Immunopipe Configuration Generator (Main Skill)

Purpose: Master skill for generating immunopipe pipeline configurations. Routes to individual process skills and determines pipeline architecture based on analysis requirements.

When to Use This Skill

• User wants to create/modify immunopipe configuration files
• Need to determine which processes to enable based on analysis goals
• Need to configure pipeline-level options (name, outdir, forks, scheduler)
• Need routing to specific process configuration skills

Pipeline Architecture Decision Tree

Step 1: Data Type Assessment

Ask the user about their data:

1. Do you have scRNA-seq data?

   • If YES → RNA analysis processes needed
   • If NO → Cannot proceed (RNA data required)

2. Do you have scTCR-seq or scBCR-seq data?

   • If YES → Enable TCR/BCR processes (TCR route)
   • If NO → RNA-only analysis (No-TCR route)

3. Is your RNA data already processed in a Seurat object?

   • If YES → Use LoadingRNAFromSeurat instead of SampleInfo + SeuratPreparing
   • If NO → Use standard input via SampleInfo

Step 2: Analysis Goals

Ask what analyses they want to perform:

Step 3: Essential vs Optional Processes

Essential Processes (always needed for TCR route):

• SampleInfo (or LoadingRNAFromSeurat)
• ScRepLoading (if TCR/BCR data present)
• SeuratPreparing (unless loading from prepared Seurat object)
• SeuratClustering
• SeuratClusterStats

Essential Processes (RNA-only route):

• SampleInfo (or LoadingRNAFromSeurat)
• SeuratPreparing
• SeuratClustering
• SeuratClusterStats

Optional Processes (enable only if requested):

• TOrBCellSelection - T/B cell separation
• SeuratClusteringOfAllCells - Clustering before T/B selection
• ClusterMarkersOfAllCells - Markers before T/B selection
• TopExpressingGenesOfAllCells - Top genes before T/B selection
• CellTypeAnnotation - Automated cell type annotation
• SeuratMap2Ref - Reference-based annotation
• SeuratSubClustering - Sub-clustering analysis
• ClusterMarkers - Differential expression between clusters
• TopExpressingGenes - Top expressed genes per cluster
• MarkersFinder - Flexible marker finding
• ModuleScoreCalculator - Module/pathway scoring
• ScRepCombiningExpression - TCR + RNA integration
• CDR3Clustering - TCR CDR3 clustering
• TESSA - TCR-specific analysis
• CDR3AAPhyschem - CDR3 physicochemical properties
• ClonalStats - Clonality statistics
• CellCellCommunication - Ligand-receptor analysis
• CellCellCommunicationPlots - Communication plots
• ScFGSEA - Fast gene set enrichment
• PseudoBulkDEG - Pseudo-bulk differential expression
• ScrnaMetabolicLandscape - Comprehensive metabolic analysis

Pipeline-Level Configuration

Basic Pipeline Options

name = "my_pipeline"           # Pipeline name (affects workdir and outdir)
outdir = "./output"            # Output directory (default: ./<name>-output)
loglevel = "info"              # Logging level: debug, info, warning, error
forks = 4                      # Number of parallel jobs (adjust based on CPU cores)
cache = true                   # Enable caching (recommended)
error_strategy = "halt"        # halt, ignore, or retry
num_retries = 3                # Number of retries if error_strategy = "retry"

Scheduler Configuration

Local execution (default):

scheduler = "local"

SLURM cluster:

scheduler = "slurm"

[scheduler_opts]
qsub_opts = "-p general -q general -N {job.name} -t {job.index}"

SGE cluster:

scheduler = "sge"

[scheduler_opts]
qsub_opts = "-V -cwd -j yes"

Google Cloud Batch:

# Use: immunopipe gbatch instead of immunopipe
# See gbatch skill for configuration

Plugin Options

[plugin_opts.report]
filters = ["name:Filter"]  # Filter processes in report

[plugin_opts.runinfo]
# Runinfo plugin enabled by default

Routing to Process Skills

When user needs specific process configuration, route to the appropriate skill:

Core Input Processes

• SampleInfo: Use sampleinfo skill
• LoadingRNAFromSeurat: Use loadingrnafromseurat skill
• ScRepLoading: Use screploading skill

Preprocessing Processes

• SeuratPreparing: Use seuratpreparing skill

Clustering Processes

• SeuratClustering: Use seuratclustering skill
• SeuratClusteringOfAllCells: Use seuratclusteringofallcells skill
• SeuratSubClustering: Use seuratsubclustering skill

Cell Selection

• TOrBCellSelection: Use torbcellselection skill

Annotation Processes

• CellTypeAnnotation: Use celltypeannotation skill
• SeuratMap2Ref: Use seuratmap2ref skill

Marker Analysis

• ClusterMarkers: Use clustermarkers skill
• ClusterMarkersOfAllCells: Use clustermarkersofallcells skill
• MarkersFinder: Use markersfinder skill
• TopExpressingGenes: Use topexpressinggenes skill
• TopExpressingGenesOfAllCells: Use topexpressinggenesofallcells skill

TCR/BCR Analysis

• ScRepCombiningExpression: Use screpcombiningexpression skill
• CDR3Clustering: Use cdr3clustering skill
• TESSA: Use tessa skill
• CDR3AAPhyschem: Use cdr3aaphyschem skill
• ClonalStats: Use clonalstats skill

Downstream Analysis

• ModuleScoreCalculator: Use modulescorecalculator skill
• CellCellCommunication: Use cellcellcommunication skill
• CellCellCommunicationPlots: Use cellcellcommunicationplots skill
• SeuratClusterStats: Use seuratclusterstats skill
• ScFGSEA: Use scfgsea skill
• PseudoBulkDEG: Use pseudobulkdeg skill

Metabolic Analysis

• ScrnaMetabolicLandscape: Use scrnametaboliclandscape skill

Configuration File Structure

A complete TOML configuration file has three sections:

# 1. PIPELINE-LEVEL OPTIONS
name = "my_pipeline"
outdir = "./output"
forks = 4

# 2. PROCESS-LEVEL OPTIONS
[ProcessName]
cache = true
forks = 2  # Override pipeline-level forks for this process

[ProcessName.in]
# Input files specification

[ProcessName.envs]
# Environment variables (process parameters)

# 3. GOOGLE BATCH OPTIONS (if using immunopipe gbatch)
[cli-gbatch]
project = "my-gcp-project"
region = "us-central1"

Example Workflows

Example 1: Basic TCR Analysis

User request: "I have scRNA-seq and scTCR-seq data. I want basic analysis with T cell selection."

Response:

1. Enable essential TCR processes: SampleInfo, ScRepLoading, SeuratPreparing, SeuratClustering, SeuratClusterStats
2. Enable T cell selection: SeuratClusteringOfAllCells, TOrBCellSelection
3. Route to sampleinfo skill to configure input files
4. Route to each process skill for configuration

Minimal config:

name = "tcr_analysis"
forks = 4

[SampleInfo.in]
infile = ["sample_info.txt"]

[SeuratClusteringOfAllCells]
[TOrBCellSelection]

Example 2: Advanced RNA-only Analysis

User request: "RNA-only data. I need clustering, cell type annotation, marker finding, and pathway enrichment."

Response:

1. Enable essential RNA processes: SampleInfo, SeuratPreparing, SeuratClustering, SeuratClusterStats
2. Add requested analyses: CellTypeAnnotation, ClusterMarkers, ScFGSEA
3. Route to individual skills for configuration

Example 3: Loading from Prepared Seurat Object

User request: "I already have a processed Seurat object. I want to run TCR analysis."

Response:

1. Use LoadingRNAFromSeurat instead of SampleInfo + SeuratPreparing
2. Enable TCR processes: ScRepLoading, SeuratClustering, etc.
3. Set prepared = true in LoadingRNAFromSeurat to skip preprocessing

Important Notes

Process Dependencies

Some processes have dependencies:

• ScRepCombiningExpression requires both ScRepLoading and RNA input
• ClusterMarkers requires SeuratClustering
• TOrBCellSelection usually follows SeuratClusteringOfAllCells
• CellCellCommunication requires clustering to be complete

Mutually Exclusive Options

• Use EITHER SampleInfo OR LoadingRNAFromSeurat as entry point (not both)
• If using TOrBCellSelection, typically enable SeuratClusteringOfAllCells first
• CellTypeAnnotation and SeuratMap2Ref serve similar purposes (can use both, but one usually sufficient)

Cache Strategy

• Set cache = "force" at pipeline level to reuse all previous results
• Set cache = false for specific process to force re-run
• Useful when tweaking visualization parameters without re-running analysis

Configuration Validation

After generating configuration, validate with:

python -m immunopipe.validate_config config.toml

External References

When process options reference external packages, expand them:

Seurat Functions

• When seeing Seurat::FunctionName, check: https://satijalab.org/seurat/reference/
• Common functions: FindMarkers(), FindClusters(), SCTransform(), RunUMAP()

Plotthis Functions

• Plot types map to functions: bar → BarPlot, box → BoxPlot
• Full reference: https://pwwang.github.io/plotthis/reference/

DESeq2 Design

• For PseudoBulkDEG, design formulas use DESeq2 syntax
• Reference: https://bioconductor.org/packages/release/bioc/html/DESeq2.html

GSEA Databases

• For ScFGSEA, GMT files from MSigDB
• Reference: https://www.gsea-msigdb.org/gsea/msigdb/

CellChat Database

• For CellCellCommunication, CellChat databases
• Reference: http://www.cellchat.org/

Workflow Summary

1. Assess data type (RNA-only vs TCR/BCR)
2. Determine analysis goals (clustering, annotation, TCR analysis, etc.)
3. Select essential processes based on data type
4. Add optional processes based on goals
5. Configure pipeline-level options (name, forks, scheduler)
6. Route to individual process skills for detailed configuration
7. Generate complete TOML file
8. Validate configuration before running

Quick Start Templates

For quick starts, use these templates:

• Basic TCR: basic-tcr template skill
• Basic RNA-only: basic-rna template skill
• Advanced TCR: advanced-tcr template skill
• Metabolic analysis: metabolic template skill
• Cell communication: communication template skill

Error Prevention

Common configuration errors to avoid:

1. Missing input specification: Always set [ProcessName.in] for entry processes
2. TCR data without ScRepLoading: If TCRData/BCRData columns exist, enable ScRepLoading
3. Contradictory process enablement: Don't enable both "OfAllCells" and regular versions without TOrBCellSelection
4. Invalid gene names: Use human gene symbols (uppercase) or mouse (title case)
5. Path issues: Use absolute paths or paths relative to config file location
6. Resource limits: Set appropriate forks based on available CPU/memory

Next Steps

After generating config:

1. Save to .toml file (e.g., config.toml)
2. Run: immunopipe config.toml
3. Or use web UI: pipen board @config.toml
4. Or use Google Batch: immunopipe gbatch config.toml

For modifications, route to specific process skills based on what needs to change.