External Model Validation

Input Validation

This skill accepts: an existing prognostic gene signature (model coefficient file with Gene and Coef columns), a bulk expression matrix in CSV format (genes as rows, samples as columns), and a clinical file with OS and OS.time survival columns.

If the user's request does not involve validating a pre-existing prognostic model on an external cohort — for example, asking to train a new model, perform feature selection, build a nomogram, run calibration curves, analyze single-cell data, or process data without survival endpoints — do not proceed with the workflow. Instead respond:

"external-model-validation is designed to validate an existing prognostic risk signature on an external bulk expression cohort with survival outcomes. Your request appears to be outside this scope. Please provide a fixed model coefficient file plus expression and clinical data with OS/OS.time columns, or use a more appropriate tool for model training, nomogram construction, or single-cell analysis."

When to Read External Files

Situation	File to Read	Purpose
Need to run the analysis	scripts/main.R	Execute: Rscript scripts/main.R --exp_file ... --cli_file ... --model_file ...
Need workflow order or output generation steps	scripts/run_analysis.R	Review the 4-step orchestration of loading, scoring, plotting, and metadata export
Need risk score or sample matching logic	scripts/functions.R	Inspect core data preparation and validation logic
Need output writing or metadata export details	scripts/io.R	Inspect output directory creation and file-writing helpers
Need plotting implementation details	scripts/plotting.R	Inspect Kaplan-Meier, risk, heatmap, and ROC plot generation
Need input validation, logging, timeout, or dependency logic	scripts/utils.R	Review validation helpers, SKILL_* error handling, logging, and runtime safeguards
Need statistical assumptions or method details	references/algorithm.md	Risk score formula, group cutoff, survival analysis, ROC, and heatmap assumptions
Need troubleshooting help	references/troubleshooting.md	Common failures, warnings, and concrete fixes
Need CLI usage examples	references/cli-guide.md	Parameter explanations, examples, and command patterns
Need expected outputs or benchmark run	references/baseline-run.md	Real-data baseline command, runtime, memory checkpoints, and output inventory
Need test inputs	tests/data/	Example expression, clinical, and model files for validation
Need to refresh the retained example output	tests/refresh_example_output.R	Rebuild tests/output/ with --overwrite using the bundled test data

---

Usage

Rscript scripts/main.R \
  --exp_file ./expression.csv \
  --cli_file ./clinical.csv \
  --model_file ./model.csv \
  --output_dir ./output/ \
  --time_unit month \
  --seed 42

---

Arguments

Short	Long	Type	Default	Description
-e	--exp_file	character	required	Expression matrix CSV with genes as rows and samples as columns
-c	--cli_file	character	required	Clinical CSV with sample IDs as row names and OS, OS.time columns
-m	--model_file	character	required	Model coefficient CSV with Gene and Coef columns
-o	--output_dir	character	./output/	Output directory
	--overwrite	flag	FALSE	Allow writing into a non-empty output directory
-u	--time_unit	character	month	Survival time unit in input clinical file: day, month, year
	--col_high	character	#E64B35	Color for high-risk samples
	--col_low	character	#4DBBD5	Color for low-risk samples
	--roc_cols	character	#E64B35,#00A087,#3C5488	Comma-separated colors for ROC curves
	--roc_times	character	1,3,5	Comma-separated ROC time points always in years, regardless of --time_unit. When follow-up is in days or months, still provide --roc_times in years (e.g., 1,3,5 for 1, 3, and 5 years).
	--roc_pos	character	bottomright	ROC legend position
	--km_breaks	integer	0	Kaplan-Meier x-axis break in years; 0 selects automatically
-s	--seed	integer	42	Random seed for reproducibility
	--timeout_seconds	integer	3600	Elapsed timeout limit in seconds

---

When to Use

• You already have a fixed prognostic gene signature and coefficients.
• You need to test that model on an independent cohort with bulk expression and survival data.
• You want standard outputs for external validation: risk table, Kaplan-Meier curve, risk score plot, survival status plot, expression heatmap, and time-dependent ROC.

When Not to Use

• Do not use this skill to train or re-fit a prognostic model.
• Do not use it for nomogram construction, calibration curves, DCA, or diagnostic classification.
• Do not use it for single-cell expression matrices or cohorts without survival endpoints.
• Do not use identifiable patient data without de-identification and local compliance approval.
• Do not use for cohorts with very few events (fewer than 5 events may produce unreliable Kaplan-Meier and ROC results).

Research Use Notice

• This skill is for research and validation workflows only.
• It does not provide diagnosis, treatment recommendations, or clinical decision support.
• Use de-identified data and follow IRB, ethics, and data-use requirements before running on human cohorts.

---

Input Format

Expression Matrix (exp_file)

CSV with genes as rows and samples as columns. The first column must contain gene identifiers.

"","Sample_1","Sample_2","Sample_3"
"TSPAN6",3.87,4.54,8.12
"TNMD",9.98,5.86,5.38
"DPM1",7.95,6.11,5.41

Clinical File (cli_file)

CSV with sample IDs as row names and at least OS and OS.time columns.

,Age,OS,OS.time
Sample_1,59,0,133.5
Sample_2,60,0,49.13
Sample_3,59,1,22.40

• OS must use 0/1 encoding.
• OS.time must be positive and interpretable under --time_unit.

Model Coefficient File (model_file)

CSV with two required columns: Gene and Coef.

Gene,Coef
TSPAN6,-0.25
TNMD,0.15
DPM1,0.32

---

Output Files

File	Description
data/risk_data.rds	Serialized analysis dataset containing survival data, model gene expression, risk scores, and risk groups
table/out_varifyRisk.txt	Tab-delimited risk table for all matched samples
plot/out_varifySurv.pdf	Kaplan-Meier survival curve with risk table
plot/out_varify.riskScore.pdf	Ordered risk score plot
plot/out_varify.survStat.pdf	Survival status plot
plot/out_varify.heatmap.pdf	Heatmap of model genes across ordered samples
plot/out_varify.ROC.pdf	Time-dependent ROC curve PDF
analysis.log	Runtime log including memory checkpoints and processing steps
run_parameters.tsv	Exact parameter values used for the run
session_info.txt	R version, platform, and package session information

---

Workflow

Step 1: Validate Inputs

• Check required files and CSV extensions.
• Validate color strings, timeout, seed, KM break setting, and time unit choice.
• Parse --roc_times and --roc_cols.

Step 2: Build Matched Validation Dataset

• Read expression, clinical, and model files.
• Match samples shared by expression columns and clinical row names.
• Check all model genes exist in the expression matrix.
• Remove incomplete cases before downstream analysis.

Step 3: Calculate Risk Scores and Groups

• Compute risk scores with the supplied linear predictor.
• Convert follow-up time into years.
• Split patients into low and high groups using the median risk score.

Step 4: Generate Validation Outputs

• Save the full risk table and RDS object.
• Produce Kaplan-Meier, risk score, survival status, heatmap, and time-dependent ROC plots.
• Save session metadata and exact run parameters.

---

Methods

Risk Score Formula

For sample i, the skill computes:

riskScore_i = sum(expression_ig * coefficient_g)

using all genes listed in model_file.

Risk Stratification

• Samples are ordered by riskScore.
• The median risk score is used as the cutoff.
• Samples with scores above the median are labeled high; the others are labeled low.

Survival Analysis

• Kaplan-Meier curves are fit with survival::survfit.
• Group difference is shown with the default log-rank p-value in survminer::ggsurvplot.

Time-Dependent ROC

• ROC analysis is performed with timeROC::timeROC using follow-up time in years.
• All --roc_times values must be smaller than the maximum observed follow-up time.
• --roc_times is always interpreted in years, regardless of --time_unit.

---

Examples

Basic Usage

Rscript scripts/main.R \
  -e tests/data/BRCA_data.csv \
  -c tests/data/BRCA_clinic.csv \
  -m tests/data/BRCA_coef.csv \
  -o ./output/

Input Follow-up Recorded in Days

Rscript scripts/main.R \
  -e expression.csv \
  -c clinical.csv \
  -m model.csv \
  -o ./output \
  -u day \
  --roc_times 1,2,3

Note: --roc_times 1,2,3 means 1, 2, and 3 years — even though --time_unit day was supplied. The skill converts OS.time from days to years internally before ROC computation.

Custom Plot Colors and ROC Settings

Rscript scripts/main.R \
  -e expression.csv \
  -c clinical.csv \
  -m model.csv \
  -o ./output \
  --col_high '#B2182B' \
  --col_low '#2166AC' \
  --roc_cols '#B2182B,#4D9221,#2166AC' \
  --roc_pos topleft \
  --km_breaks 2

---

Error Handling

Common Errors

Error	Cause	Solution
SKILL_FILE_NOT_FOUND	Input path is missing or wrong	Check file path and permissions
SKILL_MISSING_COLUMNS	Clinical or model file lacks required columns	Ensure OS, OS.time, Gene, and Coef exist
SKILL_SAMPLE_MISMATCH	No overlapping samples between expression and clinical data	Align sample IDs exactly
SKILL_EMPTY_DATA	An input file is empty after loading	Verify the CSV contains at least one row and one column of usable data
SKILL_INVALID_DATA	Duplicate genes, empty data, non-numeric coefficients, or invalid survival values. For duplicate genes: deduplicate with dplyr::distinct() or keep the row with highest mean expression (e.g., mat[order(-rowMeans(mat[,-1])),] %>% distinct(Gene, .keep_all=TRUE))	Clean input tables and verify formats
SKILL_ANALYSIS_ERROR	Risk groups collapse or event count is too low	Use a valid signature and cohort with enough events (minimum ~5)
SKILL_INVALID_PARAMETER	Bad --time_unit, invalid color, or impossible ROC time point	Correct the parameter value
SKILL_DEPENDENCY_MISSING	Required R package is not installed	Install the missing package
SKILL_PKG_VERSION	Installed package version is below the required minimum	Upgrade the package to the required version

IF error persists, READ: references/troubleshooting.md

---

Testing

Test with Included Data

# Check CLI
Rscript scripts/main.R --help

# Run with bundled test data in a fresh output directory
Rscript scripts/main.R \
  -e tests/data/BRCA_data.csv \
  -c tests/data/BRCA_clinic.csv \
  -m tests/data/BRCA_coef.csv \
  -o ./output/

Validation Commands

# Run R tests
Rscript tests/testthat.R

# Refresh the retained example output bundle
Rscript tests/refresh_example_output.R

# Inspect the generated risk table
wc -l tests/output/table/out_varifyRisk.txt

# Review the retained example outputs
ls -la tests/output/

Real-data Baseline

The repository stores a documented real-data baseline summary in references/baseline-run.md.

IF you need exact benchmark outputs or runtime expectations, READ: references/baseline-run.md

→ Directory structure and implementation details: references/project-structure.md