_Version/Sample_Tags_Version (Optional) Sample Tags Version

The sample multiplexing kit version. This option should only be set for a multiplexed experiment.

Exclude_Intronic_Reads Boolean (Optional) Exclude Intronic Reads

By default, reads aligned to exons and introns are considered and represented in molecule counts. Including intronic reads may increase sensitivity, resulting in an increase in molecule counts and the number of genes per cell for both cellular and nuclei samples. Intronic reads may indicate unspliced mRNAs and are also useful, for example, in the study of nuclei and RNA velocity. When set to true, intronic reads will be excluded.

Supplemental_Reference File[] (Optional) Supplemental Reference
Transcriptome_Annotation File Transcriptome Annotation

Steps

ID Runs Label Doc
AlignR2
workflow1_11.cwl#AlignR2.cwl (CommandLineTool)
Metrics
workflow1_11.cwl#Metrics.cwl (CommandLineTool)
Version
workflow1_11.cwl#Version.cwl (CommandLineTool)
AddtoBam
workflow1_11.cwl#AddtoBam.cwl (CommandLineTool)
IndexBAM
workflow1_11.cwl#IndexBAM.cwl (CommandLineTool)
MergeBAM
workflow1_11.cwl#MergeBAM.cwl (CommandLineTool)
AnnotateR1
workflow1_11.cwl#AnnotateR1.cwl (CommandLineTool)
AnnotateR2
workflow1_11.cwl#AnnotateR2.cwl (CommandLineTool)
BundleLogs
workflow1_11.cwl#BundleLogs.cwl (ExpressionTool)
Start_Time
workflow1_11.cwl#main/Start_Time/1338f67e-9793-46e2-8591-eb8b7ed5c84c (ExpressionTool)
CheckFastqs
workflow1_11.cwl#CheckFastqs.cwl (CommandLineTool)

CheckFastqs does several quality control routines including: (1) ensuring that read pair file names are formatted correctly and contain a read pair mate; (2) disambiguating the \"Subsample Reads\" input and; (3) if not provided, generating a subsampling seed that the downstream instances can use.

GetDataTable
workflow1_11.cwl#GetDataTable.cwl (CommandLineTool)
VDJ_Settings
workflow1_11.cwl#VDJ_Settings.cwl (ExpressionTool)
AnnotateReads
workflow1_11.cwl#AnnotateReads.cwl (CommandLineTool)
Name_Settings
workflow1_11.cwl#NameSettings.cwl (ExpressionTool)
PairReadFiles
workflow1_11.cwl#PairReadFiles.cwl (ExpressionTool)

PairReadFiles takes an array of split files and pairs them, such that an R1 file is transferred to the QualityFilter with its corresponding R2 file. The original FASTQ files are paired in CheckFastqs and then split and sub-sampled in SplitAndSubsample. The pairing information is taken from CheckFastqs.

CheckReference
workflow1_11.cwl#CheckReference.cwl (CommandLineTool)
MergeMultiplex
workflow1_11.cwl#main/MergeMultiplex/d1ec0f7a-6dfb-436f-9992-3e33d7aab5bc (ExpressionTool)
AnnotateMolecules
workflow1_11.cwl#AnnotateMolecules.cwl (CommandLineTool)
Internal_Settings
workflow1_11.cwl#InternalSettings.cwl (ExpressionTool)
Metadata_Settings
workflow1_11.cwl#Metadata.cwl (CommandLineTool)
SplitAndSubsample

SplitAndSubsample splits, subsamples and formats read files to be deposited in QualityFilter.

VDJ_GatherIGCalls

VDJ_GatherCalls collect the outputs from the multi-processed VDJ step into one file.

QualityFilterOuter
Subsample_Settings
workflow1_11.cwl#SubsampleSettings.cwl (ExpressionTool)
VDJ_GatherTCRCalls

VDJ_GatherCalls collect the outputs from the multi-processed VDJ step into one file.

VDJ_Compile_Results
workflow1_11.cwl#VDJ_Compile_Results.cwl (CommandLineTool)
Dense_to_Sparse_File
workflow1_11.cwl#DensetoSparseFile.cwl (CommandLineTool)
Multiplexing_Settings
workflow1_11.cwl#MultiplexingSettings.cwl (ExpressionTool)
Uncompress_Datatables
Intronic_Reads_Settings
workflow1_11.cwl#IntronicReadsSettings.cwl (ExpressionTool)
VDJ_Preprocess_Reads_IG
VDJ_Preprocess_Reads_TCR
Dense_to_Sparse_Datatable
workflow1_11.cwl#DensetoSparse.cwl (CommandLineTool)
Putative_Cell_Calling_Settings
workflow1_11.cwl#PutativeCellSettings.cwl (ExpressionTool)
Dense_to_Sparse_Datatable_Unfiltered
workflow1_11.cwl#DensetoSparse.cwl (CommandLineTool)
VDJ_Assemble_and_Annotate_Contigs_IG
VDJ_Assemble_and_Annotate_Contigs_TCR

Outputs

ID Type Label Doc
Logs Directory Pipeline Logs
Final_Bam File Final BAM File
Multiplex File[] (Optional)
Data_Tables File[] (Optional) Data Tables
vdjMetricsCsv File (Optional) vdjMetricsCsv
Expression_Data File (Optional) Expression Matrix
Final_Bam_Index File Final BAM Index
Metrics_Summary File Metrics Summary
Bioproduct_Stats File (Optional) Bioproduct Statistics
Cell_Label_Filter File[] (Optional) Cell Label Filter
vdjCellsDatatable File (Optional) vdjCellsDatatable
vdjDominantContigs File (Optional) vdjDominantContigs
Dim_Reduction_Coord File (Optional) Dimensionality Reduction Coordinates
Visual_Metrics_html File (Optional) Pipeline Report HTML
vdjUnfilteredContigs File (Optional) vdjUnfilteredContigs
Putative_Cells_Origin File (Optional) Putative Cells Origin
Data_Tables_Unfiltered File[] (Optional) Unfiltered Data Tables
vdjDominantContigsAIRR File (Optional) vdjDominantContigsAIRR
vdjUnfilteredContigsAIRR File (Optional) vdjUnfilteredContigsAIRR
Expression_Data_Unfiltered File (Optional) Unfiltered Expression Matrix
vdjCellsDatatableUncorrected File (Optional) vdjCellsDatatableUncorrected
Protein_Aggregates_Experimental File (Optional) Protein Aggregates (Experimental)
Immune_Cell_Classification(Experimental) File (Optional) Immune Cell Classification (Experimental)
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