Source: https://github.com/aipoch/medical-research-skills

CIBERSORT Immune Infiltration Analysis

When to Use

• Estimate relative immune cell fractions from a bulk expression matrix.
• Compare one case group against one control group after deconvolution.
• Generate structured tables, a serialized result object, and optional PDF plots.

When Not to Use

• Single-cell RNA-seq, spatial transcriptomics, or clustering tasks.
• Absolute clinical interpretation or treatment recommendation.
• Workflows that require the original online CIBERSORT service instead of a local R implementation.

Workflow

1. Confirm that the expression matrix, group file, and signature matrix are available.
2. Run scripts/main.R with the case and control groups.
3. Review the full result table, derived summary tables, and optional plots.
4. Inspect run_record.txt and output_manifest.txt after each run, including failed validation attempts.

When to Read External Files

Situation	File to Read	Purpose
Need to run the analysis	scripts/main.R	CLI entry point
Need algorithm details	references/algorithm.md	HQ reference workflow and result interpretation
Encounter an error	references/troubleshooting.md	Error codes and environment fixes
Need CLI examples or the baseline record	references/cli-guide.md	Example commands and validation notes
Need packaged test inputs	tests/data/	Demo expression matrix, group file, and LM22 file

Usage

Rscript scripts/main.R \
  --input_file ./expression_matrix.csv \
  --group_file ./group_info.csv \
  --signature_file ./LM22.txt \
  --case_group treatment \
  --control_group control \
  --output_dir ./output \
  --qn false \
  --seed 42

Arguments

Short	Long	Type	Default	Description
-i	--input_file	file	required	Expression matrix with genes as rows and samples as columns
-g	--group_file	file	required	Group annotation table
-a	--case_group	string	required	Case group label
-b	--control_group	string	required	Control group label
-o	--output_dir	dir	./output	Output directory
	--signature_file	file	tests/data/LM22.txt when present	Signature matrix file
	--sample_col	string/int	none	Optional sample column name or 1-based index
	--group_col	string/int	none	Optional group column name or 1-based index
	--gene_id_case	string	upper	Gene ID normalization: asis, upper, or lower
	--auto_unlog	boolean	true	Apply 2^x only if the expression matrix passes a conservative log-scale heuristic
	--min_mean_expression	numeric	1	Minimum mean expression before deconvolution
	--perm	integer	1000	Permutation count for empirical p-value estimation; 0 keeps the run lightweight but records P-value as NA
	--qn	boolean	true	Apply quantile normalization to the mixture matrix
	--svm_cores	integer	1	Worker count for the nu-SVR model selection step
	--make_plots	boolean	true	Generate PDF plots
	--plot_width	numeric	16	Default plot width in inches
	--plot_height	numeric	10	Default plot height in inches
-s	--seed	integer	42	Random seed
-t	--timeout_seconds	integer	0	Optional timeout in seconds; 0 disables it
	--verbose	boolean	true	Print progress logs

Input Format

Expression Matrix

CSV or TSV. The first column must contain gene identifiers. Remaining columns must be numeric sample-level expression values.

When --auto_unlog=true, the workflow reports summary statistics and applies 2^x only if the matrix passes a conservative log-scale heuristic. If the matrix is ambiguous, the values are left unchanged and the startup log explains why.

If duplicate gene identifiers are present, they are consolidated after gene-ID normalization by taking the per-sample maximum before downstream filtering and deconvolution.

gene,Sample1,Sample2,Sample3
TP53,10.2,8.5,9.1
CXCL9,4.3,6.1,5.7

Group File

CSV or TSV with one sample column and one group column.

sample,group
Sample1,control
Sample2,treatment
Sample3,treatment

Signature Matrix

The packaged default is tests/data/LM22.txt. A custom signature matrix must contain one gene column followed by immune-cell signature columns.

All immune-cell signature columns must be numeric and finite. If duplicate gene identifiers are present, they are consolidated by taking the per-cell-type maximum before gene intersection.

Output Files

File	Description
data/cibersort_input.rds	Serialized aligned input matrices used by the local algorithm
data/cibersort_null_distribution.rds	Serialized permutation null distribution
data/cibersort_result.rds	Serialized result object with cell fractions, metrics, runtime settings, and heatmap rendering metadata
table/CIBERSORT_Results.csv	Full result table in CSV format
table/CIBERSORT-Results.txt	Full result table in tab-delimited text format
table/cibersort_cell_fractions_wide.csv	Wide-format immune cell fraction table
table/cibersort_cell_fractions_long.csv	Long-format immune cell fraction table
table/cibersort_group_compare.csv	Case-vs-control comparison summary
table/cibersort_quality_metrics.csv	Sample-level P-value, Correlation, and RMSE table
table/immune_cell_correlation_matrix.csv	Spearman correlation matrix across immune cell types
table/immune_cell_correlation_pvalue.csv	P-value matrix aligned to the correlation matrix
plot/immune_cell_composition_sample.pdf	Sample-level stacked composition plot when --make_plots=true
plot/immune_group_boxplot.pdf	Group comparison boxplot when --make_plots=true
plot/immune_correlation_heatmap.pdf	Immune-cell correlation heatmap when --make_plots=true
session_info.txt	R session information
output_manifest.txt	Append-only output manifest for successful and failed runs
run_record.txt	Append-only structured run record, including runtime notes and failed-run summaries

When --make_plots=false, the plot/ directory may still exist as part of the standard output layout, but no PDF plot files are written.

When --perm=0, the workflow logs a warning and completes without empirical permutation testing, so the P-value column is recorded as NA.

When a rerun targets an existing --output_dir and then fails validation or execution, the previous successful payload is preserved and the failure is appended to run_record.txt and output_manifest.txt.

Error Handling

Error Code	Meaning	Solution
SKILL_FILE_NOT_FOUND	An input file or signature matrix was not found	Check the file path and rerun
SKILL_MISSING_COLUMNS	A required column is missing	Fix the input schema
SKILL_EMPTY_DATA	No usable genes, samples, or deconvolution outputs remain	Check the data, filtering, or signature overlap
SKILL_INVALID_PARAMETER	A CLI parameter is missing or invalid	Review the argument table and input values
SKILL_SAMPLE_MISMATCH	Expression samples and group annotations do not align	Harmonize sample identifiers
SKILL_PACKAGE_NOT_FOUND	A required R package is missing	Install the missing package
SKILL_TIMEOUT	The configured time limit was exceeded	Increase --timeout_seconds or set it to 0

If the error persists, READ: references/troubleshooting.md

Input Validation

This skill accepts:

• A bulk expression matrix file in CSV or TSV format with one gene column and numeric sample columns.
• A group annotation file in CSV or TSV format with one sample column and one group column.
• Exactly one case group label and one control group label for comparison.
• An optional custom signature matrix compatible with the documented LM22-style schema.

Do not use this skill for:

• Single-cell RNA-seq, spatial transcriptomics, or cell clustering workflows.
• Clinical diagnosis, treatment recommendation, or patient-level medical decision making.
• Requests that need the hosted CIBERSORT web service rather than this local R implementation.
• Multi-group study designs that require more than one case group versus one control group in a single run.

If the user's request is outside this scope, do not proceed with the workflow. Instead respond:

"cibersort-immune-infiltration-analysis is designed for local CIBERSORT-style immune deconvolution from a bulk expression matrix with one case group and one control group. Your request appears to be outside this scope. Please provide compatible bulk-expression inputs and group labels, or use a more appropriate tool for your task."

Testing

Rscript scripts/main.R --help

Rscript tests/run_tests.R

Rscript tests/test_skill.R

Validated packaged test path:

Rscript scripts/main.R \
  --input_file tests/data/expression_matrix.csv \
  --group_file tests/data/group_info.csv \
  --signature_file tests/data/LM22.txt \
  --case_group Tumor \
  --control_group Healthy \
  --output_dir tests/output \
  --perm 25 \
  --qn false \
  --svm_cores 1 \
  --seed 42

Container note:

• The packaged test path uses --qn false because preprocessCore::normalize.quantiles() may trigger environment-level thread failures in some containers.
• If you need a quantile-normalized run, validate that environment first and record the result in references/cli-guide.md.
• tests/run_tests.R also checks that a failed rerun does not erase an existing successful payload directory.