Workflow: BD Rhapsody™ Sequence Analysis Pipeline

Fetched 2025-05-08 17:48:27 GMT

The BD Rhapsody™ assays are used to create sequencing libraries from single cell transcriptomes. After sequencing, the analysis pipeline takes the FASTQ files and a reference file for gene alignment. The pipeline generates molecular counts per cell, read counts per cell, metrics, and an alignment file.

children parents
Workflow as SVG
  • Selected
  • Default Values
  • Nested Workflows
  • Tools
  • Inputs/Outputs

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.

AbSeq_UMI Integer (Optional)
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/50ed14112f9db254034dd5530cf1a768e04eb7ff/rhapsody_pipeline_2.0.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.

Generate_Bam Boolean (Optional) Generate Bam Output

Default: false. A Bam read alignment file contains reads from all the input libraries, but creating it can consume a lot of compute and disk resources. By setting this field to true, the Bam file will be created.

AbSeq_Reference File[] (Optional) AbSeq Reference
Maximum_Threads Integer (Optional) Maximum Number of Threads

The maximum number of threads to use in the pipeline. By default, all available cores are used.

Target_analysis Boolean (Optional)
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

VDJ_JGene_Evalue Float (Optional) e-value threshold for J gene

The e-value threshold for J gene call by IgBlast/PyIR, default is set as 0.001

VDJ_VGene_Evalue Float (Optional) e-value threshold for V gene

The e-value threshold for V gene call by IgBlast/PyIR, default is set as 0.001

Reference_Archive File (Optional) Reference Files Archive
Putative_Cell_Call https://w3id.org/cwl/view/git/50ed14112f9db254034dd5530cf1a768e04eb7ff/rhapsody_pipeline_2.0.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.

Targeted_Reference File[] (Optional) Targeted Reference
Expected_Cell_Count Integer (Optional) Expected Cell Count

Optional. Guide the basic putative cell calling algorithm by providing an estimate of the number of cells expected. Usually this can be the number of cells loaded into the Rhapsody cartridge. If there are multiple inflection points on the second derivative cumulative curve, this will ensure the one selected is near the expected.

Sample_Tags_Version https://w3id.org/cwl/view/git/50ed14112f9db254034dd5530cf1a768e04eb7ff/rhapsody_pipeline_2.0.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
Enable_Refined_Cell_Call Boolean (Optional) Enable Refined Putative Cell Calling

By default, putative cells are determined using only the basic algorithm (minimum second derivative along the cumulative reads curve). The refined algorithm builds on the basic cell call, by analyzing expression patterns to remove false positives and recover false negatives. The refined algoritm may not be suitable for noisy datasets, and certain mixtures of cell types. Does not apply if Exact Cell Count is set.

Steps

ID Runs Label Doc
Metrics
rhapsody_pipeline_2.0.cwl#Metrics.cwl (CommandLineTool)
Version
rhapsody_pipeline_2.0.cwl#Version.cwl (CommandLineTool)
AddtoBam
rhapsody_pipeline_2.0.cwl#AddtoBam.cwl (CommandLineTool)
MergeBAM
rhapsody_pipeline_2.0.cwl#MergeBAM.cwl (CommandLineTool)
BundleLogs
rhapsody_pipeline_2.0.cwl#BundleLogs.cwl (ExpressionTool)
Start_Time
706388dcf09e89cd782f6fa2e44d0f0a (ExpressionTool)
QualCLAlign
rhapsody_pipeline_2.0.cwl#QualCLAlign.cwl (CommandLineTool)

CheckFastqs does several quality control routines including: ensuring that read pair file names are formatted correctly and contain a read pair mate; QualCLAlign stage of the Rain pipeline overlaps read pairs and then performs a series of filters and mappings to reduce valid reads into a single FastQ file to be fed into the aligner. The R2 reads are annotated with cell index and UMI information derived from the R1 read.

Bam_Settings
rhapsody_pipeline_2.0.cwl#BamSettings.cwl (ExpressionTool)
GenerateH5AD
rhapsody_pipeline_2.0.cwl#GenerateH5AD.cwl (CommandLineTool)
GetDataTable
rhapsody_pipeline_2.0.cwl#GetDataTable.cwl (CommandLineTool)
VDJ_Settings
rhapsody_pipeline_2.0.cwl#VDJ_Settings.cwl (ExpressionTool)
Name_Settings
rhapsody_pipeline_2.0.cwl#NameSettings.cwl (ExpressionTool)
Assay_Settings
rhapsody_pipeline_2.0.cwl#Assay_Settings.cwl (ExpressionTool)
CheckReference
rhapsody_pipeline_2.0.cwl#CheckReference.cwl (CommandLineTool)
GenerateSeurat
rhapsody_pipeline_2.0.cwl#GenerateSeurat.cwl (CommandLineTool)
MergeMultiplex
69e05751a29f5110bcb4e7004c464353 (ExpressionTool)
AlignmentAnalysis
rhapsody_pipeline_2.0.cwl#AlignmentAnalysis.cwl (CommandLineTool)

AlignmentAnalysis stage of the Rain pipeline annotates aligned reads and collects a myriad of metrics on the aligned reads. Additional annotation is performed to the reads

AnnotateMolecules
rhapsody_pipeline_2.0.cwl#AnnotateMolecules.cwl (CommandLineTool)
Internal_Settings
rhapsody_pipeline_2.0.cwl#InternalSettings.cwl (ExpressionTool)
Metadata_Settings
rhapsody_pipeline_2.0.cwl#Metadata.cwl (CommandLineTool)
VDJ_Compile_Results
rhapsody_pipeline_2.0.cwl#VDJ_Compile_Results.cwl (CommandLineTool)
VDJ_Analyze_Reads_IG
Multiplexing_Settings
rhapsody_pipeline_2.0.cwl#MultiplexingSettings.cwl (ExpressionTool)
VDJ_Analyze_Reads_TCR
Intronic_Reads_Settings
rhapsody_pipeline_2.0.cwl#IntronicReadsSettings.cwl (ExpressionTool)
Putative_Cell_Calling_Settings
rhapsody_pipeline_2.0.cwl#PutativeCellSettings.cwl (ExpressionTool)

Outputs

ID Type Label Doc
Bam File[] (Optional) BAM file and index
H5AD File (Optional) Scanpy H5AD File
Logs Directory Pipeline Logs
Seurat File (Optional) Seurat RDS File
Multiplex File[] (Optional)
Data_Tables File[] (Optional) Data Tables
vdjMetricsCsv File (Optional) vdjMetricsCsv
Metrics_Summary File Metrics Summary
Bioproduct_Stats File (Optional) Bioproduct Statistics
vdjCellsDatatable File (Optional) vdjCellsDatatable
Dim_Reduction_Coord File (Optional) Dimensionality Reduction Coordinates
Visual_Metrics_html File (Optional) Pipeline Report HTML
vdjDominantContigsAIRR File (Optional) vdjDominantContigsAIRR
vdjUnfilteredContigsAIRR File (Optional) vdjUnfilteredContigsAIRR
vdjCellsDatatableUncorrected File (Optional) vdjCellsDatatableUncorrected
Protein_Aggregates_Experimental File (Optional) Protein Aggregates (Experimental)
Immune_Cell_Classification(Experimental) File (Optional) Immune Cell Classification (Experimental)
Permalink: https://w3id.org/cwl/view/git/50ed14112f9db254034dd5530cf1a768e04eb7ff/rhapsody_pipeline_2.0.cwl?part=main