Workflow: BD Rhapsody™ Sequence Analysis Pipeline
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.
- Selected
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- Default Values
- Nested Workflows
- Tools
- Inputs/Outputs
Inputs
ID | Type | Title | Doc |
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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)
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Version |
rhapsody_pipeline_2.0.cwl#Version.cwl
(CommandLineTool)
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AddtoBam |
rhapsody_pipeline_2.0.cwl#AddtoBam.cwl
(CommandLineTool)
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MergeBAM |
rhapsody_pipeline_2.0.cwl#MergeBAM.cwl
(CommandLineTool)
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BundleLogs |
rhapsody_pipeline_2.0.cwl#BundleLogs.cwl
(ExpressionTool)
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Start_Time |
706388dcf09e89cd782f6fa2e44d0f0a
(ExpressionTool)
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QualCLAlign |
rhapsody_pipeline_2.0.cwl#QualCLAlign.cwl
(CommandLineTool)
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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. |
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Bam_Settings |
rhapsody_pipeline_2.0.cwl#BamSettings.cwl
(ExpressionTool)
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GenerateH5AD |
rhapsody_pipeline_2.0.cwl#GenerateH5AD.cwl
(CommandLineTool)
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GetDataTable |
rhapsody_pipeline_2.0.cwl#GetDataTable.cwl
(CommandLineTool)
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VDJ_Settings |
rhapsody_pipeline_2.0.cwl#VDJ_Settings.cwl
(ExpressionTool)
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Name_Settings |
rhapsody_pipeline_2.0.cwl#NameSettings.cwl
(ExpressionTool)
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Assay_Settings |
rhapsody_pipeline_2.0.cwl#Assay_Settings.cwl
(ExpressionTool)
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CheckReference |
rhapsody_pipeline_2.0.cwl#CheckReference.cwl
(CommandLineTool)
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GenerateSeurat |
rhapsody_pipeline_2.0.cwl#GenerateSeurat.cwl
(CommandLineTool)
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MergeMultiplex |
69e05751a29f5110bcb4e7004c464353
(ExpressionTool)
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AlignmentAnalysis |
rhapsody_pipeline_2.0.cwl#AlignmentAnalysis.cwl
(CommandLineTool)
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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 |
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AnnotateMolecules |
rhapsody_pipeline_2.0.cwl#AnnotateMolecules.cwl
(CommandLineTool)
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Internal_Settings |
rhapsody_pipeline_2.0.cwl#InternalSettings.cwl
(ExpressionTool)
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Metadata_Settings |
rhapsody_pipeline_2.0.cwl#Metadata.cwl
(CommandLineTool)
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VDJ_Compile_Results |
rhapsody_pipeline_2.0.cwl#VDJ_Compile_Results.cwl
(CommandLineTool)
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VDJ_Analyze_Reads_IG | |||
Multiplexing_Settings |
rhapsody_pipeline_2.0.cwl#MultiplexingSettings.cwl
(ExpressionTool)
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VDJ_Analyze_Reads_TCR | |||
Intronic_Reads_Settings |
rhapsody_pipeline_2.0.cwl#IntronicReadsSettings.cwl
(ExpressionTool)
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Putative_Cell_Calling_Settings |
rhapsody_pipeline_2.0.cwl#PutativeCellSettings.cwl
(ExpressionTool)
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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) |
https://w3id.org/cwl/view/git/50ed14112f9db254034dd5530cf1a768e04eb7ff/rhapsody_pipeline_2.0.cwl?part=main