diff --git a/.github/workflows/tests.yml b/.github/workflows/tests.yml new file mode 100644 index 0000000..9853ba3 --- /dev/null +++ b/.github/workflows/tests.yml @@ -0,0 +1,127 @@ +name: tests + +on: + push: + pull_request: + workflow_dispatch: + inputs: + run_conda_validation: + description: "Create workflow conda envs and run package import checks" + required: false + default: false + type: boolean + run_container_validation: + description: "Pull workflow containers" + required: false + default: false + type: boolean + run_doubletfinder_install: + description: "Run the networked DoubletFinder GitHub install rule" + required: false + default: false + type: boolean + +jobs: + pytest: + runs-on: ubuntu-latest + defaults: + run: + shell: bash -el {0} + env: + SNAKEMAKE_CONDA_FRONTEND: mamba + steps: + - name: Check out repository + uses: actions/checkout@v4 + + - name: Create test environment + uses: mamba-org/setup-micromamba@v2 + with: + environment-file: environment.yml + cache-environment: true + cache-downloads: true + + - name: Cache Snakemake rule environments + uses: actions/cache@v4 + with: + path: .snakemake/conda + key: snakemake-conda-${{ runner.os }}-${{ hashFiles('workflow/envs/*.yml') }} + restore-keys: | + snakemake-conda-${{ runner.os }}- + + - name: Run tests + run: pytest tests -q + + conda-validation: + if: github.event_name == 'workflow_dispatch' && inputs.run_conda_validation + runs-on: ubuntu-latest + strategy: + fail-fast: false + matrix: + env_name: + - cellbender.yml + - doubletfinder.yml + - emptydrops.yml + - posthocfilter.yml + - scdblfinder.yml + - soupx.yml + - tenx2seuratrds.yml + defaults: + run: + shell: bash -el {0} + env: + SNAKEMAKE_CONDA_FRONTEND: mamba + steps: + - name: Check out repository + uses: actions/checkout@v4 + + - name: Create test environment + uses: mamba-org/setup-micromamba@v2 + with: + environment-file: environment.yml + cache-environment: true + cache-downloads: true + + - name: Run conda validation for ${{ matrix.env_name }} + run: pytest tests/test_conda_container_validation.py --run-conda-validation --conda-env-name "${{ matrix.env_name }}" -q + + container-validation: + if: github.event_name == 'workflow_dispatch' && inputs.run_container_validation + runs-on: ubuntu-latest + defaults: + run: + shell: bash -el {0} + steps: + - name: Check out repository + uses: actions/checkout@v4 + + - name: Create test environment + uses: mamba-org/setup-micromamba@v2 + with: + environment-file: environment.yml + cache-environment: true + cache-downloads: true + + - name: Run container validation + run: pytest tests/test_conda_container_validation.py --run-container-validation -q + + doubletfinder-install: + if: github.event_name == 'workflow_dispatch' && inputs.run_doubletfinder_install + runs-on: ubuntu-latest + defaults: + run: + shell: bash -el {0} + env: + SNAKEMAKE_CONDA_FRONTEND: mamba + steps: + - name: Check out repository + uses: actions/checkout@v4 + + - name: Create test environment + uses: mamba-org/setup-micromamba@v2 + with: + environment-file: environment.yml + cache-environment: true + cache-downloads: true + + - name: Run DoubletFinder install validation + run: pytest tests/test_conda_container_validation.py --run-doubletfinder-install -q diff --git a/.gitignore b/.gitignore new file mode 100644 index 0000000..cb76481 --- /dev/null +++ b/.gitignore @@ -0,0 +1,21 @@ +__pycache__/ +*.py[cod] +.pytest_cache/ +.cache/ +Rplots.pdf +logs/ +.snakemake/ +.jupyter/ +.vscode/ +.ipython/ +scratch/ +*err +*out +results/ +testdata/results/ +# Keep golden test outputs trackable even though results/ is ignored. +!tests/reference_outputs/ +!tests/reference_outputs/** +!tests/reference_outputs/testdata/ +!tests/reference_outputs/testdata/results/ +!tests/reference_outputs/testdata/results/** diff --git a/config/config.yaml b/config/config.yaml index 9f649ed..6ac8b47 100644 --- a/config/config.yaml +++ b/config/config.yaml @@ -1,6 +1,7 @@ conda-channel-priority: "strict" sampleTable: "samplesheet.tsv" +workflow_seed: 12345 emptydrop_removal_methods: ["tenx","emptydrops"] ambient_decon_methods: ["soupx","cellbender_fromraw"] diff --git a/environment.yml b/environment.yml new file mode 100644 index 0000000..67be718 --- /dev/null +++ b/environment.yml @@ -0,0 +1,13 @@ +name: scrnaseq-preprocessing-tests +channels: + - conda-forge + - bioconda +channel_priority: strict +dependencies: + - python=3.12 + - pytest + - pyyaml + - snakemake=9.16.3 + - conda + - mamba + - hdf5 diff --git a/testdata/filtered_feature_bc_matrix/barcodes.tsv.gz b/testdata/filtered_feature_bc_matrix/barcodes.tsv.gz new file mode 100644 index 0000000..caff0a3 Binary files /dev/null and b/testdata/filtered_feature_bc_matrix/barcodes.tsv.gz differ diff --git a/testdata/filtered_feature_bc_matrix/features.tsv.gz b/testdata/filtered_feature_bc_matrix/features.tsv.gz new file mode 100644 index 0000000..b4ba99b Binary files /dev/null and b/testdata/filtered_feature_bc_matrix/features.tsv.gz differ diff --git a/testdata/filtered_feature_bc_matrix/matrix.mtx.gz b/testdata/filtered_feature_bc_matrix/matrix.mtx.gz new file mode 100644 index 0000000..10c2d9d Binary files /dev/null and b/testdata/filtered_feature_bc_matrix/matrix.mtx.gz differ diff --git a/testdata/raw_feature_bc_matrix.h5 b/testdata/raw_feature_bc_matrix.h5 new file mode 100644 index 0000000..98a30e1 Binary files /dev/null and b/testdata/raw_feature_bc_matrix.h5 differ diff --git a/testdata/raw_feature_bc_matrix/barcodes.tsv.gz b/testdata/raw_feature_bc_matrix/barcodes.tsv.gz new file mode 100644 index 0000000..b428a33 Binary files /dev/null and b/testdata/raw_feature_bc_matrix/barcodes.tsv.gz differ diff --git a/testdata/raw_feature_bc_matrix/features.tsv.gz b/testdata/raw_feature_bc_matrix/features.tsv.gz new file mode 100644 index 0000000..b4ba99b Binary files /dev/null and b/testdata/raw_feature_bc_matrix/features.tsv.gz differ diff --git a/testdata/raw_feature_bc_matrix/matrix.mtx.gz b/testdata/raw_feature_bc_matrix/matrix.mtx.gz new file mode 100644 index 0000000..b2fb32f Binary files /dev/null and b/testdata/raw_feature_bc_matrix/matrix.mtx.gz differ diff --git a/testdata/samplesheet_test.tsv b/testdata/samplesheet_test.tsv new file mode 100644 index 0000000..b36d643 --- /dev/null +++ b/testdata/samplesheet_test.tsv @@ -0,0 +1,2 @@ +sampleid tenx_datadir +test testdata diff --git a/tests/README.md b/tests/README.md new file mode 100644 index 0000000..bffdefe --- /dev/null +++ b/tests/README.md @@ -0,0 +1,64 @@ +# Tests + +These tests are intended to run from the repository root in any environment where `snakemake` and `pytest` are available on `PATH`. How that environment is created or activated is site-specific; for example, an HPC may require loading a module before `conda activate` is available. + +Example: + +```bash +conda env create -f environment.yml +conda activate scrnaseq-preprocessing-tests +pytest tests +``` + +`python -m pytest tests` is also fine when `python` resolves to the same environment that provides `pytest` and `snakemake`. If it does not, the active shell is likely resolving commands from different environments. Confirm with: + +```bash +which python +which pytest +which snakemake +``` + +The dry-run DAG test uses the small CellRanger-style input data in `testdata/` and the test-specific sample sheet `testdata/samplesheet_test.tsv`. The tests override `sampleTable` on the Snakemake command line, so they do not use the normal workflow `samplesheet.tsv`. They do not require `--conda-prefix`, a cluster profile, or any site-specific paths. Sample-sheet validation tests cover malformed headers, duplicate sample IDs, missing or incomplete 10x data paths, absolute `tenx_datadir` values, and multi-sample DAG expansion. Config validation tests cover required keys, allowed method values, threshold types/ranges, invalid-config failures, and dry-run DAG variants driven by method-list config changes. The default suite also runs `snakemake --lint` against the test-data configuration, which catches workflow-structure problems such as missing `log:` directives, helper functions embedded in rule files, long `run:` blocks that should live in scripts, path-composition warnings, missing rule-level conda/container declarations, and shell commands that directly interpolate global workflow variables instead of passing values through `params`. + +GitHub Actions runs the default test suite via `.github/workflows/tests.yml`, using the same top-level `environment.yml` test runner environment and caching Snakemake-created rule environments under `.snakemake/conda`. + +The default test suite also includes a focused local rule-execution smoke test. It runs the real `tenx2seuratrds`, `find_markers`, and `combine_markers` rule chain against `testdata/`, using Snakemake's `--use-conda` support and writing outputs under pytest's temporary directory. This catches broken R package imports, script argument drift, invalid Seurat object creation, and marker CSV schema changes without submitting to SLURM. + +The R output validator also checks that the Seurat object has at least 100 features and 100 cells; metadata rows match the cell count; barcode row names are present, unique, and nonempty; `orig.ident`, `nCount_RNA`, `nFeature_RNA`, `percent.mt`, and `seurat_clusters` metadata columns exist; RNA count and feature-count metadata values are finite and positive; mitochondrial percentages are finite and within `[0, 100]`; at least two clusters are present; PCA and UMAP reductions exist; the marker table is nonempty and has the expected columns; marker gene symbols are present and nonempty; marker numeric columns are finite; marker p-value and percent columns are within `[0, 1]`; marker clusters are present in the Seurat metadata; markers are reported for at least two clusters; and the marker `workflow` column matches the expected test workflow label. The test runner environment is defined in the repository-level `environment.yml`; the rule-specific R environment is still created by Snakemake from `workflow/envs/tenx2seuratrds.yml`. A separate lightweight checkpoint-expansion test uses a fake `Rscript` to materialize the `marker_manifest` checkpoint, verify that dynamic `find_markers` jobs are generated for each cluster id, and confirm that `combine_markers` receives the expected marker chunks. + + +## Optional conda and container validation + +The default suite validates that workflow conda environment files are well formed, that rule-level conda references resolve to existing files, and that container declarations are recognizable. Expensive network-dependent validation is opt-in: + +```bash +pytest tests --run-conda-validation +pytest tests --run-container-validation +pytest tests --run-doubletfinder-install +``` + +`--run-conda-validation` creates each `workflow/envs/*.yml` environment in a pytest temporary directory and checks key R/Python package imports. To validate only one environment, pass `--conda-env-name ENV_FILE`, for example `--conda-env-name soupx.yml`; GitHub Actions uses this selector to run conda validation as one matrix job per env. `--run-container-validation` pulls the CellBender container with Docker, Apptainer, or Singularity. `--run-doubletfinder-install` runs the networked Snakemake `install_doubletfinder` rule and confirms that `DoubletFinder` can be imported from the created rule environment. + + +## Optional full workflow run + +The default tests build and inspect the DAG and run a focused local R-rule smoke test. To submit the full test-data workflow and verify all declared outputs against the reference snapshot, opt in explicitly: + +```bash +pytest tests --run-workflow +``` + +The full-run test calls `tests/run_test_workflow.sh`, which uses `testdata/samplesheet_test.tsv` and overrides the workflow output directory with `resultsDir=testdata/results`. The manifest in `tests/test_sample_rule_output_files.txt` is therefore written with paths under `testdata/results/`. + +For testing, omit `--snakemake-conda-prefix` so Snakemake uses its default `.snakemake/conda` location under the repository root. The runner assumes that the current environment already provides `snakemake` on `PATH`. + + +## Reference outputs + +The full workflow test compares regenerated files in `testdata/results/` against reference files under `tests/reference_outputs/`. The compared file list is in `tests/test_reference_output_files.txt`. Seurat `.rds` files are compared at the metadata-table level, marker CSVs are compared by columns and `(cluster, genesymbol)` rows with numeric tolerance, emptyDrops matrix files are compared after gzip decompression, CellBender H5 outputs are compared with `h5diff`, and remaining durable outputs are compared byte-for-byte. + +To refresh the reference snapshot after intentionally changing workflow behavior, first run the full test workflow so `testdata/results/` contains the desired outputs, then run: + +```bash +python tests/update_reference_outputs.py +``` diff --git a/tests/compare_seurat_metadata.R b/tests/compare_seurat_metadata.R new file mode 100644 index 0000000..6a2e638 --- /dev/null +++ b/tests/compare_seurat_metadata.R @@ -0,0 +1,97 @@ +args <- commandArgs(trailingOnly = TRUE) +if (length(args) == 0 || length(args) %% 2 != 0) { + stop("Provide current/reference RDS path pairs", call. = FALSE) +} + +suppressPackageStartupMessages(library("Seurat")) + +numeric_tolerance <- as.numeric(Sys.getenv("SEURAT_METADATA_NUMERIC_TOLERANCE", "1e-8")) +if (is.na(numeric_tolerance) || length(numeric_tolerance) != 1) { + stop("SEURAT_METADATA_NUMERIC_TOLERANCE must be numeric", call. = FALSE) +} + +fail <- function(path, message) { + stop(sprintf("%s: %s", path, message), call. = FALSE) +} + +compare_metadata_column <- function(current_path, column, current_values, reference_values) { + current_na <- is.na(current_values) + reference_na <- is.na(reference_values) + if (!identical(current_na, reference_na)) { + fail(current_path, sprintf("metadata column %s has different NA positions", column)) + } + + both_numeric <- is.numeric(current_values) && is.numeric(reference_values) + both_integer <- is.integer(current_values) && is.integer(reference_values) + if (both_numeric || both_integer) { + current_numeric <- as.numeric(current_values) + reference_numeric <- as.numeric(reference_values) + comparable <- !current_na & !reference_na + if (any(comparable)) { + diff <- abs(current_numeric[comparable] - reference_numeric[comparable]) + scale <- pmax(abs(current_numeric[comparable]), abs(reference_numeric[comparable]), 1) + bad <- diff > numeric_tolerance * scale + if (any(bad)) { + fail( + current_path, + sprintf( + "metadata column %s differs numerically; max abs diff %.12g", + column, + max(diff) + ) + ) + } + } + return(invisible(TRUE)) + } + + current_character <- as.character(current_values) + reference_character <- as.character(reference_values) + comparable <- !current_na & !reference_na + if (!identical(current_character[comparable], reference_character[comparable])) { + fail(current_path, sprintf("metadata column %s differs", column)) + } + + invisible(TRUE) +} + +compare_metadata_pair <- function(current_path, reference_path) { + current <- readRDS(current_path) + reference <- readRDS(reference_path) + + current_metadata <- current@meta.data + reference_metadata <- reference@meta.data + + if (!identical(colnames(current_metadata), colnames(reference_metadata))) { + fail( + current_path, + paste0( + "metadata columns differ\ncurrent: ", + paste(colnames(current_metadata), collapse = ","), + "\nreference: ", + paste(colnames(reference_metadata), collapse = ",") + ) + ) + } + + if (!identical(rownames(current_metadata), rownames(reference_metadata))) { + fail(current_path, "metadata barcodes differ") + } + + for (column in colnames(current_metadata)) { + compare_metadata_column( + current_path, + column, + current_metadata[[column]], + reference_metadata[[column]] + ) + } + + invisible(TRUE) +} + +for (i in seq(1, length(args), by = 2)) { + compare_metadata_pair(args[[i]], args[[i + 1]]) +} + +cat(sprintf("Compared Seurat metadata for %d object(s)\n", length(args) / 2)) diff --git a/tests/conftest.py b/tests/conftest.py new file mode 100644 index 0000000..4e69f78 --- /dev/null +++ b/tests/conftest.py @@ -0,0 +1,36 @@ +def pytest_addoption(parser): + parser.addoption( + "--run-workflow", + action="store_true", + default=False, + help="run the full Snakemake workflow on the test data", + ) + parser.addoption( + "--snakemake-conda-prefix", + default=None, + help="optional value to pass to Snakemake --conda-prefix for --run-workflow", + ) + parser.addoption( + "--run-conda-validation", + action="store_true", + default=False, + help="create workflow conda environments and validate key package imports", + ) + parser.addoption( + "--conda-env-name", + default=None, + help="optional workflow/envs/*.yml filename to validate with --run-conda-validation", + ) + parser.addoption( + "--run-container-validation", + action="store_true", + default=False, + help="pull workflow containers with docker, apptainer, or singularity", + ) + parser.addoption( + "--run-doubletfinder-install", + action="store_true", + default=False, + help="execute the DoubletFinder GitHub install rule on test outputs", + ) + diff --git a/tests/reference_outputs/testdata/results/cellbender/cellbender_test.h5 b/tests/reference_outputs/testdata/results/cellbender/cellbender_test.h5 new file mode 100644 index 0000000..b8d668b Binary files /dev/null and b/tests/reference_outputs/testdata/results/cellbender/cellbender_test.h5 differ diff --git a/tests/reference_outputs/testdata/results/cellbender/cellbender_test_filtered.h5 b/tests/reference_outputs/testdata/results/cellbender/cellbender_test_filtered.h5 new file mode 100644 index 0000000..9be81a1 Binary files /dev/null and b/tests/reference_outputs/testdata/results/cellbender/cellbender_test_filtered.h5 differ diff --git a/tests/reference_outputs/testdata/results/cellbender_fromraw/seurat_cellbender_fromraw_test.rds b/tests/reference_outputs/testdata/results/cellbender_fromraw/seurat_cellbender_fromraw_test.rds new file mode 100644 index 0000000..36e4d3d Binary files /dev/null and b/tests/reference_outputs/testdata/results/cellbender_fromraw/seurat_cellbender_fromraw_test.rds differ diff --git a/tests/reference_outputs/testdata/results/cellbender_fromraw/seurat_cellbender_fromraw_test_markergenes.csv b/tests/reference_outputs/testdata/results/cellbender_fromraw/seurat_cellbender_fromraw_test_markergenes.csv new file mode 100644 index 0000000..89d2ea5 --- /dev/null +++ b/tests/reference_outputs/testdata/results/cellbender_fromraw/seurat_cellbender_fromraw_test_markergenes.csv @@ -0,0 +1,203 @@ +genesymbol,p_val,avg_log2FC,pct.1,pct.2,p_val_adj,cluster,workflow +Myt1l,3.971037213949109e-28,-3.854800385471793,0.357,0.991,3.260221552652219e-25,3,seurat_cellbender_fromraw_test +Slco1c1,9.359392871918687e-21,4.185000321904411,0.518,0.041,7.684061547845243e-18,3,seurat_cellbender_fromraw_test +Rdm1,3.6952356820901395e-19,4.510622900349515,0.446,0.027,3.033788494996005e-16,3,seurat_cellbender_fromraw_test +Nde1,1.4117230537785938e-18,4.288230479013067,0.5,0.05,1.1590246271522255e-15,3,seurat_cellbender_fromraw_test 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a/tests/reference_outputs/testdata/results/emptydrops/filtered_seurat_emptydrops_test.rds b/tests/reference_outputs/testdata/results/emptydrops/filtered_seurat_emptydrops_test.rds new file mode 100644 index 0000000..8ab1f56 Binary files /dev/null and b/tests/reference_outputs/testdata/results/emptydrops/filtered_seurat_emptydrops_test.rds differ diff --git a/tests/reference_outputs/testdata/results/emptydrops/filtered_seurat_emptydrops_test_markergenes.csv b/tests/reference_outputs/testdata/results/emptydrops/filtered_seurat_emptydrops_test_markergenes.csv new file mode 100644 index 0000000..6b3cdcb --- /dev/null +++ b/tests/reference_outputs/testdata/results/emptydrops/filtered_seurat_emptydrops_test_markergenes.csv @@ -0,0 +1,195 @@ +genesymbol,p_val,avg_log2FC,pct.1,pct.2,p_val_adj,cluster,workflow +Myt1l,5.601970901555697e-28,-3.502275092181775,0.424,0.986,4.5992181101772275e-25,3,filtered_seurat_emptydrops_test 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a/tests/reference_outputs/testdata/results/posthocfilter/seurat_posthocfilt_threshold_doubletfinder_cellbender_fromraw_test.rds b/tests/reference_outputs/testdata/results/posthocfilter/seurat_posthocfilt_threshold_doubletfinder_cellbender_fromraw_test.rds new file mode 100644 index 0000000..897ac44 Binary files /dev/null and b/tests/reference_outputs/testdata/results/posthocfilter/seurat_posthocfilt_threshold_doubletfinder_cellbender_fromraw_test.rds differ diff --git a/tests/reference_outputs/testdata/results/posthocfilter/seurat_posthocfilt_threshold_doubletfinder_cellbender_fromraw_test_markergenes.csv b/tests/reference_outputs/testdata/results/posthocfilter/seurat_posthocfilt_threshold_doubletfinder_cellbender_fromraw_test_markergenes.csv new file mode 100644 index 0000000..eccbbe7 --- /dev/null +++ b/tests/reference_outputs/testdata/results/posthocfilter/seurat_posthocfilt_threshold_doubletfinder_cellbender_fromraw_test_markergenes.csv @@ -0,0 +1,93 @@ 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a/tests/reference_outputs/testdata/results/posthocfilter/seurat_posthocfilt_threshold_doubletfinder_soupx_emptydrops_test.rds b/tests/reference_outputs/testdata/results/posthocfilter/seurat_posthocfilt_threshold_doubletfinder_soupx_emptydrops_test.rds new file mode 100644 index 0000000..154d9be Binary files /dev/null and b/tests/reference_outputs/testdata/results/posthocfilter/seurat_posthocfilt_threshold_doubletfinder_soupx_emptydrops_test.rds differ diff --git a/tests/reference_outputs/testdata/results/posthocfilter/seurat_posthocfilt_threshold_doubletfinder_soupx_emptydrops_test_markergenes.csv b/tests/reference_outputs/testdata/results/posthocfilter/seurat_posthocfilt_threshold_doubletfinder_soupx_emptydrops_test_markergenes.csv new file mode 100644 index 0000000..eea057f --- /dev/null +++ b/tests/reference_outputs/testdata/results/posthocfilter/seurat_posthocfilt_threshold_doubletfinder_soupx_emptydrops_test_markergenes.csv @@ -0,0 +1,107 @@ 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a/tests/reference_outputs/testdata/results/scdblfinder/seurat_scdblfinder_cellbender_fromraw_test.rds b/tests/reference_outputs/testdata/results/scdblfinder/seurat_scdblfinder_cellbender_fromraw_test.rds new file mode 100644 index 0000000..93113aa Binary files /dev/null and b/tests/reference_outputs/testdata/results/scdblfinder/seurat_scdblfinder_cellbender_fromraw_test.rds differ diff --git a/tests/reference_outputs/testdata/results/scdblfinder/seurat_scdblfinder_cellbender_fromraw_test_markergenes.csv b/tests/reference_outputs/testdata/results/scdblfinder/seurat_scdblfinder_cellbender_fromraw_test_markergenes.csv new file mode 100644 index 0000000..e2c4d90 --- /dev/null +++ b/tests/reference_outputs/testdata/results/scdblfinder/seurat_scdblfinder_cellbender_fromraw_test_markergenes.csv @@ -0,0 +1,194 @@ +genesymbol,p_val,avg_log2FC,pct.1,pct.2,p_val_adj,cluster,workflow 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+Hivep3,3.9823660847893635e-5,-1.023569403458554,0.57,0.704,0.03245628359103331,0,seurat_soupx_tenx_test diff --git a/tests/run_test_workflow.sh b/tests/run_test_workflow.sh new file mode 100755 index 0000000..f3136a4 --- /dev/null +++ b/tests/run_test_workflow.sh @@ -0,0 +1,74 @@ +#!/usr/bin/env bash +#SBATCH -p sapphire,shared +#SBATCH -e testdata_workflow_%A.err +#SBATCH -o testdata_workflow_%A.out +#SBATCH -J testdata_workflow +#SBATCH -N 1 +#SBATCH -c 1 +#SBATCH --mem 8000 +#SBATCH -t 72:00:00 + +set -euo pipefail + +usage() { + echo "Usage: $0 [--conda-prefix PATH] [additional snakemake args...]" +} + +script_dir=$(cd "$(dirname "${BASH_SOURCE[0]}")" && pwd) +repo_root=$(cd "${script_dir}/.." && pwd) +cd "${repo_root}" + +snakemake_conda_prefix="" +extra_snakemake_args=() + +while [[ $# -gt 0 ]]; do + case "$1" in + --conda-prefix) + if [[ $# -lt 2 ]]; then + usage >&2 + exit 2 + fi + snakemake_conda_prefix="$2" + shift 2 + ;; + -h|--help) + usage + exit 0 + ;; + *) + extra_snakemake_args+=("$1") + shift + ;; + esac +done + +if ! command -v snakemake >/dev/null 2>&1; then + echo "snakemake is not available on PATH" >&2 + exit 127 +fi + +common_args=( + --snakefile workflow/Snakefile + --configfile config/config.yaml + --config sampleTable=testdata/samplesheet_test.tsv resultsDir=testdata/results + --use-conda + --workflow-profile profiles/slurm + --profile cannon +) + +conda_prefix_args=() +if [[ -n "${snakemake_conda_prefix}" ]]; then + conda_prefix_args=(--conda-prefix "${snakemake_conda_prefix}") +fi + +snakemake --unlock "${common_args[@]}" "${conda_prefix_args[@]}" +rm -rf testdata/results + +snakemake \ + "${common_args[@]}" \ + "${conda_prefix_args[@]}" \ + --rerun-incomplete \ + --retries 2 \ + --jobs 200 \ + --latency-wait 600 \ + "${extra_snakemake_args[@]}" diff --git a/tests/test_cellbender_rule.py b/tests/test_cellbender_rule.py new file mode 100644 index 0000000..9aa2e4d --- /dev/null +++ b/tests/test_cellbender_rule.py @@ -0,0 +1,127 @@ +import os +import shutil +import subprocess +from pathlib import Path + + +TEST_SAMPLE_SHEET = Path("testdata/samplesheet_test.tsv") + + +FAKE_CELLBENDER = r"""#!/usr/bin/env bash +set -euo pipefail + +if [[ "${1:-}" == "remove-background" && "${2:-}" == "--help" ]]; then + printf '%s\\n' "cellbender remove-background" " --seed INTEGER" + exit 0 +fi + +if [[ "${1:-}" != "remove-background" ]]; then + echo "unexpected cellbender invocation: $*" >&2 + exit 2 +fi +shift + +input="" +output="" +while [[ $# -gt 0 ]]; do + case "$1" in + --input) + input="$2" + shift 2 + ;; + --output) + output="$2" + shift 2 + ;; + --seed) + shift 2 + ;; + --cuda) + shift + ;; + *) + shift + ;; + esac +done + +if [[ -z "${input}" ]]; then + echo "missing --input" >&2 + exit 3 +fi +if [[ -z "${output}" ]]; then + echo "missing --output" >&2 + exit 3 +fi +if [[ ! -s "${input}" ]]; then + echo "input does not exist or is empty: ${input}" >&2 + exit 4 +fi + +mkdir -p "$(dirname "${output}")" +printf 'fake raw cellbender output for %s\\n' "${input}" > "${output}" +filtered="${output%.h5}_filtered.h5" +printf 'fake filtered cellbender output for %s\\n' "${input}" > "${filtered}" +""" + + +def write_fake_cellbender(tmp_path): + fake_bin = tmp_path / "bin" + fake_bin.mkdir() + fake_cellbender = fake_bin / "cellbender" + fake_cellbender.write_text(FAKE_CELLBENDER) + fake_cellbender.chmod(0o755) + return fake_bin + + +def test_cellbender_rule_uses_cellbender_filtered_output_convention(tmp_path): + repo_root = Path(__file__).resolve().parents[1] + sample_sheet = repo_root / TEST_SAMPLE_SHEET + assert sample_sheet.exists(), f"missing test sample sheet: {sample_sheet}" + + snakemake = shutil.which("snakemake") + assert snakemake is not None, "snakemake is not available on PATH" + + fake_bin = write_fake_cellbender(tmp_path) + results_dir = tmp_path / "results" + base_output = results_dir / "cellbender" / "cellbender_test.h5" + filtered_output = results_dir / "cellbender" / "cellbender_test_filtered.h5" + + env = os.environ.copy() + env["PATH"] = f"{fake_bin}{os.pathsep}{env.get('PATH', '')}" + + cmd = [ + snakemake, + str(base_output), + str(filtered_output), + "--profile", + "none", + "--workflow-profile", + "none", + "--executor", + "local", + "--cores", + "1", + "--latency-wait", + "5", + "--snakefile", + "workflow/Snakefile", + "--configfile", + "config/config.yaml", + "--config", + f"sampleTable={TEST_SAMPLE_SHEET.as_posix()}", + f"resultsDir={results_dir.as_posix()}", + ] + + result = subprocess.run( + cmd, + cwd=repo_root, + env=env, + text=True, + capture_output=True, + check=False, + ) + + assert result.returncode == 0, result.stdout + result.stderr + assert base_output.read_text().startswith("fake raw cellbender output") + assert filtered_output.read_text().startswith("fake filtered cellbender output") diff --git a/tests/test_conda_container_validation.py b/tests/test_conda_container_validation.py new file mode 100644 index 0000000..ceb5140 --- /dev/null +++ b/tests/test_conda_container_validation.py @@ -0,0 +1,245 @@ +import os +import re +import shutil +import subprocess +from pathlib import Path + +import pytest +import yaml + + +ENV_DIR = Path("workflow/envs") +RULE_DIR = Path("workflow/rules") +TEST_SAMPLE_SHEET = Path("testdata/samplesheet_test.tsv") +EXPECTED_ENV_FILES = { + "cellbender.yml", + "doubletfinder.yml", + "emptydrops.yml", + "posthocfilter.yml", + "scdblfinder.yml", + "soupx.yml", + "tenx2seuratrds.yml", +} +CONDA_REFERENCE_RE = re.compile(r"conda:\s*\n\s*['\"]\.\./envs/([^'\"]+)['\"]") +CONTAINER_RE = re.compile(r"container:\s*\n\s*['\"]([^'\"]+)['\"]") + +R_IMPORTS_BY_ENV = { + "cellbender.yml": ["Seurat", "tidyverse", "bluster"], + "doubletfinder.yml": ["Seurat", "tidyverse", "remotes", "fields", "Matrix", "KernSmooth", "ROCR", "igraph", "glmGamPoi", "bluster"], + "emptydrops.yml": ["Seurat", "tidyverse", "DropletUtils", "scater", "glmGamPoi", "bluster", "Matrix", "R.utils"], + "posthocfilter.yml": ["Seurat", "tidyverse", "glmGamPoi", "scater", "bluster"], + "scdblfinder.yml": ["Seurat", "tidyverse", "scDblFinder", "glmGamPoi", "bluster"], + "soupx.yml": ["Seurat", "tidyverse", "glmGamPoi", "bluster", "SoupX"], + "tenx2seuratrds.yml": ["Seurat", "tidyverse", "scCustomize", "hdf5r", "glmGamPoi", "bluster", "presto"], +} +PYTHON_IMPORTS_BY_ENV = { + "cellbender.yml": ["cellbender"], +} + + +def repo_root(): + return Path(__file__).resolve().parents[1] + + +def read_yaml(path): + with path.open() as handle: + return yaml.safe_load(handle) + + +def combined_output(result): + return result.stdout + result.stderr + + +def run_command(cmd, cwd, env=None, timeout=120): + return subprocess.run( + cmd, + cwd=cwd, + env=env, + text=True, + capture_output=True, + check=False, + timeout=timeout, + ) + + +def available_conda_frontend(): + requested = os.environ.get("SNAKEMAKE_CONDA_FRONTEND") + if requested and shutil.which(requested): + return shutil.which(requested) + for candidate in ("mamba", "conda"): + path = shutil.which(candidate) + if path: + return path + raise AssertionError("neither mamba nor conda is available on PATH") + + +def r_require_namespace_expr(packages): + package_vector = ", ".join(repr(package) for package in packages) + return ( + f"packages <- c({package_vector}); " + "missing <- packages[!vapply(packages, requireNamespace, logical(1), quietly = TRUE)]; " + "if (length(missing)) { stop(paste('missing R packages:', paste(missing, collapse = ',')), call. = FALSE) }" + ) + + +def test_selected_conda_env_name_is_known(pytestconfig): + selected = pytestconfig.getoption("--conda-env-name") + if selected is not None: + assert selected in EXPECTED_ENV_FILES, f"unknown workflow env requested: {selected}" + + +def test_workflow_conda_env_files_are_well_formed_and_referenced_envs_exist(): + root = repo_root() + env_paths = sorted((root / ENV_DIR).glob("*.yml")) + observed = {path.name for path in env_paths} + assert observed == EXPECTED_ENV_FILES + + for env_path in env_paths: + env = read_yaml(env_path) + assert env["channels"] == ["conda-forge", "bioconda"], env_path + assert env.get("channel_priority") == "strict", env_path + assert env.get("dependencies"), env_path + + referenced = set() + for rule_path in sorted((root / RULE_DIR).glob("*.smk")): + referenced.update(CONDA_REFERENCE_RE.findall(rule_path.read_text())) + + missing_references = sorted(name for name in referenced if not (root / ENV_DIR / name).exists()) + assert not missing_references, "rule conda env references are missing: " + ", ".join(missing_references) + + +def test_workflow_container_declarations_are_explicit_and_recognized(): + root = repo_root() + containers = [] + for rule_path in sorted((root / RULE_DIR).glob("*.smk")): + for uri in CONTAINER_RE.findall(rule_path.read_text()): + containers.append((rule_path, uri)) + + assert containers, "no workflow container declarations found" + assert containers == [(root / "workflow/rules/cellbender.smk", "docker://us.gcr.io/broad-dsde-methods/cellbender:latest")] + for _, uri in containers: + assert uri.startswith("docker://") + assert ":" in uri.removeprefix("docker://"), f"container URI is missing a tag: {uri}" + + +@pytest.mark.parametrize("env_name", sorted(EXPECTED_ENV_FILES)) +def test_conda_env_solves_and_key_packages_import(tmp_path, pytestconfig, env_name): + if not pytestconfig.getoption("--run-conda-validation"): + pytest.skip("use --run-conda-validation to create and import-check workflow conda envs") + selected = pytestconfig.getoption("--conda-env-name") + if selected is not None and env_name != selected: + pytest.skip(f"only validating requested workflow env: {selected}") + + root = repo_root() + env_path = root / ENV_DIR / env_name + prefix = tmp_path / env_name.removesuffix(".yml") + conda = available_conda_frontend() + + create = run_command( + [conda, "env", "create", "--yes", "--prefix", str(prefix), "--file", str(env_path)], + root, + timeout=1800, + ) + assert create.returncode == 0, combined_output(create) + + r_packages = R_IMPORTS_BY_ENV.get(env_name, []) + if r_packages: + rscript = prefix / "bin" / "Rscript" + assert rscript.exists(), f"missing Rscript in {prefix}" + imports = run_command([str(rscript), "-e", r_require_namespace_expr(r_packages)], root, timeout=300) + assert imports.returncode == 0, combined_output(imports) + + python_packages = PYTHON_IMPORTS_BY_ENV.get(env_name, []) + if python_packages: + python = prefix / "bin" / "python" + assert python.exists(), f"missing python in {prefix}" + statements = "; ".join(f"import {package}" for package in python_packages) + imports = run_command([str(python), "-c", statements], root, timeout=300) + assert imports.returncode == 0, combined_output(imports) + + +def test_cellbender_container_can_be_pulled(tmp_path, pytestconfig): + if not pytestconfig.getoption("--run-container-validation"): + pytest.skip("use --run-container-validation to pull workflow containers") + + root = repo_root() + uri = "docker://us.gcr.io/broad-dsde-methods/cellbender:latest" + docker_image = uri.removeprefix("docker://") + + if shutil.which("docker"): + result = run_command(["docker", "pull", docker_image], root, timeout=1800) + elif shutil.which("apptainer"): + result = run_command(["apptainer", "pull", str(tmp_path / "cellbender.sif"), uri], root, timeout=1800) + elif shutil.which("singularity"): + result = run_command(["singularity", "pull", str(tmp_path / "cellbender.sif"), uri], root, timeout=1800) + else: + pytest.skip("docker, apptainer, or singularity is required for container validation") + + assert result.returncode == 0, combined_output(result) + + +def test_doubletfinder_github_install_rule_executes(tmp_path, pytestconfig): + if not pytestconfig.getoption("--run-doubletfinder-install"): + pytest.skip("use --run-doubletfinder-install to test the networked GitHub install rule") + + root = repo_root() + sample_sheet = root / TEST_SAMPLE_SHEET + assert sample_sheet.exists(), f"missing test sample sheet: {sample_sheet}" + + snakemake = shutil.which("snakemake") + assert snakemake is not None, "snakemake is not available on PATH" + + results_dir = tmp_path / "results" + conda_prefix = tmp_path / "snakemake-conda" + target = results_dir / "doubletfinder_installed.txt" + env = os.environ.copy() + conda_frontend = os.environ.get("SNAKEMAKE_CONDA_FRONTEND") + if conda_frontend is None and shutil.which("mamba"): + conda_frontend = "mamba" + + cmd = [ + snakemake, + str(target), + "--profile", + "none", + "--workflow-profile", + "none", + "--executor", + "local", + "--cores", + "1", + "--jobs", + "1", + "--latency-wait", + "30", + "--use-conda", + "--conda-prefix", + str(conda_prefix), + "--show-failed-logs", + "--snakefile", + "workflow/Snakefile", + "--configfile", + "config/config.yaml", + "--config", + f"sampleTable={TEST_SAMPLE_SHEET.as_posix()}", + f"resultsDir={results_dir.as_posix()}", + ] + if conda_frontend: + cmd.extend(["--conda-frontend", conda_frontend]) + + result = run_command(cmd, root, env=env, timeout=1800) + assert result.returncode == 0, combined_output(result) + assert target.exists(), f"missing DoubletFinder install sentinel: {target}" + + rscript_candidates = sorted(conda_prefix.glob("*/bin/Rscript")) + assert rscript_candidates, f"no Snakemake conda Rscript found under {conda_prefix}" + import_result = None + for rscript in rscript_candidates: + import_result = run_command( + [str(rscript), "-e", "quit(status = ifelse(requireNamespace('DoubletFinder', quietly = TRUE), 0, 1))"], + root, + timeout=120, + ) + if import_result.returncode == 0: + break + assert import_result is not None and import_result.returncode == 0, combined_output(import_result) diff --git a/tests/test_config_validation.py b/tests/test_config_validation.py new file mode 100644 index 0000000..bef5076 --- /dev/null +++ b/tests/test_config_validation.py @@ -0,0 +1,182 @@ +import copy +import shutil +import subprocess +from pathlib import Path + +import pytest +import yaml + + +CONFIG_FILE = Path("config/config.yaml") +TEST_SAMPLE_SHEET = Path("testdata/samplesheet_test.tsv") +REQUIRED_CONFIG_KEYS = { + "conda-channel-priority", + "sampleTable", + "workflow_seed", + "emptydrop_removal_methods", + "ambient_decon_methods", + "doublet_removal_methods", + "posthoc_methods", + "min_nfeature", + "min_ncount", + "max_mtdna", +} +ALLOWED_EMPTYDROP_METHODS = {"tenx", "emptydrops"} +ALLOWED_DECON_METHODS = {"soupx", "cellbender_fromraw"} +ALLOWED_DOUBLET_METHODS = {"doubletfinder", "scdblfinder"} +ALLOWED_POSTHOC_METHODS = {"threshold", "mad"} + + +def load_default_config(repo_root): + with (repo_root / CONFIG_FILE).open() as handle: + return yaml.safe_load(handle) + + +def write_config(path, config): + with path.open("w") as handle: + yaml.safe_dump(config, handle, sort_keys=False) + return path + + +def results_dir_path(tmp_path): + return tmp_path / "results" + + +def run_snakemake(repo_root, config_file, results_dir, *extra_args): + snakemake = shutil.which("snakemake") + assert snakemake is not None, "snakemake is not available on PATH" + + cmd = [ + snakemake, + *extra_args, + "--profile", + "none", + "--workflow-profile", + "none", + "--snakefile", + "workflow/Snakefile", + "--configfile", + str(config_file), + ] + if results_dir is not None: + cmd.extend(["--config", f"resultsDir={results_dir}"]) + return subprocess.run( + cmd, + cwd=repo_root, + text=True, + capture_output=True, + check=False, + timeout=120, + ) + + +def combined_output(result): + return result.stdout + result.stderr + + +def baseline_test_config(repo_root): + config = copy.deepcopy(load_default_config(repo_root)) + config["sampleTable"] = TEST_SAMPLE_SHEET.as_posix() + return config + + +def assert_no_duplicate_values(config, key): + values = config[key] + assert len(values) == len(set(values)), f"{key} contains duplicates" + + +def test_default_config_has_required_keys_and_valid_values(): + repo_root = Path(__file__).resolve().parents[1] + config = load_default_config(repo_root) + + assert REQUIRED_CONFIG_KEYS <= set(config) + assert config["conda-channel-priority"] == "strict" + assert isinstance(config["sampleTable"], str) and config["sampleTable"] + assert isinstance(config["workflow_seed"], int) and not isinstance(config["workflow_seed"], bool) + + for key, allowed_values in [ + ("emptydrop_removal_methods", ALLOWED_EMPTYDROP_METHODS), + ("ambient_decon_methods", ALLOWED_DECON_METHODS), + ("doublet_removal_methods", ALLOWED_DOUBLET_METHODS), + ("posthoc_methods", ALLOWED_POSTHOC_METHODS), + ]: + assert isinstance(config[key], list) and config[key] + assert set(config[key]) <= allowed_values + assert_no_duplicate_values(config, key) + + assert isinstance(config["min_nfeature"], int) and config["min_nfeature"] > 0 + assert isinstance(config["min_ncount"], int) and config["min_ncount"] > 0 + assert isinstance(config["max_mtdna"], (int, float)) and 0 <= config["max_mtdna"] <= 100 + + +@pytest.mark.parametrize( + "mutate, expected_message, override_results_dir", + [ + (lambda cfg: cfg.pop("posthoc_methods"), "missing required key(s): posthoc_methods", True), + (lambda cfg: cfg.update({"posthoc_methods": ["threshold", "bad_method"]}), "posthoc_methods contains invalid value(s): bad_method", True), + (lambda cfg: cfg.update({"doublet_removal_methods": ["scdblfinder", "scdblfinder"]}), "doublet_removal_methods contains duplicate value(s): scdblfinder", True), + (lambda cfg: cfg.update({"ambient_decon_methods": []}), "ambient_decon_methods must be a non-empty list", True), + (lambda cfg: cfg.update({"workflow_seed": "12345"}), "workflow_seed must be an integer", True), + (lambda cfg: cfg.update({"min_nfeature": 0}), "min_nfeature must be a positive integer", True), + (lambda cfg: cfg.update({"max_mtdna": 101}), "max_mtdna must be a number between 0 and 100", True), + (lambda cfg: cfg.update({"resultsDir": ""}), "resultsDir must be a non-empty string", False), + ], +) +def test_invalid_config_fails_early_with_clear_message(tmp_path, mutate, expected_message, override_results_dir): + repo_root = Path(__file__).resolve().parents[1] + config = baseline_test_config(repo_root) + mutate(config) + config_file = write_config(tmp_path / "invalid_config.yaml", config) + + results_dir = results_dir_path(tmp_path) if override_results_dir else None + result = run_snakemake(repo_root, config_file, results_dir, "-np") + output = combined_output(result) + + assert result.returncode != 0 + assert "Invalid workflow config" in output + assert expected_message in output + + +def run_variant_dry_run(tmp_path, **overrides): + repo_root = Path(__file__).resolve().parents[1] + config = baseline_test_config(repo_root) + config.update(overrides) + config_file = write_config(tmp_path / "variant_config.yaml", config) + results_dir = results_dir_path(tmp_path) + result = run_snakemake(repo_root, config_file, results_dir, "-np") + output = combined_output(result) + assert result.returncode == 0, output + return output, results_dir + + +def test_posthoc_method_variant_changes_rule_all_targets(tmp_path): + output, results_dir = run_variant_dry_run(tmp_path, posthoc_methods=["threshold"]) + + assert str(results_dir / "posthocfilter" / "seurat_posthocfilt_threshold_scdblfinder_cellbender_fromraw_test_markergenes.csv") in output + assert str(results_dir / "posthocfilter" / "seurat_posthocfilt_threshold_doubletfinder_soupx_tenx_test_markergenes.csv") in output + assert "seurat_posthocfilt_mad" not in output + + +def test_doublet_method_variant_changes_rule_all_targets(tmp_path): + output, results_dir = run_variant_dry_run(tmp_path, doublet_removal_methods=["scdblfinder"]) + + assert str(results_dir / "scdblfinder" / "seurat_scdblfinder_soupx_tenx_test_markergenes.csv") in output + assert str(results_dir / "scdblfinder" / "seurat_scdblfinder_cellbender_fromraw_test_markergenes.csv") in output + assert "seurat_doubletfinder" not in output + assert "doubletfinder_installed.txt" not in output + + +def test_decon_method_variant_can_exclude_cellbender_targets(tmp_path): + output, results_dir = run_variant_dry_run(tmp_path, ambient_decon_methods=["soupx"]) + + assert str(results_dir / "soupx" / "seurat_soupx_tenx_test_markergenes.csv") in output + assert str(results_dir / "soupx" / "seurat_soupx_emptydrops_test_markergenes.csv") in output + assert "cellbender" not in output + + +def test_emptydrop_method_variant_excludes_emptydrops_specific_targets(tmp_path): + output, results_dir = run_variant_dry_run(tmp_path, emptydrop_removal_methods=["tenx"]) + + assert str(results_dir / "seurat_filtered" / "filtered_seurat_tenx_test_markergenes.csv") in output + assert str(results_dir / "soupx" / "seurat_soupx_tenx_test_markergenes.csv") in output + assert "emptydrops" not in output diff --git a/tests/test_marker_checkpoint_expansion.py b/tests/test_marker_checkpoint_expansion.py new file mode 100644 index 0000000..4772daa --- /dev/null +++ b/tests/test_marker_checkpoint_expansion.py @@ -0,0 +1,157 @@ +import csv +import os +import shutil +import subprocess +from pathlib import Path + + +TEST_SAMPLE_SHEET = Path("testdata/samplesheet_test.tsv") +CLUSTERS = ["0", "1", "3"] + + +FAKE_RSCRIPT = r"""#!/usr/bin/env bash +set -euo pipefail + +script="${1:?missing script path}" +shift +log="${FAKE_RSCRIPT_LOG:?missing FAKE_RSCRIPT_LOG}" + +case "$(basename "${script}")" in + find_markers.R) + rds="${1:?missing input rds}" + cluster="${2:?missing cluster id}" + output="${3:?missing output csv}" + mkdir -p "$(dirname "${output}")" + printf 'find_markers\t%s\t%s\t%s\n' "${cluster}" "${rds}" "${output}" >> "${log}" + { + printf 'genesymbol,p_val,avg_log2FC,pct.1,pct.2,p_val_adj,cluster,workflow\n' + printf 'Gene%s,0.01,1.0,0.5,0.1,0.05,%s,checkpoint_test\n' "${cluster}" "${cluster}" + } > "${output}" + ;; + combine_markers.R) + output="${1:?missing output csv}" + shift + mkdir -p "$(dirname "${output}")" + printf 'combine_markers\t%s\n' "$*" >> "${log}" + : > "${output}" + header_written=0 + for marker_csv in "$@"; do + if [[ "${header_written}" -eq 0 ]]; then + cat "${marker_csv}" >> "${output}" + header_written=1 + else + tail -n +2 "${marker_csv}" >> "${output}" + fi + done + ;; + *) + echo "unexpected Rscript target: ${script}" >&2 + exit 2 + ;; +esac +""" + + +def write_fake_rscript(tmp_path): + fake_bin = tmp_path / "bin" + fake_bin.mkdir() + fake_rscript = fake_bin / "Rscript" + fake_rscript.write_text(FAKE_RSCRIPT) + fake_rscript.chmod(0o755) + return fake_bin + + +def run_command(cmd, repo_root, env): + return subprocess.run( + cmd, + cwd=repo_root, + env=env, + text=True, + capture_output=True, + check=False, + timeout=120, + ) + + +def combined_output(result): + return result.stdout + result.stderr + + +def read_marker_rows(path): + with path.open(newline="") as handle: + return list(csv.DictReader(handle)) + + +def test_marker_checkpoint_expands_find_markers_for_materialized_clusters(tmp_path): + repo_root = Path(__file__).resolve().parents[1] + sample_sheet = repo_root / TEST_SAMPLE_SHEET + assert sample_sheet.exists(), f"missing test sample sheet: {sample_sheet}" + + snakemake = shutil.which("snakemake") + assert snakemake is not None, "snakemake is not available on PATH" + + fake_bin = write_fake_rscript(tmp_path) + rscript_log = tmp_path / "fake_rscript.log" + results_dir = tmp_path / "results" + prefix = Path("checkpoint_marker") / "seurat_checkpoint_marker_test" + prefix_path = results_dir / prefix + marker_csv = prefix_path.with_name(prefix_path.name + "_markergenes.csv") + cluster_ids = prefix_path.with_name(prefix_path.name + "_cluster_ids.txt") + input_rds = prefix_path.with_suffix(".rds") + manifest_dir = prefix_path.with_name(prefix_path.name + "_marker_manifest") + + prefix_path.parent.mkdir(parents=True) + input_rds.write_text("dummy rds path consumed only by fake Rscript\n") + cluster_ids.write_text("\n".join(CLUSTERS) + "\n") + + env = os.environ.copy() + env["PATH"] = f"{fake_bin}{os.pathsep}{env.get('PATH', '')}" + env["FAKE_RSCRIPT_LOG"] = str(rscript_log) + + cmd = [ + snakemake, + str(marker_csv), + "--profile", + "none", + "--workflow-profile", + "none", + "--executor", + "local", + "--cores", + "1", + "--jobs", + "1", + "--latency-wait", + "5", + "--printshellcmds", + "--snakefile", + "workflow/Snakefile", + "--configfile", + "config/config.yaml", + "--config", + f"sampleTable={TEST_SAMPLE_SHEET.as_posix()}", + f"resultsDir={results_dir.as_posix()}", + ] + + result = run_command(cmd, repo_root, env) + output = combined_output(result) + assert result.returncode == 0, output + assert "marker_manifest" in output + assert "find_markers" in output + assert "combine_markers" in output + + rows = read_marker_rows(marker_csv) + observed_clusters = sorted(row["cluster"] for row in rows) + assert observed_clusters == CLUSTERS + assert [row["workflow"] for row in rows] == ["checkpoint_test"] * len(CLUSTERS) + + log_lines = rscript_log.read_text().splitlines() + find_lines = [line for line in log_lines if line.startswith("find_markers\t")] + combine_lines = [line for line in log_lines if line.startswith("combine_markers\t")] + assert sorted(line.split("\t")[1] for line in find_lines) == CLUSTERS + assert len(combine_lines) == 1 + for cluster in CLUSTERS: + assert f"_markergenes_cluster{cluster}.csv" in combine_lines[0] + + assert not cluster_ids.exists(), "combine_markers should remove cluster id temp input" + assert not manifest_dir.exists(), "combine_markers should remove checkpoint manifest directory" diff --git a/tests/test_r_rule_execution.py b/tests/test_r_rule_execution.py new file mode 100644 index 0000000..52603b0 --- /dev/null +++ b/tests/test_r_rule_execution.py @@ -0,0 +1,115 @@ +import os +import shutil +import subprocess +from pathlib import Path + + +TEST_SAMPLE_SHEET = Path("testdata/samplesheet_test.tsv") +VALIDATOR = Path(__file__).with_name("validate_r_rule_outputs.R") + + +def run_command(cmd, repo_root, env=None, timeout=None): + return subprocess.run( + cmd, + cwd=repo_root, + env=env, + text=True, + capture_output=True, + check=False, + timeout=timeout, + ) + + +def combined_output(result): + return result.stdout + result.stderr + + +def find_rscript_with_seuratobject(repo_root): + candidates = [] + path_rscript = shutil.which("Rscript") + if path_rscript: + candidates.append(Path(path_rscript)) + candidates.extend(sorted((repo_root / ".snakemake" / "conda").glob("*/bin/Rscript"))) + + seen = set() + for candidate in candidates: + if candidate in seen or not candidate.exists(): + continue + seen.add(candidate) + result = subprocess.run( + [ + str(candidate), + "-e", + "quit(status = ifelse(requireNamespace('SeuratObject', quietly = TRUE), 0, 1))", + ], + text=True, + capture_output=True, + check=False, + ) + if result.returncode == 0: + return candidate + + raise AssertionError("could not find an Rscript with the SeuratObject package installed") + + +def test_tenx_marker_rule_chain_executes_r_scripts_and_produces_valid_outputs(tmp_path): + repo_root = Path(__file__).resolve().parents[1] + sample_sheet = repo_root / TEST_SAMPLE_SHEET + assert sample_sheet.exists(), f"missing test sample sheet: {sample_sheet}" + assert VALIDATOR.exists(), f"missing R output validator: {VALIDATOR}" + + snakemake = shutil.which("snakemake") + assert snakemake is not None, "snakemake is not available on PATH" + + results_dir = tmp_path / "results" + seurat_rds = results_dir / "seurat_filtered" / "filtered_seurat_tenx_test.rds" + marker_csv = results_dir / "seurat_filtered" / "filtered_seurat_tenx_test_markergenes.csv" + + env = os.environ.copy() + env["SCRNASEQ_PREPROCESS_SEED"] = "12345" + + conda_frontend = os.environ.get("SNAKEMAKE_CONDA_FRONTEND") + if conda_frontend is None and shutil.which("mamba"): + conda_frontend = "mamba" + + cmd = [ + snakemake, + str(marker_csv), + "--profile", + "none", + "--workflow-profile", + "none", + "--executor", + "local", + "--cores", + "1", + "--jobs", + "1", + "--latency-wait", + "30", + "--use-conda", + "--show-failed-logs", + "--snakefile", + "workflow/Snakefile", + "--configfile", + "config/config.yaml", + "--config", + f"sampleTable={TEST_SAMPLE_SHEET.as_posix()}", + f"resultsDir={results_dir.as_posix()}", + ] + if conda_frontend: + cmd.extend(["--conda-frontend", conda_frontend]) + + result = run_command(cmd, repo_root, env=env, timeout=600) + assert result.returncode == 0, combined_output(result) + assert seurat_rds.exists(), f"missing Seurat RDS: {seurat_rds}" + assert marker_csv.exists(), f"missing marker CSV: {marker_csv}" + + rscript = find_rscript_with_seuratobject(repo_root) + validation = run_command( + [str(rscript), str(VALIDATOR), str(seurat_rds), str(marker_csv)], + repo_root, + env=env, + timeout=120, + ) + assert validation.returncode == 0, combined_output(validation) diff --git a/tests/test_reference_output_files.txt b/tests/test_reference_output_files.txt new file mode 100644 index 0000000..37e2115 --- /dev/null +++ b/tests/test_reference_output_files.txt @@ -0,0 +1,52 @@ +testdata/results/cellbender/cellbender_test.h5 +testdata/results/cellbender/cellbender_test_filtered.h5 +testdata/results/cellbender_fromraw/seurat_cellbender_fromraw_test.rds +testdata/results/cellbender_fromraw/seurat_cellbender_fromraw_test_markergenes.csv +testdata/results/doubletfinder/seurat_doubletfinder_cellbender_fromraw_test.rds +testdata/results/doubletfinder/seurat_doubletfinder_cellbender_fromraw_test_markergenes.csv +testdata/results/doubletfinder/seurat_doubletfinder_soupx_emptydrops_test.rds +testdata/results/doubletfinder/seurat_doubletfinder_soupx_emptydrops_test_markergenes.csv +testdata/results/doubletfinder/seurat_doubletfinder_soupx_tenx_test.rds +testdata/results/doubletfinder/seurat_doubletfinder_soupx_tenx_test_markergenes.csv +testdata/results/doubletfinder_installed.txt +testdata/results/emptydrops/filtered_seurat_emptydrops_test.rds +testdata/results/emptydrops/filtered_seurat_emptydrops_test_markergenes.csv +testdata/results/emptydrops/test_emptydrops_filtered_matrix/barcodes.tsv.gz +testdata/results/emptydrops/test_emptydrops_filtered_matrix/features.tsv.gz +testdata/results/emptydrops/test_emptydrops_filtered_matrix/matrix.mtx.gz +testdata/results/posthocfilter/seurat_posthocfilt_mad_doubletfinder_cellbender_fromraw_test.rds +testdata/results/posthocfilter/seurat_posthocfilt_mad_doubletfinder_cellbender_fromraw_test_markergenes.csv +testdata/results/posthocfilter/seurat_posthocfilt_mad_doubletfinder_soupx_emptydrops_test.rds +testdata/results/posthocfilter/seurat_posthocfilt_mad_doubletfinder_soupx_emptydrops_test_markergenes.csv +testdata/results/posthocfilter/seurat_posthocfilt_mad_doubletfinder_soupx_tenx_test.rds +testdata/results/posthocfilter/seurat_posthocfilt_mad_doubletfinder_soupx_tenx_test_markergenes.csv +testdata/results/posthocfilter/seurat_posthocfilt_mad_scdblfinder_cellbender_fromraw_test.rds +testdata/results/posthocfilter/seurat_posthocfilt_mad_scdblfinder_cellbender_fromraw_test_markergenes.csv +testdata/results/posthocfilter/seurat_posthocfilt_mad_scdblfinder_soupx_emptydrops_test.rds +testdata/results/posthocfilter/seurat_posthocfilt_mad_scdblfinder_soupx_emptydrops_test_markergenes.csv +testdata/results/posthocfilter/seurat_posthocfilt_mad_scdblfinder_soupx_tenx_test.rds +testdata/results/posthocfilter/seurat_posthocfilt_mad_scdblfinder_soupx_tenx_test_markergenes.csv +testdata/results/posthocfilter/seurat_posthocfilt_threshold_doubletfinder_cellbender_fromraw_test.rds +testdata/results/posthocfilter/seurat_posthocfilt_threshold_doubletfinder_cellbender_fromraw_test_markergenes.csv +testdata/results/posthocfilter/seurat_posthocfilt_threshold_doubletfinder_soupx_emptydrops_test.rds +testdata/results/posthocfilter/seurat_posthocfilt_threshold_doubletfinder_soupx_emptydrops_test_markergenes.csv +testdata/results/posthocfilter/seurat_posthocfilt_threshold_doubletfinder_soupx_tenx_test.rds +testdata/results/posthocfilter/seurat_posthocfilt_threshold_doubletfinder_soupx_tenx_test_markergenes.csv +testdata/results/posthocfilter/seurat_posthocfilt_threshold_scdblfinder_cellbender_fromraw_test.rds +testdata/results/posthocfilter/seurat_posthocfilt_threshold_scdblfinder_cellbender_fromraw_test_markergenes.csv +testdata/results/posthocfilter/seurat_posthocfilt_threshold_scdblfinder_soupx_emptydrops_test.rds +testdata/results/posthocfilter/seurat_posthocfilt_threshold_scdblfinder_soupx_emptydrops_test_markergenes.csv +testdata/results/posthocfilter/seurat_posthocfilt_threshold_scdblfinder_soupx_tenx_test.rds +testdata/results/posthocfilter/seurat_posthocfilt_threshold_scdblfinder_soupx_tenx_test_markergenes.csv +testdata/results/scdblfinder/seurat_scdblfinder_cellbender_fromraw_test.rds +testdata/results/scdblfinder/seurat_scdblfinder_cellbender_fromraw_test_markergenes.csv +testdata/results/scdblfinder/seurat_scdblfinder_soupx_emptydrops_test.rds +testdata/results/scdblfinder/seurat_scdblfinder_soupx_emptydrops_test_markergenes.csv +testdata/results/scdblfinder/seurat_scdblfinder_soupx_tenx_test.rds +testdata/results/scdblfinder/seurat_scdblfinder_soupx_tenx_test_markergenes.csv +testdata/results/seurat_filtered/filtered_seurat_tenx_test.rds +testdata/results/seurat_filtered/filtered_seurat_tenx_test_markergenes.csv +testdata/results/soupx/seurat_soupx_emptydrops_test.rds +testdata/results/soupx/seurat_soupx_emptydrops_test_markergenes.csv +testdata/results/soupx/seurat_soupx_tenx_test.rds +testdata/results/soupx/seurat_soupx_tenx_test_markergenes.csv diff --git a/tests/test_sample_rule_output_files.txt b/tests/test_sample_rule_output_files.txt new file mode 100644 index 0000000..460fad6 --- /dev/null +++ b/tests/test_sample_rule_output_files.txt @@ -0,0 +1,51 @@ +testdata/results/cellbender/cellbender_test.h5 +testdata/results/cellbender/cellbender_test_filtered.h5 +testdata/results/cellbender_fromraw/seurat_cellbender_fromraw_test.rds +testdata/results/cellbender_fromraw/seurat_cellbender_fromraw_test_markergenes.csv +testdata/results/doubletfinder/seurat_doubletfinder_cellbender_fromraw_test.rds +testdata/results/doubletfinder/seurat_doubletfinder_cellbender_fromraw_test_markergenes.csv +testdata/results/doubletfinder/seurat_doubletfinder_soupx_emptydrops_test.rds +testdata/results/doubletfinder/seurat_doubletfinder_soupx_emptydrops_test_markergenes.csv +testdata/results/doubletfinder/seurat_doubletfinder_soupx_tenx_test.rds +testdata/results/doubletfinder/seurat_doubletfinder_soupx_tenx_test_markergenes.csv +testdata/results/emptydrops/filtered_seurat_emptydrops_test.rds +testdata/results/emptydrops/filtered_seurat_emptydrops_test_markergenes.csv +testdata/results/emptydrops/test_emptydrops_filtered_matrix/barcodes.tsv.gz +testdata/results/emptydrops/test_emptydrops_filtered_matrix/features.tsv.gz +testdata/results/emptydrops/test_emptydrops_filtered_matrix/matrix.mtx.gz +testdata/results/posthocfilter/seurat_posthocfilt_mad_doubletfinder_cellbender_fromraw_test.rds +testdata/results/posthocfilter/seurat_posthocfilt_mad_doubletfinder_cellbender_fromraw_test_markergenes.csv +testdata/results/posthocfilter/seurat_posthocfilt_mad_doubletfinder_soupx_emptydrops_test.rds +testdata/results/posthocfilter/seurat_posthocfilt_mad_doubletfinder_soupx_emptydrops_test_markergenes.csv +testdata/results/posthocfilter/seurat_posthocfilt_mad_doubletfinder_soupx_tenx_test.rds +testdata/results/posthocfilter/seurat_posthocfilt_mad_doubletfinder_soupx_tenx_test_markergenes.csv +testdata/results/posthocfilter/seurat_posthocfilt_mad_scdblfinder_cellbender_fromraw_test.rds +testdata/results/posthocfilter/seurat_posthocfilt_mad_scdblfinder_cellbender_fromraw_test_markergenes.csv +testdata/results/posthocfilter/seurat_posthocfilt_mad_scdblfinder_soupx_emptydrops_test.rds +testdata/results/posthocfilter/seurat_posthocfilt_mad_scdblfinder_soupx_emptydrops_test_markergenes.csv +testdata/results/posthocfilter/seurat_posthocfilt_mad_scdblfinder_soupx_tenx_test.rds +testdata/results/posthocfilter/seurat_posthocfilt_mad_scdblfinder_soupx_tenx_test_markergenes.csv +testdata/results/posthocfilter/seurat_posthocfilt_threshold_doubletfinder_cellbender_fromraw_test.rds +testdata/results/posthocfilter/seurat_posthocfilt_threshold_doubletfinder_cellbender_fromraw_test_markergenes.csv +testdata/results/posthocfilter/seurat_posthocfilt_threshold_doubletfinder_soupx_emptydrops_test.rds +testdata/results/posthocfilter/seurat_posthocfilt_threshold_doubletfinder_soupx_emptydrops_test_markergenes.csv +testdata/results/posthocfilter/seurat_posthocfilt_threshold_doubletfinder_soupx_tenx_test.rds +testdata/results/posthocfilter/seurat_posthocfilt_threshold_doubletfinder_soupx_tenx_test_markergenes.csv +testdata/results/posthocfilter/seurat_posthocfilt_threshold_scdblfinder_cellbender_fromraw_test.rds +testdata/results/posthocfilter/seurat_posthocfilt_threshold_scdblfinder_cellbender_fromraw_test_markergenes.csv +testdata/results/posthocfilter/seurat_posthocfilt_threshold_scdblfinder_soupx_emptydrops_test.rds +testdata/results/posthocfilter/seurat_posthocfilt_threshold_scdblfinder_soupx_emptydrops_test_markergenes.csv +testdata/results/posthocfilter/seurat_posthocfilt_threshold_scdblfinder_soupx_tenx_test.rds +testdata/results/posthocfilter/seurat_posthocfilt_threshold_scdblfinder_soupx_tenx_test_markergenes.csv +testdata/results/scdblfinder/seurat_scdblfinder_cellbender_fromraw_test.rds +testdata/results/scdblfinder/seurat_scdblfinder_cellbender_fromraw_test_markergenes.csv +testdata/results/scdblfinder/seurat_scdblfinder_soupx_emptydrops_test.rds +testdata/results/scdblfinder/seurat_scdblfinder_soupx_emptydrops_test_markergenes.csv +testdata/results/scdblfinder/seurat_scdblfinder_soupx_tenx_test.rds +testdata/results/scdblfinder/seurat_scdblfinder_soupx_tenx_test_markergenes.csv +testdata/results/seurat_filtered/filtered_seurat_tenx_test.rds +testdata/results/seurat_filtered/filtered_seurat_tenx_test_markergenes.csv +testdata/results/soupx/seurat_soupx_emptydrops_test.rds +testdata/results/soupx/seurat_soupx_emptydrops_test_markergenes.csv +testdata/results/soupx/seurat_soupx_tenx_test.rds +testdata/results/soupx/seurat_soupx_tenx_test_markergenes.csv diff --git a/tests/test_sample_sheet_validation.py b/tests/test_sample_sheet_validation.py new file mode 100644 index 0000000..f0d179d --- /dev/null +++ b/tests/test_sample_sheet_validation.py @@ -0,0 +1,180 @@ +import shutil +import subprocess +from pathlib import Path + + +REQUIRED_TENX_CHILDREN = [ + "filtered_feature_bc_matrix", + "raw_feature_bc_matrix", + "raw_feature_bc_matrix.h5", +] + + +def run_snakemake_with_sample_sheet(repo_root, sample_sheet, results_dir, *extra_args): + snakemake = shutil.which("snakemake") + assert snakemake is not None, "snakemake is not available on PATH" + + cmd = [ + snakemake, + *extra_args, + "--profile", + "none", + "--workflow-profile", + "none", + "--snakefile", + "workflow/Snakefile", + "--configfile", + "config/config.yaml", + "--config", + f"sampleTable={sample_sheet}", + f"resultsDir={results_dir}", + ] + return subprocess.run( + cmd, + cwd=repo_root, + text=True, + capture_output=True, + check=False, + timeout=120, + ) + + +def combined_output(result): + return result.stdout + result.stderr + + +def write_sample_sheet(path, rows, header="sampleid\ttenx_datadir"): + lines = [header] + lines.extend(f"{sampleid}\t{tenx_datadir}" for sampleid, tenx_datadir in rows) + path.write_text("\n".join(lines) + "\n") + return path + + +def test_malformed_sample_sheet_missing_required_column_fails(tmp_path): + repo_root = Path(__file__).resolve().parents[1] + sample_sheet = tmp_path / "malformed_samplesheet.tsv" + sample_sheet.write_text("sampleid\nmissing_tenx_column\n") + + result = run_snakemake_with_sample_sheet( + repo_root, + sample_sheet, + tmp_path / "results", + "-np", + ) + output = combined_output(result) + + assert result.returncode != 0 + assert "Invalid sample sheet" in output + assert "missing required column(s): tenx_datadir" in output + + +def test_duplicate_sample_ids_fail_validation(tmp_path): + repo_root = Path(__file__).resolve().parents[1] + sample_sheet = write_sample_sheet( + tmp_path / "duplicate_samplesheet.tsv", + [("duplicate", "testdata"), ("duplicate", "testdata")], + ) + + result = run_snakemake_with_sample_sheet( + repo_root, + sample_sheet, + tmp_path / "results", + "-np", + ) + output = combined_output(result) + + assert result.returncode != 0 + assert "Invalid sample sheet" in output + assert "duplicate sampleid value(s): duplicate" in output + + +def test_missing_tenx_datadir_fails_validation(tmp_path): + repo_root = Path(__file__).resolve().parents[1] + missing_data_dir = tmp_path / "does_not_exist" + sample_sheet = write_sample_sheet( + tmp_path / "missing_data_samplesheet.tsv", + [("missing_path", missing_data_dir)], + ) + + result = run_snakemake_with_sample_sheet( + repo_root, + sample_sheet, + tmp_path / "results", + "-np", + ) + output = combined_output(result) + + assert result.returncode != 0 + assert "Invalid sample sheet" in output + assert "tenx_datadir does not exist" in output + assert str(missing_data_dir) in output + + +def test_incomplete_tenx_datadir_fails_with_missing_required_children(tmp_path): + repo_root = Path(__file__).resolve().parents[1] + incomplete_data_dir = tmp_path / "incomplete_testdata" + incomplete_data_dir.mkdir() + (incomplete_data_dir / "filtered_feature_bc_matrix").mkdir() + + sample_sheet = write_sample_sheet( + tmp_path / "incomplete_data_samplesheet.tsv", + [("incomplete", incomplete_data_dir)], + ) + + result = run_snakemake_with_sample_sheet( + repo_root, + sample_sheet, + tmp_path / "results", + "-np", + ) + output = combined_output(result) + + assert result.returncode != 0 + assert "Invalid sample sheet" in output + assert "missing raw_feature_bc_matrix" in output + assert "missing raw_feature_bc_matrix.h5" in output + + +def test_absolute_tenx_datadir_is_normalized_and_used_for_rule_inputs(tmp_path): + repo_root = Path(__file__).resolve().parents[1] + absolute_testdata = (repo_root / "testdata").resolve() + sample_sheet = write_sample_sheet( + tmp_path / "absolute_data_samplesheet.tsv", + [("absolute_path", absolute_testdata)], + ) + + result = run_snakemake_with_sample_sheet( + repo_root, + sample_sheet, + tmp_path / "results", + "-np", + ) + output = combined_output(result) + + assert result.returncode == 0, output + for child in REQUIRED_TENX_CHILDREN: + assert str(absolute_testdata / child) in output + + +def test_sample_sheet_with_multiple_samples_expands_outputs_for_each_sample(tmp_path): + repo_root = Path(__file__).resolve().parents[1] + sample_sheet = write_sample_sheet( + tmp_path / "multi_sample_samplesheet.tsv", + [("test_a", "testdata"), ("test_b", "testdata")], + ) + results_dir = tmp_path / "results" + + result = run_snakemake_with_sample_sheet( + repo_root, + sample_sheet, + results_dir, + "-np", + ) + output = combined_output(result) + + assert result.returncode == 0, output + for sample_id in ["test_a", "test_b"]: + assert f"wildcards: sample={sample_id}" in output + assert str(results_dir / "seurat_filtered" / f"filtered_seurat_tenx_{sample_id}_markergenes.csv") in output + assert str(results_dir / "cellbender" / f"cellbender_{sample_id}.h5") in output + assert str(results_dir / "cellbender" / f"cellbender_{sample_id}_filtered.h5") in output diff --git a/tests/test_snakemake_dry_run.py b/tests/test_snakemake_dry_run.py new file mode 100644 index 0000000..c03186c --- /dev/null +++ b/tests/test_snakemake_dry_run.py @@ -0,0 +1,126 @@ +import csv +import shutil +import subprocess +from pathlib import Path + + + +SAMPLE_ID = "test" +TEST_SAMPLE_SHEET = Path("testdata/samplesheet_test.tsv") +RESULTS_DIR = "testdata/results" +EXPECTED_OUTPUTS = Path(__file__).with_name("test_sample_rule_output_files.txt") + + +def run_snakemake(repo_root, *extra_args): + sample_sheet = repo_root / TEST_SAMPLE_SHEET + assert sample_sheet.exists(), f"missing test sample sheet: {sample_sheet}" + + snakemake = shutil.which("snakemake") + assert snakemake is not None, "snakemake is not available on PATH" + + cmd = [ + snakemake, + *extra_args, + "--snakefile", + "workflow/Snakefile", + "--configfile", + "config/config.yaml", + "--config", + f"sampleTable={TEST_SAMPLE_SHEET.as_posix()}", + f"resultsDir={RESULTS_DIR}", + ] + return subprocess.run( + cmd, + cwd=repo_root, + text=True, + capture_output=True, + check=False, + ) + + +def combined_output(result): + return result.stdout + result.stderr + + +def test_testdata_dag_builds_with_dry_run(): + repo_root = Path(__file__).resolve().parents[1] + result = run_snakemake(repo_root, "-np") + output = combined_output(result) + + assert result.returncode == 0, output + assert "Building DAG of jobs" in output + assert "This was a dry-run" in output + for rule_name in [ + "tenx2seuratrds", + "emptydrops", + "soupx", + "soupx_emptydrops", + "cellbender", + "cellbender2seurat", + "doubletfinder", + "scdblfinder", + "posthocfilter_threshold", + "posthocfilter_mad", + "combine_markers", + ]: + assert rule_name in output + + +def is_internal_workflow_output(output): + return ( + "_markergenes_cluster" in output + or output.endswith("_cluster_ids.txt") + or output.endswith("_nclusters.txt") + or output.endswith("_marker_manifest") + ) + + +def declared_sample_output_files(summary_text, expected_manifest): + lines = summary_text.splitlines() + header_index = next( + i for i, line in enumerate(lines) if line.startswith("output_file\t") + ) + reader = csv.DictReader(lines[header_index:], delimiter="\t") + + outputs = [] + for row in reader: + output = row["output_file"] + if f"_{SAMPLE_ID}" not in output and f"/{SAMPLE_ID}_" not in output: + continue + if row["status"] == "removed temp file": + continue + if is_internal_workflow_output(output): + continue + + directory_children = [ + expected + for expected in expected_manifest + if expected.startswith(f"{output}/") + ] + if directory_children: + outputs.extend(directory_children) + else: + outputs.append(output) + + return sorted(set(outputs)) + + +def test_declared_test_sample_outputs_match_manifest(): + repo_root = Path(__file__).resolve().parents[1] + expected = EXPECTED_OUTPUTS.read_text().splitlines() + + result = run_snakemake(repo_root, "--summary") + output = combined_output(result) + + assert result.returncode == 0, output + + observed = declared_sample_output_files(output, expected) + + missing = sorted(set(expected) - set(observed)) + unexpected = sorted(set(observed) - set(expected)) + + assert observed == expected, ( + "declared outputs differ from manifest\n" + f"missing: {missing}\n" + f"unexpected: {unexpected}" + ) diff --git a/tests/test_snakemake_lint.py b/tests/test_snakemake_lint.py new file mode 100644 index 0000000..ba252d8 --- /dev/null +++ b/tests/test_snakemake_lint.py @@ -0,0 +1,33 @@ +import shutil +import subprocess +from pathlib import Path + + +TEST_SAMPLE_SHEET = Path("testdata/samplesheet_test.tsv") + + +def test_snakemake_lint_has_no_findings(): + repo_root = Path(__file__).resolve().parents[1] + snakemake = shutil.which("snakemake") + assert snakemake is not None, "snakemake is not available on PATH" + + cmd = [ + snakemake, + "--lint", + "--snakefile", + "workflow/Snakefile", + "--configfile", + "config/config.yaml", + "--config", + f"sampleTable={TEST_SAMPLE_SHEET.as_posix()}", + "resultsDir=testdata/results", + ] + result = subprocess.run( + cmd, + cwd=repo_root, + text=True, + capture_output=True, + check=False, + timeout=120, + ) + assert result.returncode == 0, result.stdout + result.stderr diff --git a/tests/test_snakemake_workflow_run.py b/tests/test_snakemake_workflow_run.py new file mode 100644 index 0000000..256f0cc --- /dev/null +++ b/tests/test_snakemake_workflow_run.py @@ -0,0 +1,273 @@ +import csv +import gzip +import hashlib +import math +import shutil +import subprocess +from pathlib import Path + +import pytest + + +EXPECTED_OUTPUTS = Path(__file__).with_name("test_sample_rule_output_files.txt") +REFERENCE_OUTPUTS = Path(__file__).with_name("test_reference_output_files.txt") +REFERENCE_ROOT = Path(__file__).with_name("reference_outputs") +SEURAT_METADATA_COMPARATOR = Path(__file__).with_name("compare_seurat_metadata.R") +MARKER_ABS_TOLERANCE = 1e-8 +MARKER_REL_TOLERANCE = 1e-6 + + +def read_manifest(path): + return [line.strip() for line in path.read_text().splitlines() if line.strip()] + + +def file_sha256(path): + digest = hashlib.sha256() + with path.open("rb") as handle: + for chunk in iter(lambda: handle.read(1024 * 1024), b""): + digest.update(chunk) + return digest.hexdigest() + + +def is_seurat_object(path): + return path.endswith(".rds") + + +def is_marker_table(path): + return path.endswith("_markergenes.csv") + + +def is_emptydrops_matrix_component(path): + return "/emptydrops/test_emptydrops_filtered_matrix/" in path and path.endswith(".gz") + + +def is_cellbender_h5(path): + return path.startswith("testdata/results/cellbender/") and path.endswith(".h5") + + +def find_rscript_with_seurat(repo_root): + candidates = [] + path_rscript = shutil.which("Rscript") + if path_rscript: + candidates.append(Path(path_rscript)) + candidates.extend(sorted((repo_root / ".snakemake" / "conda").glob("*/bin/Rscript"))) + + checked = [] + for candidate in candidates: + if candidate in checked or not candidate.exists(): + continue + checked.append(candidate) + result = subprocess.run( + [ + str(candidate), + "-e", + "quit(status = ifelse(requireNamespace('Seurat', quietly = TRUE), 0, 1))", + ], + text=True, + capture_output=True, + check=False, + ) + if result.returncode == 0: + return candidate + + raise AssertionError("could not find an Rscript with the Seurat package installed") + + +def assert_seurat_metadata_matches(repo_root, paths): + if not paths: + return + + rscript = find_rscript_with_seurat(repo_root) + cmd = [str(rscript), str(SEURAT_METADATA_COMPARATOR)] + for rel_path in paths: + cmd.extend([str(repo_root / rel_path), str(REFERENCE_ROOT / rel_path)]) + + result = subprocess.run(cmd, text=True, capture_output=True, check=False) + assert result.returncode == 0, result.stdout + result.stderr + + +def read_csv_table(path): + with path.open(newline="") as handle: + reader = csv.DictReader(handle) + rows = list(reader) + return reader.fieldnames or [], rows + + +def numeric_value(value): + if value == "": + return None + try: + parsed = float(value) + except ValueError: + return None + if math.isnan(parsed): + return None + return parsed + + +def assert_marker_value_matches(path, column, current, reference): + current_numeric = numeric_value(current) + reference_numeric = numeric_value(reference) + if current_numeric is not None and reference_numeric is not None: + assert math.isclose( + current_numeric, + reference_numeric, + rel_tol=MARKER_REL_TOLERANCE, + abs_tol=MARKER_ABS_TOLERANCE, + ), ( + f"{path}: marker value differs for column {column}: " + f"{current} != {reference}" + ) + return + + assert current == reference, ( + f"{path}: marker value differs for column {column}: {current!r} != {reference!r}" + ) + + +def marker_rows_by_cluster_gene(path, rows): + keys = [] + indexed = {} + for row in rows: + key = (row.get("cluster"), row.get("genesymbol")) + keys.append(key) + indexed[key] = row + + assert len(indexed) == len(rows), f"{path}: duplicate cluster/genesymbol rows found" + return keys, indexed + + +def assert_marker_tables_match(repo_root, paths): + for rel_path in paths: + current_path = repo_root / rel_path + reference_path = REFERENCE_ROOT / rel_path + current_columns, current_rows = read_csv_table(current_path) + reference_columns, reference_rows = read_csv_table(reference_path) + + assert current_columns == reference_columns, ( + f"{rel_path}: marker columns differ\n" + f"current: {current_columns}\nreference: {reference_columns}" + ) + assert "cluster" in current_columns, f"{rel_path}: missing cluster column" + assert "genesymbol" in current_columns, f"{rel_path}: missing genesymbol column" + + current_keys, current_index = marker_rows_by_cluster_gene(rel_path, current_rows) + reference_keys, reference_index = marker_rows_by_cluster_gene(rel_path, reference_rows) + + assert sorted(current_keys) == sorted(reference_keys), ( + f"{rel_path}: marker genes differ by cluster" + ) + + for key in sorted(reference_keys): + current_row = current_index[key] + reference_row = reference_index[key] + for column in current_columns: + assert_marker_value_matches( + rel_path, + column, + current_row[column], + reference_row[column], + ) + + +def read_gzip_contents(path): + with gzip.open(path, "rb") as handle: + return handle.read() + + +def assert_emptydrops_matrix_contents_match(repo_root, paths): + for rel_path in paths: + current = read_gzip_contents(repo_root / rel_path) + reference = read_gzip_contents(REFERENCE_ROOT / rel_path) + assert current == reference, f"{rel_path}: decompressed matrix component differs" + + +def assert_h5_contents_match(repo_root, paths): + for rel_path in paths: + result = subprocess.run( + ["h5diff", "-q", str(repo_root / rel_path), str(REFERENCE_ROOT / rel_path)], + text=True, + capture_output=True, + check=False, + ) + assert result.returncode == 0, ( + f"{rel_path}: HDF5 contents differ\n" + result.stdout + result.stderr + ) + + +def assert_byte_identical(repo_root, paths): + mismatches = [] + for rel_path in paths: + current = repo_root / rel_path + reference = REFERENCE_ROOT / rel_path + if file_sha256(current) != file_sha256(reference): + mismatches.append(rel_path) + + assert not mismatches, ( + "current outputs differ byte-for-byte from reference outputs:\n" + + "\n".join(mismatches[:50]) + ) + + +def assert_current_outputs_match_reference(repo_root): + expected = read_manifest(REFERENCE_OUTPUTS) + + missing_current = [path for path in expected if not (repo_root / path).exists()] + missing_reference = [ + path for path in expected if not (REFERENCE_ROOT / path).exists() + ] + assert not missing_current, ( + "missing current outputs listed in reference manifest:\n" + + "\n".join(missing_current[:50]) + ) + assert not missing_reference, ( + "missing reference outputs listed in reference manifest:\n" + + "\n".join(missing_reference[:50]) + ) + + seurat_objects = [path for path in expected if is_seurat_object(path)] + marker_tables = [path for path in expected if is_marker_table(path)] + matrix_components = [ + path for path in expected if is_emptydrops_matrix_component(path) + ] + h5_outputs = [path for path in expected if is_cellbender_h5(path)] + byte_identical = [ + path + for path in expected + if path not in set(seurat_objects + marker_tables + matrix_components + h5_outputs) + ] + + assert_seurat_metadata_matches(repo_root, seurat_objects) + assert_marker_tables_match(repo_root, marker_tables) + assert_emptydrops_matrix_contents_match(repo_root, matrix_components) + assert_h5_contents_match(repo_root, h5_outputs) + assert_byte_identical(repo_root, byte_identical) + + +def test_testdata_workflow_run(pytestconfig): + if not pytestconfig.getoption("--run-workflow"): + pytest.skip("use --run-workflow to run the full workflow on testdata") + + repo_root = Path(__file__).resolve().parents[1] + cmd = ["bash", "tests/run_test_workflow.sh"] + + conda_prefix = pytestconfig.getoption("--snakemake-conda-prefix") + if conda_prefix: + cmd.extend(["--conda-prefix", conda_prefix]) + + result = subprocess.run( + cmd, + cwd=repo_root, + text=True, + capture_output=True, + check=False, + ) + + assert result.returncode == 0, result.stdout + result.stderr + + expected = read_manifest(EXPECTED_OUTPUTS) + missing = [path for path in expected if not (repo_root / path).exists()] + + assert not missing, "missing expected workflow outputs:\n" + "\n".join(missing[:50]) + + assert_current_outputs_match_reference(repo_root) diff --git a/tests/update_reference_outputs.py b/tests/update_reference_outputs.py new file mode 100755 index 0000000..a701510 --- /dev/null +++ b/tests/update_reference_outputs.py @@ -0,0 +1,34 @@ +#!/usr/bin/env python3 +import shutil +from pathlib import Path + + +REPO_ROOT = Path(__file__).resolve().parents[1] +MANIFEST = Path(__file__).with_name("test_reference_output_files.txt") +REFERENCE_ROOT = Path(__file__).with_name("reference_outputs") + + +def read_manifest(path): + return [line.strip() for line in path.read_text().splitlines() if line.strip()] + + +def main(): + outputs = read_manifest(MANIFEST) + missing = [path for path in outputs if not (REPO_ROOT / path).exists()] + if missing: + raise SystemExit( + "Cannot update reference outputs; missing current outputs:\n" + + "\n".join(missing) + ) + + for rel_path in outputs: + source = REPO_ROOT / rel_path + target = REFERENCE_ROOT / rel_path + target.parent.mkdir(parents=True, exist_ok=True) + shutil.copy2(source, target) + + print(f"Copied {len(outputs)} reference output files to {REFERENCE_ROOT}") + + +if __name__ == "__main__": + main() diff --git a/tests/validate_r_rule_outputs.R b/tests/validate_r_rule_outputs.R new file mode 100644 index 0000000..bb38f25 --- /dev/null +++ b/tests/validate_r_rule_outputs.R @@ -0,0 +1,122 @@ +args <- commandArgs(trailingOnly = TRUE) +if (length(args) != 2) { + stop("usage: validate_r_rule_outputs.R SEURAT_RDS MARKER_CSV", call. = FALSE) +} + +rds_path <- args[[1]] +marker_path <- args[[2]] + +suppressPackageStartupMessages({ + library(SeuratObject) +}) + +fail <- function(message) { + stop(message, call. = FALSE) +} + +if (!file.exists(rds_path) || file.info(rds_path)$size == 0) { + fail(paste("missing or empty Seurat RDS:", rds_path)) +} +if (!file.exists(marker_path) || file.info(marker_path)$size == 0) { + fail(paste("missing or empty marker CSV:", marker_path)) +} + +obj <- readRDS(rds_path) +if (!inherits(obj, "Seurat")) { + fail(paste("RDS is not a Seurat object; classes:", paste(class(obj), collapse = ","))) +} + +n_features <- nrow(obj) +n_cells <- ncol(obj) +if (n_features < 100) { + fail(paste("Seurat object has too few features:", n_features)) +} +if (n_cells < 100) { + fail(paste("Seurat object has too few cells:", n_cells)) +} + +metadata <- obj@meta.data +if (nrow(metadata) != n_cells) { + fail("metadata row count does not match Seurat cell count") +} +if (anyDuplicated(rownames(metadata))) { + fail("metadata contains duplicate cell barcodes") +} +if (any(is.na(rownames(metadata))) || any(rownames(metadata) == "")) { + fail("metadata contains missing or empty cell barcodes") +} + +required_metadata <- c("orig.ident", "nCount_RNA", "nFeature_RNA", "percent.mt", "seurat_clusters") +missing_metadata <- setdiff(required_metadata, colnames(metadata)) +if (length(missing_metadata) > 0) { + fail(paste("metadata missing columns:", paste(missing_metadata, collapse = ","))) +} + +if (any(!is.finite(metadata$nCount_RNA)) || any(metadata$nCount_RNA <= 0)) { + fail("metadata nCount_RNA contains non-positive or non-finite values") +} +if (any(!is.finite(metadata$nFeature_RNA)) || any(metadata$nFeature_RNA <= 0)) { + fail("metadata nFeature_RNA contains non-positive or non-finite values") +} +if (any(!is.finite(metadata$percent.mt)) || any(metadata$percent.mt < 0 | metadata$percent.mt > 100)) { + fail("metadata percent.mt contains values outside [0, 100]") +} + +cluster_ids <- as.character(metadata$seurat_clusters) +if (length(unique(cluster_ids)) < 2) { + fail("Seurat object has fewer than two clusters") +} + +required_reductions <- c("pca", "umap") +missing_reductions <- setdiff(required_reductions, names(obj@reductions)) +if (length(missing_reductions) > 0) { + fail(paste("Seurat object missing reductions:", paste(missing_reductions, collapse = ","))) +} + +markers <- read.csv(marker_path, stringsAsFactors = FALSE, check.names = FALSE) +expected_marker_columns <- c("genesymbol", "p_val", "avg_log2FC", "pct.1", "pct.2", "p_val_adj", "cluster", "workflow") +if (!identical(colnames(markers), expected_marker_columns)) { + fail(paste( + "marker CSV columns differ; observed:", paste(colnames(markers), collapse = ","), + "expected:", paste(expected_marker_columns, collapse = ",") + )) +} +if (nrow(markers) == 0) { + fail("marker CSV has no rows") +} +if (any(is.na(markers$genesymbol)) || any(markers$genesymbol == "")) { + fail("marker CSV contains missing or empty genesymbol values") +} + +numeric_columns <- c("p_val", "avg_log2FC", "pct.1", "pct.2", "p_val_adj") +for (column in numeric_columns) { + values <- markers[[column]] + if (any(!is.finite(values))) { + fail(paste("marker CSV column has non-finite values:", column)) + } +} +for (column in c("p_val", "pct.1", "pct.2", "p_val_adj")) { + values <- markers[[column]] + if (any(values < 0 | values > 1)) { + fail(paste("marker CSV column has values outside [0, 1]:", column)) + } +} + +marker_clusters <- as.character(markers$cluster) +if (!all(marker_clusters %in% cluster_ids)) { + fail("marker CSV contains clusters not present in Seurat metadata") +} +if (length(unique(marker_clusters)) < 2) { + fail("marker CSV has markers for fewer than two clusters") +} +if (!all(markers$workflow == "filtered_seurat_tenx_test")) { + fail("marker CSV workflow column does not match filtered_seurat_tenx_test") +} + +cat(sprintf( + "validated R rule outputs: %d features, %d cells, %d marker rows, %d clusters\n", + n_features, + n_cells, + nrow(markers), + length(unique(cluster_ids)) +)) diff --git a/workflow/Snakefile b/workflow/Snakefile index 1403532..44b8d5e 100644 --- a/workflow/Snakefile +++ b/workflow/Snakefile @@ -1,32 +1,111 @@ import pandas as pd -import os import yaml -sampleinfo = pd.read_table(config["sampleTable"],dtype={"sampleid": str}) +include: "rules/common.smk" -SAMPLES=list(sampleinfo.loc[:,"sampleid"]) +validated_config = validate_workflow_config(config) +sampleinfo = pd.read_table(config["sampleTable"], dtype={"sampleid": str}) +sampleinfo = validate_sample_sheet(sampleinfo, config["sampleTable"]) + +SAMPLES = list(sampleinfo.loc[:, "sampleid"]) with open("profiles/slurm/config.yaml") as f: slurm_profile_config = yaml.safe_load(f) -DECON_METHODS = config["ambient_decon_methods"] -DOUBLET_METHODS = config["doublet_removal_methods"] -EMPTYDROP_METHODS = config["emptydrop_removal_methods"] -POSTHOC_METHODS = config["posthoc_methods"] +DECON_METHODS = validated_config["decon_methods"] +DOUBLET_METHODS = validated_config["doublet_methods"] +EMPTYDROP_METHODS = validated_config["emptydrop_methods"] +POSTHOC_METHODS = validated_config["posthoc_methods"] +RESULTS_DIR = config.get("resultsDir", "results").rstrip("/") +WORKFLOW_SEED = int(config["workflow_seed"]) + +TENX_TARGETS = expand( + f"{RESULTS_DIR}/seurat_filtered/filtered_seurat_tenx_{{sample}}_markergenes.csv", + sample=SAMPLES, +) +EMPTYDROPS_TARGETS = ( + expand( + f"{RESULTS_DIR}/emptydrops/filtered_seurat_emptydrops_{{sample}}_markergenes.csv", + sample=SAMPLES, + ) + if "emptydrops" in EMPTYDROP_METHODS + else [] +) +SOUPX_TARGETS = ( + expand( + f"{RESULTS_DIR}/soupx/seurat_soupx_{{empty_method}}_{{sample}}_markergenes.csv", + sample=SAMPLES, + empty_method=EMPTYDROP_METHODS, + ) + if "soupx" in DECON_METHODS + else [] +) +CELLBENDER_TARGETS = ( + expand( + f"{RESULTS_DIR}/cellbender_fromraw/seurat_cellbender_fromraw_{{sample}}_markergenes.csv", + sample=SAMPLES, + ) + if "cellbender_fromraw" in DECON_METHODS + else [] +) +SOUPX_DOUBLET_TARGETS = ( + expand( + f"{RESULTS_DIR}/{{doublet_method}}/seurat_{{doublet_method}}_soupx_{{empty_method}}_{{sample}}_markergenes.csv", + sample=SAMPLES, + doublet_method=DOUBLET_METHODS, + empty_method=EMPTYDROP_METHODS, + ) + if "soupx" in DECON_METHODS + else [] +) +CELLBENDER_DOUBLET_TARGETS = ( + expand( + f"{RESULTS_DIR}/{{doublet_method}}/seurat_{{doublet_method}}_cellbender_fromraw_{{sample}}_markergenes.csv", + sample=SAMPLES, + doublet_method=DOUBLET_METHODS, + ) + if "cellbender_fromraw" in DECON_METHODS + else [] +) +SOUPX_POSTHOC_TARGETS = ( + expand( + f"{RESULTS_DIR}/posthocfilter/seurat_posthocfilt_{{posthoc_method}}_{{doublet_method}}_soupx_{{empty_method}}_{{sample}}_markergenes.csv", + sample=SAMPLES, + posthoc_method=POSTHOC_METHODS, + doublet_method=DOUBLET_METHODS, + empty_method=EMPTYDROP_METHODS, + ) + if "soupx" in DECON_METHODS + else [] +) +CELLBENDER_POSTHOC_TARGETS = ( + expand( + f"{RESULTS_DIR}/posthocfilter/seurat_posthocfilt_{{posthoc_method}}_{{doublet_method}}_cellbender_fromraw_{{sample}}_markergenes.csv", + sample=SAMPLES, + posthoc_method=POSTHOC_METHODS, + doublet_method=DOUBLET_METHODS, + ) + if "cellbender_fromraw" in DECON_METHODS + else [] +) +ALL_TARGETS = ( + TENX_TARGETS + + EMPTYDROPS_TARGETS + + SOUPX_TARGETS + + CELLBENDER_TARGETS + + SOUPX_DOUBLET_TARGETS + + CELLBENDER_DOUBLET_TARGETS + + SOUPX_POSTHOC_TARGETS + + CELLBENDER_POSTHOC_TARGETS +) default_target: True rule all: input: - expand("results/seurat_filtered/filtered_seurat_tenx_" + "{sample}" + "_markergenes.csv",sample=SAMPLES), - expand("results/emptydrops/filtered_seurat_emptydrops_{sample}_markergenes.csv",sample=SAMPLES), - expand("results/soupx/seurat_soupx_{empty_method}_" + "{sample}" + "_markergenes.csv",sample=SAMPLES,empty_method=EMPTYDROP_METHODS), - expand("results/cellbender_fromraw/seurat_cellbender_fromraw_{sample}_markergenes.csv",sample=SAMPLES), - expand("results/{doublet_method}/seurat_{doublet_method}_soupx_{empty_method}_{sample}_markergenes.csv",sample=SAMPLES,doublet_method=DOUBLET_METHODS,empty_method=EMPTYDROP_METHODS), - expand("results/{doublet_method}/seurat_{doublet_method}_cellbender_fromraw_{sample}_markergenes.csv",sample=SAMPLES,doublet_method=DOUBLET_METHODS), - expand("results/posthocfilter/seurat_posthocfilt_{posthoc_method}_{doublet_method}_cellbender_fromraw_{sample}_markergenes.csv",sample=SAMPLES,posthoc_method=POSTHOC_METHODS,doublet_method=DOUBLET_METHODS), - expand("results/posthocfilter/seurat_posthocfilt_{posthoc_method}_{doublet_method}_soupx_{empty_method}_{sample}_markergenes.csv",sample=SAMPLES,posthoc_method=POSTHOC_METHODS,doublet_method=DOUBLET_METHODS,empty_method=EMPTYDROP_METHODS) + ALL_TARGETS + include: "rules/tenx2seuratrds.smk" include: "rules/soupx.smk" diff --git a/workflow/envs/cellbender.yml b/workflow/envs/cellbender.yml index 2437bc9..da1aef2 100644 --- a/workflow/envs/cellbender.yml +++ b/workflow/envs/cellbender.yml @@ -2,6 +2,8 @@ channels: - conda-forge - bioconda +channel_priority: strict + dependencies: - r-tidyverse - python=3.9 diff --git a/workflow/rules/cellbender.smk b/workflow/rules/cellbender.smk index 5c3f9c0..5b9d8de 100644 --- a/workflow/rules/cellbender.smk +++ b/workflow/rules/cellbender.smk @@ -1,21 +1,15 @@ -def input_function(wildcards): - tenx_dir = sampleinfo.loc[ - sampleinfo["sampleid"] == wildcards.sample, - "tenx_datadir" - ].values[0] - input_path = os.path.join(tenx_dir, "raw_feature_bc_matrix.h5") - return input_path - - rule cellbender: input: - input_function + tenx_raw_h5_input output: - base = "results/cellbender/cellbender_{sample}.h5", - filtered = "results/cellbender/cellbender_{sample}_filtered.h5" + base=f"{RESULTS_DIR}/cellbender/cellbender_{{sample}}.h5", + filtered=f"{RESULTS_DIR}/cellbender/cellbender_{{sample}}_filtered.h5" + log: + f"{RESULTS_DIR}/logs/cellbender/cellbender_{{sample}}.log" params: # per-sample directory ONLY for checkpoints / temp - workdir = "scratch/cellbender/{sample}" + workdir="scratch/cellbender/{sample}", + seed=WORKFLOW_SEED container: "docker://us.gcr.io/broad-dsde-methods/cellbender:latest" resources: @@ -25,21 +19,51 @@ rule cellbender: runtime = 2880 shell: r""" + set -euo pipefail + # base_dir is the directory from which Snakemake was launched + exec > {log} 2>&1 base_dir=$(pwd) + export PYTHONHASHSEED={params.seed} + + seed_args=() + if cellbender remove-background --help 2>&1 | grep -q -- "--seed"; then + seed_args=(--seed {params.seed}) + fi + + input_h5="{input}" + case "${{input_h5}}" in + /*) ;; + *) input_h5="${{base_dir}}/${{input_h5}}" ;; + esac + + base_output="{output.base}" + case "${{base_output}}" in + /*) ;; + *) base_output="${{base_dir}}/${{base_output}}" ;; + esac + + filtered_output="{output.filtered}" + case "${{filtered_output}}" in + /*) ;; + *) filtered_output="${{base_dir}}/${{filtered_output}}" ;; + esac + + expected_filtered_output="${{base_output%.h5}}_filtered.h5" + if [ "${{expected_filtered_output}}" != "${{filtered_output}}" ]; then + echo "CellBender derives filtered output from --output as ${{expected_filtered_output}}, but the rule declares ${{filtered_output}}" >&2 + exit 1 + fi # Make sure output and scratch dirs exist on the host - mkdir -p "$(dirname "{output.base}")" + mkdir -p "$(dirname "${{base_output}}")" mkdir -p "{params.workdir}" - # Run cellbender in the per-sample scratch dir so ckpt.tar.gz is unique + # Run CellBender in the per-sample scratch dir so ckpt.tar.gz is unique. cd "{params.workdir}" - cellbender remove-background \ - --cuda \ - --input "{input}" \ - --output "${{base_dir}}/{output.base}" + cellbender remove-background --cuda "${{seed_args[@]}}" --input "${{input_h5}}" --output "${{base_output}}" - # If you actually generate a separate filtered file, add that command here, - # and ensure it writes to ${{base_dir}}/{output.filtered} + test -s "${{base_output}}" + test -s "${{filtered_output}}" """ diff --git a/workflow/rules/cellbender2seurat.smk b/workflow/rules/cellbender2seurat.smk index b41dc8b..4a536cc 100644 --- a/workflow/rules/cellbender2seurat.smk +++ b/workflow/rules/cellbender2seurat.smk @@ -1,18 +1,22 @@ rule cellbender2seurat: input: - data="results/cellbender/cellbender_{sample}_filtered.h5", + data=f"{RESULTS_DIR}/cellbender/cellbender_{{sample}}_filtered.h5", script="workflow/scripts/cellbender2seurat.R", helper="workflow/scripts/silhouette_utils.R" output: - rds="results/cellbender_fromraw/seurat_cellbender_fromraw_{sample}.rds", - nclusters=temp("results/cellbender_fromraw/seurat_cellbender_fromraw_{sample}_nclusters.txt"), - cluster_ids=temp("results/cellbender_fromraw/seurat_cellbender_fromraw_{sample}_cluster_ids.txt") + rds=f"{RESULTS_DIR}/cellbender_fromraw/seurat_cellbender_fromraw_{{sample}}.rds", + nclusters=temp(f"{RESULTS_DIR}/cellbender_fromraw/seurat_cellbender_fromraw_{{sample}}_nclusters.txt"), + cluster_ids=temp(f"{RESULTS_DIR}/cellbender_fromraw/seurat_cellbender_fromraw_{{sample}}_cluster_ids.txt") + log: + f"{RESULTS_DIR}/logs/cellbender2seurat/cellbender2seurat_{{sample}}.log" conda: "../envs/tenx2seuratrds.yml" resources: mem_mb = lambda wildcards, attempt: int(24000 * (2 ** (attempt - 1))), runtime = lambda wildcards, attempt: int(480* (2 ** (attempt - 1))) + params: + seed=WORKFLOW_SEED shell: """ - Rscript {input.script} {input.data} {output.rds} {output.nclusters} {output.cluster_ids} + SCRNASEQ_PREPROCESS_SEED={params.seed} Rscript {input.script} {input.data} {output.rds} {output.nclusters} {output.cluster_ids} > {log} 2>&1 """ diff --git a/workflow/rules/common.smk b/workflow/rules/common.smk new file mode 100644 index 0000000..023708c --- /dev/null +++ b/workflow/rules/common.smk @@ -0,0 +1,189 @@ +import os +from pathlib import Path + + +REQUIRED_CONFIG_KEYS = { + "conda-channel-priority", + "sampleTable", + "workflow_seed", + "emptydrop_removal_methods", + "ambient_decon_methods", + "doublet_removal_methods", + "posthoc_methods", + "min_nfeature", + "min_ncount", + "max_mtdna", +} +ALLOWED_EMPTYDROP_METHODS = {"tenx", "emptydrops"} +ALLOWED_DECON_METHODS = {"soupx", "cellbender_fromraw"} +ALLOWED_DOUBLET_METHODS = {"doubletfinder", "scdblfinder"} +ALLOWED_POSTHOC_METHODS = {"threshold", "mad"} + + +def require_non_empty_string(config_values, key, errors): + value = config_values.get(key) + if not isinstance(value, str) or not value.strip(): + errors.append(f"{key} must be a non-empty string") + + +def require_positive_int(config_values, key, errors): + value = config_values.get(key) + if isinstance(value, bool) or not isinstance(value, int) or value <= 0: + errors.append(f"{key} must be a positive integer") + + +def validate_method_list(config_values, key, allowed_values, errors): + value = config_values.get(key) + if not isinstance(value, list) or not value: + errors.append(f"{key} must be a non-empty list") + return [] + + normalized = [] + for item in value: + if not isinstance(item, str) or not item.strip(): + errors.append(f"{key} contains a non-string or empty value") + continue + normalized.append(item) + + duplicates = sorted({item for item in normalized if normalized.count(item) > 1}) + if duplicates: + errors.append(f"{key} contains duplicate value(s): " + ", ".join(duplicates)) + + invalid = sorted(set(normalized) - allowed_values) + if invalid: + errors.append( + f"{key} contains invalid value(s): " + + ", ".join(invalid) + + "; allowed values are: " + + ", ".join(sorted(allowed_values)) + ) + + return normalized + + +def validate_workflow_config(config_values): + errors = [] + missing_keys = sorted(REQUIRED_CONFIG_KEYS - set(config_values.keys())) + if missing_keys: + errors.append("missing required key(s): " + ", ".join(missing_keys)) + + require_non_empty_string(config_values, "sampleTable", errors) + if config_values.get("conda-channel-priority") != "strict": + errors.append("conda-channel-priority must be 'strict'") + + workflow_seed = config_values.get("workflow_seed") + if isinstance(workflow_seed, bool) or not isinstance(workflow_seed, int): + errors.append("workflow_seed must be an integer") + + emptydrop_methods = validate_method_list(config_values, "emptydrop_removal_methods", ALLOWED_EMPTYDROP_METHODS, errors) + decon_methods = validate_method_list(config_values, "ambient_decon_methods", ALLOWED_DECON_METHODS, errors) + doublet_methods = validate_method_list(config_values, "doublet_removal_methods", ALLOWED_DOUBLET_METHODS, errors) + posthoc_methods = validate_method_list(config_values, "posthoc_methods", ALLOWED_POSTHOC_METHODS, errors) + + require_positive_int(config_values, "min_nfeature", errors) + require_positive_int(config_values, "min_ncount", errors) + max_mtdna = config_values.get("max_mtdna") + if isinstance(max_mtdna, bool) or not isinstance(max_mtdna, (int, float)) or not 0 <= max_mtdna <= 100: + errors.append("max_mtdna must be a number between 0 and 100") + + if "resultsDir" in config_values: + require_non_empty_string(config_values, "resultsDir", errors) + + if errors: + raise ValueError("Invalid workflow config: " + "; ".join(errors)) + + return { + "emptydrop_methods": emptydrop_methods, + "decon_methods": decon_methods, + "doublet_methods": doublet_methods, + "posthoc_methods": posthoc_methods, + } + + +REQUIRED_SAMPLE_COLUMNS = {"sampleid", "tenx_datadir"} +REQUIRED_TENX_PATHS = ( + "filtered_feature_bc_matrix", + "raw_feature_bc_matrix", + "raw_feature_bc_matrix.h5", +) + + +def resolve_tenx_datadir(path_value): + path = Path(str(path_value)).expanduser() + if not path.is_absolute(): + path = Path.cwd() / path + return path + + +def validate_sample_sheet(sampleinfo, sample_table): + errors = [] + missing_columns = sorted(REQUIRED_SAMPLE_COLUMNS - set(sampleinfo.columns)) + if missing_columns: + errors.append("missing required column(s): " + ", ".join(missing_columns)) + raise ValueError(f"Invalid sample sheet {sample_table}: " + "; ".join(errors)) + + if sampleinfo.empty: + errors.append("sample sheet has no rows") + + sample_ids = sampleinfo["sampleid"].astype("string") + missing_sample_ids = sampleinfo.index[sample_ids.isna() | (sample_ids.str.strip() == "")].tolist() + if missing_sample_ids: + errors.append("sampleid is missing or empty on row(s): " + ", ".join(str(i + 2) for i in missing_sample_ids)) + + duplicate_sample_ids = sorted(sample_ids[sample_ids.duplicated(keep=False)].dropna().unique()) + if duplicate_sample_ids: + errors.append("duplicate sampleid value(s): " + ", ".join(duplicate_sample_ids)) + + data_dirs = sampleinfo["tenx_datadir"].astype("string") + missing_data_dirs = sampleinfo.index[data_dirs.isna() | (data_dirs.str.strip() == "")].tolist() + if missing_data_dirs: + errors.append("tenx_datadir is missing or empty on row(s): " + ", ".join(str(i + 2) for i in missing_data_dirs)) + + if errors: + raise ValueError(f"Invalid sample sheet {sample_table}: " + "; ".join(errors)) + + resolved_data_dirs = [] + for row_number, (sample_id, data_dir_value) in enumerate(zip(sample_ids, data_dirs), start=2): + data_dir = resolve_tenx_datadir(data_dir_value) + resolved_data_dirs.append(str(data_dir)) + if not data_dir.exists(): + errors.append(f"sample {sample_id} row {row_number}: tenx_datadir does not exist: {data_dir}") + continue + for required_path in REQUIRED_TENX_PATHS: + candidate = data_dir / required_path + if not candidate.exists(): + errors.append(f"sample {sample_id} row {row_number}: missing {required_path}: {candidate}") + + if errors: + raise ValueError(f"Invalid sample sheet {sample_table}: " + "; ".join(errors)) + + validated = sampleinfo.copy() + validated["sampleid"] = sample_ids.astype(str) + validated["tenx_datadir"] = resolved_data_dirs + return validated + + +def sample_tenx_dir(wildcards): + return sampleinfo.loc[sampleinfo["sampleid"] == wildcards.sample, "tenx_datadir"].values[0] + + +def tenx_filtered_matrix_input(wildcards): + return os.path.join(sample_tenx_dir(wildcards), "filtered_feature_bc_matrix") + + +def tenx_raw_matrix_input(wildcards): + return os.path.join(sample_tenx_dir(wildcards), "raw_feature_bc_matrix") + + +def tenx_raw_h5_input(wildcards): + return os.path.join(sample_tenx_dir(wildcards), "raw_feature_bc_matrix.h5") + + +def marker_chunk_inputs(wildcards): + manifest_dir = checkpoints.marker_manifest.get(prefix=wildcards.prefix).output.manifest + cluster_ids = glob_wildcards(str(Path(manifest_dir) / "{cluster}.txt")).cluster + return expand( + f"{RESULTS_DIR}/{{prefix}}_markergenes_cluster{{cluster}}.csv", + prefix=wildcards.prefix, + cluster=cluster_ids, + ) diff --git a/workflow/rules/doubletfinder.smk b/workflow/rules/doubletfinder.smk index cb0e802..9eb8431 100644 --- a/workflow/rules/doubletfinder.smk +++ b/workflow/rules/doubletfinder.smk @@ -1,13 +1,15 @@ rule doubletfinder: input: - install_validation="results/doubletfinder_installed.txt", - data="results/{decon_method}/seurat_{decon_method}_{empty_method}_{sample}.rds", + install_validation=f"{RESULTS_DIR}/doubletfinder_installed.txt", + data=f"{RESULTS_DIR}/{{decon_method}}/seurat_{{decon_method}}_{{empty_method}}_{{sample}}.rds", script="workflow/scripts/doubletfinder.R", helper="workflow/scripts/silhouette_utils.R" output: - rds="results/doubletfinder/seurat_doubletfinder_{decon_method}_{empty_method}_{sample}.rds", - nclusters=temp("results/doubletfinder/seurat_doubletfinder_{decon_method}_{empty_method}_{sample}_nclusters.txt"), - cluster_ids=temp("results/doubletfinder/seurat_doubletfinder_{decon_method}_{empty_method}_{sample}_cluster_ids.txt") + rds=f"{RESULTS_DIR}/doubletfinder/seurat_doubletfinder_{{decon_method}}_{{empty_method}}_{{sample}}.rds", + nclusters=temp(f"{RESULTS_DIR}/doubletfinder/seurat_doubletfinder_{{decon_method}}_{{empty_method}}_{{sample}}_nclusters.txt"), + cluster_ids=temp(f"{RESULTS_DIR}/doubletfinder/seurat_doubletfinder_{{decon_method}}_{{empty_method}}_{{sample}}_cluster_ids.txt") + log: + f"{RESULTS_DIR}/logs/doubletfinder/doubletfinder_{{decon_method}}_{{empty_method}}_{{sample}}.log" conda: "../envs/doubletfinder.yml" wildcard_constraints: @@ -16,28 +18,34 @@ rule doubletfinder: resources: mem_mb = lambda wildcards, attempt: int(24000 * (2 ** (attempt - 1))), runtime = lambda wildcards, attempt: int(480* (2 ** (attempt - 1))) + params: + seed=WORKFLOW_SEED shell: """ - Rscript {input.script} {input.data} {output.rds} {output.nclusters} {output.cluster_ids} + SCRNASEQ_PREPROCESS_SEED={params.seed} Rscript {input.script} {input.data} {output.rds} {output.nclusters} {output.cluster_ids} > {log} 2>&1 """ rule doubletfinder_cellbender: input: - install_validation="results/doubletfinder_installed.txt", - data="results/cellbender_fromraw/seurat_cellbender_fromraw_{sample}.rds", + install_validation=f"{RESULTS_DIR}/doubletfinder_installed.txt", + data=f"{RESULTS_DIR}/cellbender_fromraw/seurat_cellbender_fromraw_{{sample}}.rds", script="workflow/scripts/doubletfinder.R", helper="workflow/scripts/silhouette_utils.R" output: - rds="results/doubletfinder/seurat_doubletfinder_cellbender_fromraw_{sample}.rds", - nclusters=temp("results/doubletfinder/seurat_doubletfinder_cellbender_fromraw_{sample}_nclusters.txt"), - cluster_ids=temp("results/doubletfinder/seurat_doubletfinder_cellbender_fromraw_{sample}_cluster_ids.txt") + rds=f"{RESULTS_DIR}/doubletfinder/seurat_doubletfinder_cellbender_fromraw_{{sample}}.rds", + nclusters=temp(f"{RESULTS_DIR}/doubletfinder/seurat_doubletfinder_cellbender_fromraw_{{sample}}_nclusters.txt"), + cluster_ids=temp(f"{RESULTS_DIR}/doubletfinder/seurat_doubletfinder_cellbender_fromraw_{{sample}}_cluster_ids.txt") + log: + f"{RESULTS_DIR}/logs/doubletfinder/doubletfinder_cellbender_fromraw_{{sample}}.log" conda: "../envs/doubletfinder.yml" resources: mem_mb = lambda wildcards, attempt: int(24000 * (2 ** (attempt - 1))), runtime = lambda wildcards, attempt: int(480* (2 ** (attempt - 1))) + params: + seed=WORKFLOW_SEED shell: """ - Rscript {input.script} {input.data} {output.rds} {output.nclusters} {output.cluster_ids} + SCRNASEQ_PREPROCESS_SEED={params.seed} Rscript {input.script} {input.data} {output.rds} {output.nclusters} {output.cluster_ids} > {log} 2>&1 """ diff --git a/workflow/rules/emptydrops.smk b/workflow/rules/emptydrops.smk index 408a72e..10825bc 100644 --- a/workflow/rules/emptydrops.smk +++ b/workflow/rules/emptydrops.smk @@ -1,24 +1,23 @@ -def input_function(wildcards): - tenx_dir = sampleinfo.loc[sampleinfo["sampleid"] == wildcards.sample, "tenx_datadir"].values[0] - input_path = os.path.join(tenx_dir, "raw_feature_bc_matrix") - return input_path - rule emptydrops: input: - data=input_function, + data=tenx_raw_matrix_input, script="workflow/scripts/emptydrops.R", helper="workflow/scripts/silhouette_utils.R" output: - seurat="results/emptydrops/filtered_seurat_emptydrops_{sample}.rds", - matrixdir=directory("results/emptydrops/{sample}_emptydrops_filtered_matrix"), - nclusters=temp("results/emptydrops/filtered_seurat_emptydrops_{sample}_nclusters.txt"), - cluster_ids=temp("results/emptydrops/filtered_seurat_emptydrops_{sample}_cluster_ids.txt") + seurat=f"{RESULTS_DIR}/emptydrops/filtered_seurat_emptydrops_{{sample}}.rds", + matrixdir=directory(f"{RESULTS_DIR}/emptydrops/{{sample}}_emptydrops_filtered_matrix"), + nclusters=temp(f"{RESULTS_DIR}/emptydrops/filtered_seurat_emptydrops_{{sample}}_nclusters.txt"), + cluster_ids=temp(f"{RESULTS_DIR}/emptydrops/filtered_seurat_emptydrops_{{sample}}_cluster_ids.txt") + log: + f"{RESULTS_DIR}/logs/emptydrops/emptydrops_{{sample}}.log" conda: "../envs/emptydrops.yml" resources: mem_mb = lambda wildcards, attempt: int(24000 * (2 ** (attempt - 1))), runtime = lambda wildcards, attempt: int(480* (2 ** (attempt - 1))) + params: + seed=WORKFLOW_SEED shell: """ - Rscript {input.script} {input.data} {output.seurat} {output.matrixdir} {output.nclusters} {output.cluster_ids} + SCRNASEQ_PREPROCESS_SEED={params.seed} Rscript {input.script} {input.data} {output.seurat} {output.matrixdir} {output.nclusters} {output.cluster_ids} {wildcards.sample} > {log} 2>&1 """ diff --git a/workflow/rules/install_doubletfinder.smk b/workflow/rules/install_doubletfinder.smk index c451a89..4c5b95c 100644 --- a/workflow/rules/install_doubletfinder.smk +++ b/workflow/rules/install_doubletfinder.smk @@ -1,12 +1,14 @@ localrules: install_doubletfinder rule install_doubletfinder: - output: - "results/doubletfinder_installed.txt" - conda: - "../envs/doubletfinder.yml" - shell: - """ - Rscript -e "remotes::install_github('chris-mcginnis-ucsf/DoubletFinder', force = TRUE)" - touch {output} - """ + output: + f"{RESULTS_DIR}/doubletfinder_installed.txt" + log: + f"{RESULTS_DIR}/logs/install_doubletfinder.log" + conda: + "../envs/doubletfinder.yml" + shell: + """ + Rscript -e "remotes::install_github('chris-mcginnis-ucsf/DoubletFinder', force = TRUE)" > {log} 2>&1 + touch {output} + """ diff --git a/workflow/rules/markers.smk b/workflow/rules/markers.smk index b604d7a..3897ace 100644 --- a/workflow/rules/markers.smk +++ b/workflow/rules/markers.smk @@ -1,51 +1,34 @@ -from pathlib import Path - - -def read_cluster_ids(cluster_ids_path): - with open(cluster_ids_path) as handle: - return [line.strip() for line in handle if line.strip()] - - checkpoint marker_manifest: input: - cluster_ids="results/{prefix}_cluster_ids.txt" + cluster_ids=f"{RESULTS_DIR}/{{prefix}}_cluster_ids.txt" output: - manifest=temp(directory("results/{prefix}_marker_manifest")) - run: - manifest_dir = Path(output.manifest) - manifest_dir.mkdir(parents=True, exist_ok=True) - - for existing_path in manifest_dir.iterdir(): - if existing_path.is_file(): - existing_path.unlink() - - for cluster_id in read_cluster_ids(input.cluster_ids): - (manifest_dir / f"{cluster_id}.txt").write_text(f"{cluster_id}\n") - - -def marker_chunk_inputs(wildcards): - manifest_dir = checkpoints.marker_manifest.get(prefix=wildcards.prefix).output.manifest - cluster_ids = glob_wildcards(str(Path(manifest_dir) / "{cluster}.txt")).cluster - return expand( - "results/{prefix}_markergenes_cluster{cluster}.csv", - prefix=wildcards.prefix, - cluster=cluster_ids - ) + manifest=temp(directory(f"{RESULTS_DIR}/{{prefix}}_marker_manifest")) + log: + f"{RESULTS_DIR}/logs/markers/{{prefix}}_marker_manifest.log" + conda: + "../envs/tenx2seuratrds.yml" + script: + "../scripts/write_marker_manifest.py" rule find_markers: input: - rds="results/{prefix}.rds", - script="workflow/scripts/find_markers.R" + rds=f"{RESULTS_DIR}/{{prefix}}.rds", + script="workflow/scripts/find_markers.R", + helper="workflow/scripts/silhouette_utils.R" output: - temp("results/{prefix}_markergenes_cluster{cluster}.csv") + temp(f"{RESULTS_DIR}/{{prefix}}_markergenes_cluster{{cluster}}.csv") + log: + f"{RESULTS_DIR}/logs/markers/{{prefix}}_markergenes_cluster{{cluster}}.log" conda: "../envs/tenx2seuratrds.yml" resources: mem_mb=lambda wildcards, attempt: int(12000 * (2 ** (attempt - 1))), runtime=lambda wildcards, attempt: int(240 * (2 ** (attempt - 1))) + params: + seed=WORKFLOW_SEED shell: - "Rscript {input.script} {input.rds} {wildcards.cluster} {output}" + "SCRNASEQ_PREPROCESS_SEED={params.seed} Rscript {input.script} {input.rds} {wildcards.cluster} {output} > {log} 2>&1" rule combine_markers: @@ -53,16 +36,23 @@ rule combine_markers: markers=marker_chunk_inputs, script="workflow/scripts/combine_markers.R" output: - "results/{prefix}_markergenes.csv" + f"{RESULTS_DIR}/{{prefix}}_markergenes.csv" + log: + f"{RESULTS_DIR}/logs/markers/{{prefix}}_combine_markers.log" conda: "../envs/tenx2seuratrds.yml" resources: mem_mb=lambda wildcards, attempt: int(4000 * (2 ** (attempt - 1))), runtime=lambda wildcards, attempt: int(60 * (2 ** (attempt - 1))) + params: + seed=WORKFLOW_SEED, + cluster_ids=lambda wildcards: f"{RESULTS_DIR}/{wildcards.prefix}_cluster_ids.txt", + nclusters=lambda wildcards: f"{RESULTS_DIR}/{wildcards.prefix}_nclusters.txt", + manifest=lambda wildcards: f"{RESULTS_DIR}/{wildcards.prefix}_marker_manifest" shell: """ - Rscript {input.script} {output} {input.markers} - rm -f results/{wildcards.prefix}_cluster_ids.txt - rm -f results/{wildcards.prefix}_nclusters.txt - rm -rf results/{wildcards.prefix}_marker_manifest + SCRNASEQ_PREPROCESS_SEED={params.seed} Rscript {input.script} {output} {input.markers} > {log} 2>&1 + rm -f {params.cluster_ids} + rm -f {params.nclusters} + rm -rf {params.manifest} """ diff --git a/workflow/rules/posthocfilter_mad.smk b/workflow/rules/posthocfilter_mad.smk index 9b9ed86..53c84ae 100644 --- a/workflow/rules/posthocfilter_mad.smk +++ b/workflow/rules/posthocfilter_mad.smk @@ -1,13 +1,14 @@ rule posthocfilter_mad: input: - data="results/{doublet_method}/seurat_{doublet_method}_{decon_method}_{empty_method}_{sample}.rds", + data=f"{RESULTS_DIR}/{{doublet_method}}/seurat_{{doublet_method}}_{{decon_method}}_{{empty_method}}_{{sample}}.rds", script="workflow/scripts/posthocfilter_mad.R", helper="workflow/scripts/silhouette_utils.R" - output: - rds="results/posthocfilter/seurat_posthocfilt_mad_{doublet_method}_{decon_method}_{empty_method}_{sample}.rds", - nclusters=temp("results/posthocfilter/seurat_posthocfilt_mad_{doublet_method}_{decon_method}_{empty_method}_{sample}_nclusters.txt"), - cluster_ids=temp("results/posthocfilter/seurat_posthocfilt_mad_{doublet_method}_{decon_method}_{empty_method}_{sample}_cluster_ids.txt") + rds=f"{RESULTS_DIR}/posthocfilter/seurat_posthocfilt_mad_{{doublet_method}}_{{decon_method}}_{{empty_method}}_{{sample}}.rds", + nclusters=temp(f"{RESULTS_DIR}/posthocfilter/seurat_posthocfilt_mad_{{doublet_method}}_{{decon_method}}_{{empty_method}}_{{sample}}_nclusters.txt"), + cluster_ids=temp(f"{RESULTS_DIR}/posthocfilter/seurat_posthocfilt_mad_{{doublet_method}}_{{decon_method}}_{{empty_method}}_{{sample}}_cluster_ids.txt") + log: + f"{RESULTS_DIR}/logs/posthocfilter/posthocfilter_mad_{{doublet_method}}_{{decon_method}}_{{empty_method}}_{{sample}}.log" conda: "../envs/posthocfilter.yml" wildcard_constraints: @@ -17,20 +18,25 @@ rule posthocfilter_mad: resources: mem_mb = lambda wildcards, attempt: int(24000 * (2 ** (attempt - 1))), runtime = lambda wildcards, attempt: int(480* (2 ** (attempt - 1))) + params: + seed=WORKFLOW_SEED shell: """ - Rscript {input.script} {input.data} {output.rds} {output.nclusters} {output.cluster_ids} + SCRNASEQ_PREPROCESS_SEED={params.seed} Rscript {input.script} {input.data} {output.rds} {output.nclusters} {output.cluster_ids} > {log} 2>&1 """ rule posthocfilter_mad_cellbender: input: - data="results/{doublet_method}/seurat_{doublet_method}_cellbender_fromraw_{sample}.rds", - script="workflow/scripts/posthocfilter_mad.R" + data=f"{RESULTS_DIR}/{{doublet_method}}/seurat_{{doublet_method}}_cellbender_fromraw_{{sample}}.rds", + script="workflow/scripts/posthocfilter_mad.R", + helper="workflow/scripts/silhouette_utils.R" output: - rds="results/posthocfilter/seurat_posthocfilt_mad_{doublet_method}_cellbender_fromraw_{sample}.rds", - nclusters=temp("results/posthocfilter/seurat_posthocfilt_mad_{doublet_method}_cellbender_fromraw_{sample}_nclusters.txt"), - cluster_ids=temp("results/posthocfilter/seurat_posthocfilt_mad_{doublet_method}_cellbender_fromraw_{sample}_cluster_ids.txt") + rds=f"{RESULTS_DIR}/posthocfilter/seurat_posthocfilt_mad_{{doublet_method}}_cellbender_fromraw_{{sample}}.rds", + nclusters=temp(f"{RESULTS_DIR}/posthocfilter/seurat_posthocfilt_mad_{{doublet_method}}_cellbender_fromraw_{{sample}}_nclusters.txt"), + cluster_ids=temp(f"{RESULTS_DIR}/posthocfilter/seurat_posthocfilt_mad_{{doublet_method}}_cellbender_fromraw_{{sample}}_cluster_ids.txt") + log: + f"{RESULTS_DIR}/logs/posthocfilter/posthocfilter_mad_{{doublet_method}}_cellbender_fromraw_{{sample}}.log" conda: "../envs/posthocfilter.yml" wildcard_constraints: @@ -38,7 +44,9 @@ rule posthocfilter_mad_cellbender: resources: mem_mb = lambda wildcards, attempt: int(24000 * (2 ** (attempt - 1))), runtime = lambda wildcards, attempt: int(480* (2 ** (attempt - 1))) + params: + seed=WORKFLOW_SEED shell: """ - Rscript {input.script} {input.data} {output.rds} {output.nclusters} {output.cluster_ids} + SCRNASEQ_PREPROCESS_SEED={params.seed} Rscript {input.script} {input.data} {output.rds} {output.nclusters} {output.cluster_ids} > {log} 2>&1 """ diff --git a/workflow/rules/posthocfilter_threshold.smk b/workflow/rules/posthocfilter_threshold.smk index 2d08bce..4d4c1e0 100644 --- a/workflow/rules/posthocfilter_threshold.smk +++ b/workflow/rules/posthocfilter_threshold.smk @@ -1,13 +1,14 @@ rule posthocfilter_threshold: input: - data="results/{doublet_method}/seurat_{doublet_method}_{decon_method}_{empty_method}_{sample}.rds", + data=f"{RESULTS_DIR}/{{doublet_method}}/seurat_{{doublet_method}}_{{decon_method}}_{{empty_method}}_{{sample}}.rds", script="workflow/scripts/posthocfilter_threshold.R", helper="workflow/scripts/silhouette_utils.R" - output: - rds="results/posthocfilter/seurat_posthocfilt_threshold_{doublet_method}_{decon_method}_{empty_method}_{sample}.rds", - nclusters=temp("results/posthocfilter/seurat_posthocfilt_threshold_{doublet_method}_{decon_method}_{empty_method}_{sample}_nclusters.txt"), - cluster_ids=temp("results/posthocfilter/seurat_posthocfilt_threshold_{doublet_method}_{decon_method}_{empty_method}_{sample}_cluster_ids.txt") + rds=f"{RESULTS_DIR}/posthocfilter/seurat_posthocfilt_threshold_{{doublet_method}}_{{decon_method}}_{{empty_method}}_{{sample}}.rds", + nclusters=temp(f"{RESULTS_DIR}/posthocfilter/seurat_posthocfilt_threshold_{{doublet_method}}_{{decon_method}}_{{empty_method}}_{{sample}}_nclusters.txt"), + cluster_ids=temp(f"{RESULTS_DIR}/posthocfilter/seurat_posthocfilt_threshold_{{doublet_method}}_{{decon_method}}_{{empty_method}}_{{sample}}_cluster_ids.txt") + log: + f"{RESULTS_DIR}/logs/posthocfilter/posthocfilter_threshold_{{doublet_method}}_{{decon_method}}_{{empty_method}}_{{sample}}.log" conda: "../envs/posthocfilter.yml" wildcard_constraints: @@ -20,22 +21,25 @@ rule posthocfilter_threshold: params: min_numfeatures = config["min_nfeature"], min_umicount = config["min_ncount"], - max_mtdna_pcent = config["max_mtdna"] + max_mtdna_pcent = config["max_mtdna"], + seed=WORKFLOW_SEED shell: """ - Rscript {input.script} {input.data} {output.rds} \ - {params.min_numfeatures} {params.min_umicount} {params.max_mtdna_pcent} {output.nclusters} {output.cluster_ids} + SCRNASEQ_PREPROCESS_SEED={params.seed} Rscript {input.script} {input.data} {output.rds} {params.min_numfeatures} {params.min_umicount} {params.max_mtdna_pcent} {output.nclusters} {output.cluster_ids} > {log} 2>&1 """ rule posthocfilter_threshold_cellbender: input: - data="results/{doublet_method}/seurat_{doublet_method}_cellbender_fromraw_{sample}.rds", - script="workflow/scripts/posthocfilter_threshold.R" + data=f"{RESULTS_DIR}/{{doublet_method}}/seurat_{{doublet_method}}_cellbender_fromraw_{{sample}}.rds", + script="workflow/scripts/posthocfilter_threshold.R", + helper="workflow/scripts/silhouette_utils.R" output: - rds="results/posthocfilter/seurat_posthocfilt_threshold_{doublet_method}_cellbender_fromraw_{sample}.rds", - nclusters=temp("results/posthocfilter/seurat_posthocfilt_threshold_{doublet_method}_cellbender_fromraw_{sample}_nclusters.txt"), - cluster_ids=temp("results/posthocfilter/seurat_posthocfilt_threshold_{doublet_method}_cellbender_fromraw_{sample}_cluster_ids.txt") + rds=f"{RESULTS_DIR}/posthocfilter/seurat_posthocfilt_threshold_{{doublet_method}}_cellbender_fromraw_{{sample}}.rds", + nclusters=temp(f"{RESULTS_DIR}/posthocfilter/seurat_posthocfilt_threshold_{{doublet_method}}_cellbender_fromraw_{{sample}}_nclusters.txt"), + cluster_ids=temp(f"{RESULTS_DIR}/posthocfilter/seurat_posthocfilt_threshold_{{doublet_method}}_cellbender_fromraw_{{sample}}_cluster_ids.txt") + log: + f"{RESULTS_DIR}/logs/posthocfilter/posthocfilter_threshold_{{doublet_method}}_cellbender_fromraw_{{sample}}.log" conda: "../envs/posthocfilter.yml" wildcard_constraints: @@ -46,9 +50,9 @@ rule posthocfilter_threshold_cellbender: params: min_numfeatures = config["min_nfeature"], min_umicount = config["min_ncount"], - max_mtdna_pcent = config["max_mtdna"] + max_mtdna_pcent = config["max_mtdna"], + seed=WORKFLOW_SEED shell: """ - Rscript {input.script} {input.data} {output.rds} \ - {params.min_numfeatures} {params.min_umicount} {params.max_mtdna_pcent} {output.nclusters} {output.cluster_ids} + SCRNASEQ_PREPROCESS_SEED={params.seed} Rscript {input.script} {input.data} {output.rds} {params.min_numfeatures} {params.min_umicount} {params.max_mtdna_pcent} {output.nclusters} {output.cluster_ids} > {log} 2>&1 """ diff --git a/workflow/rules/scdblfinder.smk b/workflow/rules/scdblfinder.smk index d3765ec..e63e807 100644 --- a/workflow/rules/scdblfinder.smk +++ b/workflow/rules/scdblfinder.smk @@ -1,12 +1,14 @@ rule scdblfinder: input: - data="results/{decon_method}/seurat_{decon_method}_{empty_method}_{sample}.rds", + data=f"{RESULTS_DIR}/{{decon_method}}/seurat_{{decon_method}}_{{empty_method}}_{{sample}}.rds", script="workflow/scripts/scdblfinder.R", helper="workflow/scripts/silhouette_utils.R" output: - rds="results/scdblfinder/seurat_scdblfinder_{decon_method}_{empty_method}_{sample}.rds", - nclusters=temp("results/scdblfinder/seurat_scdblfinder_{decon_method}_{empty_method}_{sample}_nclusters.txt"), - cluster_ids=temp("results/scdblfinder/seurat_scdblfinder_{decon_method}_{empty_method}_{sample}_cluster_ids.txt") + rds=f"{RESULTS_DIR}/scdblfinder/seurat_scdblfinder_{{decon_method}}_{{empty_method}}_{{sample}}.rds", + nclusters=temp(f"{RESULTS_DIR}/scdblfinder/seurat_scdblfinder_{{decon_method}}_{{empty_method}}_{{sample}}_nclusters.txt"), + cluster_ids=temp(f"{RESULTS_DIR}/scdblfinder/seurat_scdblfinder_{{decon_method}}_{{empty_method}}_{{sample}}_cluster_ids.txt") + log: + f"{RESULTS_DIR}/logs/scdblfinder/scdblfinder_{{decon_method}}_{{empty_method}}_{{sample}}.log" conda: "../envs/scdblfinder.yml" wildcard_constraints: @@ -15,27 +17,33 @@ rule scdblfinder: resources: mem_mb = lambda wildcards, attempt: int(24000 * (2 ** (attempt - 1))), runtime = lambda wildcards, attempt: int(480* (2 ** (attempt - 1))) + params: + seed=WORKFLOW_SEED shell: """ - Rscript {input.script} {input.data} {output.rds} {output.nclusters} {output.cluster_ids} + SCRNASEQ_PREPROCESS_SEED={params.seed} Rscript {input.script} {input.data} {output.rds} {output.nclusters} {output.cluster_ids} > {log} 2>&1 """ rule scdblfinder_cellbender: input: - data="results/cellbender_fromraw/seurat_cellbender_fromraw_{sample}.rds", + data=f"{RESULTS_DIR}/cellbender_fromraw/seurat_cellbender_fromraw_{{sample}}.rds", script="workflow/scripts/scdblfinder.R", helper="workflow/scripts/silhouette_utils.R" output: - rds="results/scdblfinder/seurat_scdblfinder_cellbender_fromraw_{sample}.rds", - nclusters=temp("results/scdblfinder/seurat_scdblfinder_cellbender_fromraw_{sample}_nclusters.txt"), - cluster_ids=temp("results/scdblfinder/seurat_scdblfinder_cellbender_fromraw_{sample}_cluster_ids.txt") + rds=f"{RESULTS_DIR}/scdblfinder/seurat_scdblfinder_cellbender_fromraw_{{sample}}.rds", + nclusters=temp(f"{RESULTS_DIR}/scdblfinder/seurat_scdblfinder_cellbender_fromraw_{{sample}}_nclusters.txt"), + cluster_ids=temp(f"{RESULTS_DIR}/scdblfinder/seurat_scdblfinder_cellbender_fromraw_{{sample}}_cluster_ids.txt") + log: + f"{RESULTS_DIR}/logs/scdblfinder/scdblfinder_cellbender_fromraw_{{sample}}.log" conda: "../envs/scdblfinder.yml" resources: mem_mb = lambda wildcards, attempt: int(24000 * (2 ** (attempt - 1))), runtime = lambda wildcards, attempt: int(480* (2 ** (attempt - 1))) + params: + seed=WORKFLOW_SEED shell: """ - Rscript {input.script} {input.data} {output.rds} {output.nclusters} {output.cluster_ids} + SCRNASEQ_PREPROCESS_SEED={params.seed} Rscript {input.script} {input.data} {output.rds} {output.nclusters} {output.cluster_ids} > {log} 2>&1 """ diff --git a/workflow/rules/soupx.smk b/workflow/rules/soupx.smk index 4fb698e..2a3a431 100644 --- a/workflow/rules/soupx.smk +++ b/workflow/rules/soupx.smk @@ -1,31 +1,24 @@ -def input_function_filtered(wildcards): - tenx_dir = sampleinfo.loc[sampleinfo["sampleid"] == wildcards.sample, "tenx_datadir"].values[0] - input_path = os.path.join(tenx_dir, "filtered_feature_bc_matrix") - return input_path - -def input_function_raw(wildcards): - tenx_dir = sampleinfo.loc[sampleinfo["sampleid"] == wildcards.sample, "tenx_datadir"].values[0] - input_path = os.path.join(tenx_dir, "raw_feature_bc_matrix") - return input_path - - rule soupx: input: - raw=input_function_raw, - filtered = input_function_filtered, - seurat_base = "results/seurat_filtered/filtered_seurat_tenx_" + "{sample}" + ".rds", + raw=tenx_raw_matrix_input, + filtered=tenx_filtered_matrix_input, + seurat_base=f"{RESULTS_DIR}/seurat_filtered/filtered_seurat_tenx_{{sample}}.rds", script="workflow/scripts/soupx.R", helper="workflow/scripts/silhouette_utils.R" output: - rds="results/soupx/seurat_soupx_tenx_" + "{sample}" + ".rds", - nclusters=temp("results/soupx/seurat_soupx_tenx_" + "{sample}" + "_nclusters.txt"), - cluster_ids=temp("results/soupx/seurat_soupx_tenx_" + "{sample}" + "_cluster_ids.txt") + rds=f"{RESULTS_DIR}/soupx/seurat_soupx_tenx_{{sample}}.rds", + nclusters=temp(f"{RESULTS_DIR}/soupx/seurat_soupx_tenx_{{sample}}_nclusters.txt"), + cluster_ids=temp(f"{RESULTS_DIR}/soupx/seurat_soupx_tenx_{{sample}}_cluster_ids.txt") + log: + f"{RESULTS_DIR}/logs/soupx/soupx_tenx_{{sample}}.log" conda: "../envs/soupx.yml" resources: mem_mb = lambda wildcards, attempt: int(24000 * (2 ** (attempt - 1))), runtime = lambda wildcards, attempt: int(480* (2 ** (attempt - 1))) + params: + seed=WORKFLOW_SEED shell: """ - Rscript {input.script} {input.filtered} {input.raw} {input.seurat_base} {output.rds} {output.nclusters} {output.cluster_ids} + SCRNASEQ_PREPROCESS_SEED={params.seed} Rscript {input.script} {input.filtered} {input.raw} {input.seurat_base} {output.rds} {output.nclusters} {output.cluster_ids} > {log} 2>&1 """ diff --git a/workflow/rules/soupx_emptydrops.smk b/workflow/rules/soupx_emptydrops.smk index 18e0260..3bc9869 100644 --- a/workflow/rules/soupx_emptydrops.smk +++ b/workflow/rules/soupx_emptydrops.smk @@ -1,26 +1,24 @@ -def input_function_raw(wildcards): - tenx_dir = sampleinfo.loc[sampleinfo["sampleid"] == wildcards.sample, "tenx_datadir"].values[0] - input_path = os.path.join(tenx_dir, "raw_feature_bc_matrix") - return input_path - - rule soupx_emptydrops: input: - raw=input_function_raw, - filtered = "results/emptydrops/{sample}_emptydrops_filtered_matrix", - seurat_base = "results/emptydrops/filtered_seurat_emptydrops_{sample}.rds", + raw=tenx_raw_matrix_input, + filtered=f"{RESULTS_DIR}/emptydrops/{{sample}}_emptydrops_filtered_matrix", + seurat_base=f"{RESULTS_DIR}/emptydrops/filtered_seurat_emptydrops_{{sample}}.rds", script="workflow/scripts/soupx.R", helper="workflow/scripts/silhouette_utils.R" output: - rds="results/soupx/seurat_soupx_emptydrops_" + "{sample}" + ".rds", - nclusters=temp("results/soupx/seurat_soupx_emptydrops_" + "{sample}" + "_nclusters.txt"), - cluster_ids=temp("results/soupx/seurat_soupx_emptydrops_" + "{sample}" + "_cluster_ids.txt") + rds=f"{RESULTS_DIR}/soupx/seurat_soupx_emptydrops_{{sample}}.rds", + nclusters=temp(f"{RESULTS_DIR}/soupx/seurat_soupx_emptydrops_{{sample}}_nclusters.txt"), + cluster_ids=temp(f"{RESULTS_DIR}/soupx/seurat_soupx_emptydrops_{{sample}}_cluster_ids.txt") + log: + f"{RESULTS_DIR}/logs/soupx/soupx_emptydrops_{{sample}}.log" conda: "../envs/soupx.yml" resources: mem_mb = lambda wildcards, attempt: int(24000 * (2 ** (attempt - 1))), runtime = lambda wildcards, attempt: int(480* (2 ** (attempt - 1))) + params: + seed=WORKFLOW_SEED shell: """ - Rscript {input.script} {input.filtered} {input.raw} {input.seurat_base} {output.rds} {output.nclusters} {output.cluster_ids} + SCRNASEQ_PREPROCESS_SEED={params.seed} Rscript {input.script} {input.filtered} {input.raw} {input.seurat_base} {output.rds} {output.nclusters} {output.cluster_ids} > {log} 2>&1 """ diff --git a/workflow/rules/tenx2seuratrds.smk b/workflow/rules/tenx2seuratrds.smk index 0406426..fa5ab8a 100644 --- a/workflow/rules/tenx2seuratrds.smk +++ b/workflow/rules/tenx2seuratrds.smk @@ -1,23 +1,20 @@ -def input_function(wildcards): - tenx_dir = sampleinfo.loc[sampleinfo["sampleid"] == wildcards.sample, "tenx_datadir"].values[0] - input_path = os.path.join(tenx_dir, "filtered_feature_bc_matrix") - print(f"Input path for sample {wildcards.sample}: {input_path}") - return input_path - rule tenx2seuratrds: input: - data=input_function, + data=tenx_filtered_matrix_input, script="workflow/scripts/tenx2seuratrds.R", helper="workflow/scripts/silhouette_utils.R" output: - rds="results/seurat_filtered/filtered_seurat_tenx_" + "{sample}" + ".rds", - nclusters=temp("results/seurat_filtered/filtered_seurat_tenx_" + "{sample}" + "_nclusters.txt"), - cluster_ids=temp("results/seurat_filtered/filtered_seurat_tenx_" + "{sample}" + "_cluster_ids.txt") + rds=f"{RESULTS_DIR}/seurat_filtered/filtered_seurat_tenx_{{sample}}.rds", + nclusters=temp(f"{RESULTS_DIR}/seurat_filtered/filtered_seurat_tenx_{{sample}}_nclusters.txt"), + cluster_ids=temp(f"{RESULTS_DIR}/seurat_filtered/filtered_seurat_tenx_{{sample}}_cluster_ids.txt") + log: + f"{RESULTS_DIR}/logs/tenx2seuratrds/tenx2seuratrds_{{sample}}.log" conda: "../envs/tenx2seuratrds.yml" resources: mem_mb = lambda wildcards, attempt: int(24000 * (2 ** (attempt - 1))), runtime = lambda wildcards, attempt: int(480* (2 ** (attempt - 1))) - + params: + seed=WORKFLOW_SEED shell: - "Rscript {input.script} {input.data} {output.rds} {output.nclusters} {output.cluster_ids}" + "SCRNASEQ_PREPROCESS_SEED={params.seed} Rscript {input.script} {input.data} {output.rds} {output.nclusters} {output.cluster_ids} > {log} 2>&1" diff --git a/workflow/scripts/cellbender2seurat.R b/workflow/scripts/cellbender2seurat.R index d957e21..3400be9 100644 --- a/workflow/scripts/cellbender2seurat.R +++ b/workflow/scripts/cellbender2seurat.R @@ -9,6 +9,7 @@ library("scCustomize") options(future.globals.maxSize = 16 * 1024^3) source("workflow/scripts/silhouette_utils.R") +WORKFLOW_SEED <- set_workflow_seed() write_cluster_metadata <- function(seurat_obj, nclusters_output, cluster_ids_output) { cluster_ids <- levels(Idents(seurat_obj)) @@ -23,11 +24,11 @@ write_cluster_metadata <- function(seurat_obj, nclusters_output, cluster_ids_out mat <- Read_CellBender_h5_Mat(cellbender_h5) seurat <- CreateSeuratObject(mat) seurat[["percent.mt"]] <- PercentageFeatureSet(seurat, pattern = "(?i)^mt-") -seurat <- SCTransform(seurat, vars.to.regress = "percent.mt", verbose = FALSE) -seurat <- RunPCA(seurat, verbose = FALSE) -seurat <- RunUMAP(seurat, dims = 1:30) +seurat <- SCTransform(seurat, vars.to.regress = "percent.mt", seed.use = WORKFLOW_SEED, verbose = FALSE) +seurat <- RunPCA(seurat, seed.use = WORKFLOW_SEED, verbose = FALSE) +seurat <- RunUMAP(seurat, dims = 1:30, seed.use = WORKFLOW_SEED) seurat <- FindNeighbors(seurat, dims = 1:30) -seurat <- FindClusters(seurat) +seurat <- FindClusters(seurat, random.seed = WORKFLOW_SEED) seurat <- add_silhouette_to_metadata(seurat) saveRDS(seurat,file=output) write_cluster_metadata(seurat, nclusters_output, cluster_ids_output) diff --git a/workflow/scripts/doubletfinder.R b/workflow/scripts/doubletfinder.R index 8711afa..98ad1de 100644 --- a/workflow/scripts/doubletfinder.R +++ b/workflow/scripts/doubletfinder.R @@ -10,6 +10,7 @@ library("igraph") options(future.globals.maxSize = 16 * 1024^3) source("workflow/scripts/silhouette_utils.R") +WORKFLOW_SEED <- set_workflow_seed() write_cluster_metadata <- function(seurat_obj, nclusters_output, cluster_ids_output) { cluster_ids <- levels(Idents(seurat_obj)) @@ -23,6 +24,7 @@ write_cluster_metadata <- function(seurat_obj, nclusters_output, cluster_ids_out seurat <- readRDS(seurat) +set.seed(WORKFLOW_SEED) sweep.res.list <- paramSweep(seurat, PCs = 1:10, sct = TRUE) sweep.stats <- summarizeSweep(sweep.res.list, GT = FALSE) bcmvn <- find.pK(sweep.stats) @@ -35,6 +37,7 @@ nExp_poi <- round(0.15*nrow(seurat@meta.data)) homotypic.prop <- modelHomotypic(seurat$seurat_clusters) nExp_poi.adj <- round(nExp_poi*(1-homotypic.prop)) +set.seed(WORKFLOW_SEED) seurat <- doubletFinder(seurat, PCs = 1:10, pN = 0.25, pK = optimal_pk, nExp = nExp_poi.adj, reuse.pANN = NULL, @@ -46,11 +49,11 @@ rm(seurat) gc() seurat_singlets[["percent.mt"]] <- PercentageFeatureSet(seurat_singlets, pattern = "(?i)^mt-") -seurat_singlets <- SCTransform(seurat_singlets, vars.to.regress = "percent.mt", verbose = FALSE) -seurat_singlets <- RunPCA(seurat_singlets, verbose = FALSE) -seurat_singlets <- RunUMAP(seurat_singlets, dims = 1:30) +seurat_singlets <- SCTransform(seurat_singlets, vars.to.regress = "percent.mt", seed.use = WORKFLOW_SEED, verbose = FALSE) +seurat_singlets <- RunPCA(seurat_singlets, seed.use = WORKFLOW_SEED, verbose = FALSE) +seurat_singlets <- RunUMAP(seurat_singlets, dims = 1:30, seed.use = WORKFLOW_SEED) seurat_singlets <- FindNeighbors(seurat_singlets, dims = 1:30) -seurat_singlets <- FindClusters(seurat_singlets) +seurat_singlets <- FindClusters(seurat_singlets, random.seed = WORKFLOW_SEED) seurat_singlets <- add_silhouette_to_metadata(seurat_singlets) saveRDS(seurat_singlets,file=output) diff --git a/workflow/scripts/emptydrops.R b/workflow/scripts/emptydrops.R index 5188b16..5ac7d5d 100644 --- a/workflow/scripts/emptydrops.R +++ b/workflow/scripts/emptydrops.R @@ -4,6 +4,7 @@ seurat_output <- args[2] matrix_outdir <- args[3] nclusters_output <- args[4] cluster_ids_output <- args[5] +sample_id <- if (length(args) >= 6 && nzchar(args[6])) args[6] else basename(normalizePath(raw, mustWork = FALSE)) library("Seurat") library("DropletUtils") @@ -13,6 +14,7 @@ library("glmGamPoi") options(future.globals.maxSize = 16 * 1024^3) source("workflow/scripts/silhouette_utils.R") +WORKFLOW_SEED <- set_workflow_seed() write_cluster_metadata <- function(seurat_obj, nclusters_output, cluster_ids_output) { cluster_ids <- levels(Idents(seurat_obj)) @@ -32,12 +34,14 @@ library("R.utils") droputil_rawdata <-read10xCounts(raw) colnames(droputil_rawdata) <- droputil_rawdata$Barcode rownames(droputil_rawdata) <- make.unique(rowData(droputil_rawdata)$Symbol) +set.seed(WORKFLOW_SEED) dropletutils_out <-emptyDrops(counts(droputil_rawdata)) is.cell <-!is.na(dropletutils_out$FDR) & dropletutils_out$FDR<0.01 cell_barcodes <- colnames(droputil_rawdata)[which(is.cell)] droputil_filtered <- droputil_rawdata[,is.cell] droputil_filtered <- logNormCounts(droputil_filtered) seurat_droputil_filtered <- as.Seurat(droputil_filtered, counts = "counts",data="logcounts") +seurat_droputil_filtered$Sample <- sample_id cm <- counts(droputil_filtered) if (!inherits(cm, "dgCMatrix")) { cm <- as(cm, "dgCMatrix") @@ -48,11 +52,11 @@ rm(droputil_rawdata, dropletutils_out, droputil_filtered, is.cell, cell_barcodes gc() seurat_droputil_filtered <- RenameAssays(seurat_droputil_filtered,originalexp="RNA") seurat_droputil_filtered[["percent.mt"]] <- PercentageFeatureSet(seurat_droputil_filtered, pattern = "(?i)^mt-") -seurat_droputil_filtered <- SCTransform(seurat_droputil_filtered, vars.to.regress = "percent.mt", verbose = FALSE) -seurat_droputil_filtered <- RunPCA(seurat_droputil_filtered, verbose = FALSE) -seurat_droputil_filtered <- RunUMAP(seurat_droputil_filtered, dims = 1:30) +seurat_droputil_filtered <- SCTransform(seurat_droputil_filtered, vars.to.regress = "percent.mt", seed.use = WORKFLOW_SEED, verbose = FALSE) +seurat_droputil_filtered <- RunPCA(seurat_droputil_filtered, seed.use = WORKFLOW_SEED, verbose = FALSE) +seurat_droputil_filtered <- RunUMAP(seurat_droputil_filtered, dims = 1:30, seed.use = WORKFLOW_SEED) seurat_droputil_filtered <- FindNeighbors(seurat_droputil_filtered, dims = 1:30) -seurat_droputil_filtered <- FindClusters(seurat_droputil_filtered) +seurat_droputil_filtered <- FindClusters(seurat_droputil_filtered, random.seed = WORKFLOW_SEED) seurat_droputil_filtered <- add_silhouette_to_metadata(seurat_droputil_filtered) saveRDS(seurat_droputil_filtered,file=seurat_output) write_cluster_metadata(seurat_droputil_filtered, nclusters_output, cluster_ids_output) diff --git a/workflow/scripts/find_markers.R b/workflow/scripts/find_markers.R index 47c0337..573bafb 100644 --- a/workflow/scripts/find_markers.R +++ b/workflow/scripts/find_markers.R @@ -9,9 +9,13 @@ library("tools") options(future.globals.maxSize = 16 * 1024^3) +source("workflow/scripts/silhouette_utils.R") +WORKFLOW_SEED <- set_workflow_seed() + seurat_obj <- readRDS(rds_input) Idents(seurat_obj) <- "seurat_clusters" +set.seed(WORKFLOW_SEED) markers <- FindMarkers(seurat_obj, ident.1 = cluster_id) markers_tbl <- markers %>% as.data.frame() %>% diff --git a/workflow/scripts/posthocfilter_mad.R b/workflow/scripts/posthocfilter_mad.R index a558699..eed8c92 100644 --- a/workflow/scripts/posthocfilter_mad.R +++ b/workflow/scripts/posthocfilter_mad.R @@ -11,6 +11,7 @@ library("scater") options(future.globals.maxSize = 16 * 1024^3) source("workflow/scripts/silhouette_utils.R") +WORKFLOW_SEED <- set_workflow_seed() write_cluster_metadata <- function(seurat_obj, nclusters_output, cluster_ids_output) { cluster_ids <- levels(Idents(seurat_obj)) @@ -34,11 +35,11 @@ cells_to_keep <- colnames(seurat)[!discard] seurat_filtered<- subset(seurat, cells = cells_to_keep) seurat_filtered[["percent.mt"]] <- PercentageFeatureSet(seurat_filtered, pattern = "(?i)^mt-") -seurat_filtered <- SCTransform(seurat_filtered, vars.to.regress = "percent.mt", verbose = FALSE) -seurat_filtered <- RunPCA(seurat_filtered, verbose = FALSE) -seurat_filtered <- RunUMAP(seurat_filtered, dims = 1:30) +seurat_filtered <- SCTransform(seurat_filtered, vars.to.regress = "percent.mt", seed.use = WORKFLOW_SEED, verbose = FALSE) +seurat_filtered <- RunPCA(seurat_filtered, seed.use = WORKFLOW_SEED, verbose = FALSE) +seurat_filtered <- RunUMAP(seurat_filtered, dims = 1:30, seed.use = WORKFLOW_SEED) seurat_filtered <- FindNeighbors(seurat_filtered, dims = 1:30) -seurat_filtered <- FindClusters(seurat_filtered) +seurat_filtered <- FindClusters(seurat_filtered, random.seed = WORKFLOW_SEED) seurat_filtered <- add_silhouette_to_metadata(seurat_filtered) saveRDS(seurat_filtered,file=filtered_output) write_cluster_metadata(seurat_filtered, nclusters_output, cluster_ids_output) diff --git a/workflow/scripts/posthocfilter_threshold.R b/workflow/scripts/posthocfilter_threshold.R index 71d9db1..1680d5f 100644 --- a/workflow/scripts/posthocfilter_threshold.R +++ b/workflow/scripts/posthocfilter_threshold.R @@ -14,6 +14,7 @@ library("scater") options(future.globals.maxSize = 16 * 1024^3) source("workflow/scripts/silhouette_utils.R") +WORKFLOW_SEED <- set_workflow_seed() write_cluster_metadata <- function(seurat_obj, nclusters_output, cluster_ids_output) { cluster_ids <- levels(Idents(seurat_obj)) @@ -29,11 +30,11 @@ seurat <- readRDS(seurat) seurat_filtered <- subset(seurat, subset = nFeature_RNA > min_nfeature & nCount_RNA > min_ncount & percent.mt < max_mtdna) -seurat_filtered <- SCTransform(seurat_filtered, vars.to.regress = "percent.mt", verbose = FALSE) -seurat_filtered <- RunPCA(seurat_filtered, verbose = FALSE) -seurat_filtered <- RunUMAP(seurat_filtered, dims = 1:30) +seurat_filtered <- SCTransform(seurat_filtered, vars.to.regress = "percent.mt", seed.use = WORKFLOW_SEED, verbose = FALSE) +seurat_filtered <- RunPCA(seurat_filtered, seed.use = WORKFLOW_SEED, verbose = FALSE) +seurat_filtered <- RunUMAP(seurat_filtered, dims = 1:30, seed.use = WORKFLOW_SEED) seurat_filtered <- FindNeighbors(seurat_filtered, dims = 1:30) -seurat_filtered <- FindClusters(seurat_filtered) +seurat_filtered <- FindClusters(seurat_filtered, random.seed = WORKFLOW_SEED) seurat_filtered <- add_silhouette_to_metadata(seurat_filtered) saveRDS(seurat_filtered,file=filtered_output) write_cluster_metadata(seurat_filtered, nclusters_output, cluster_ids_output) diff --git a/workflow/scripts/scdblfinder.R b/workflow/scripts/scdblfinder.R index 490794d..e4bd4fa 100644 --- a/workflow/scripts/scdblfinder.R +++ b/workflow/scripts/scdblfinder.R @@ -10,6 +10,7 @@ library("scDblFinder") options(future.globals.maxSize = 16 * 1024^3) source("workflow/scripts/silhouette_utils.R") +WORKFLOW_SEED <- set_workflow_seed() write_cluster_metadata <- function(seurat_obj, nclusters_output, cluster_ids_output) { cluster_ids <- levels(Idents(seurat_obj)) @@ -23,7 +24,8 @@ write_cluster_metadata <- function(seurat_obj, nclusters_output, cluster_ids_out seurat <- readRDS(seurat) sce <- as.SingleCellExperiment(seurat) -sce <- scDblFinder(sce) +set.seed(WORKFLOW_SEED) +sce <- scDblFinder(sce, BPPARAM = BiocParallel::SerialParam()) seurat$scDblFinder.class <- colData(sce)$scDblFinder.class rm(sce) gc() @@ -31,11 +33,11 @@ seurat_singlets <- subset(seurat, subset = scDblFinder.class == "singlet") rm(seurat) gc() seurat_singlets[["percent.mt"]] <- PercentageFeatureSet(seurat_singlets, pattern = "(?i)^mt-") -seurat_singlets <- SCTransform(seurat_singlets, vars.to.regress = "percent.mt", verbose = FALSE) -seurat_singlets <- RunPCA(seurat_singlets, verbose = FALSE) -seurat_singlets <- RunUMAP(seurat_singlets, dims = 1:30) +seurat_singlets <- SCTransform(seurat_singlets, vars.to.regress = "percent.mt", seed.use = WORKFLOW_SEED, verbose = FALSE) +seurat_singlets <- RunPCA(seurat_singlets, seed.use = WORKFLOW_SEED, verbose = FALSE) +seurat_singlets <- RunUMAP(seurat_singlets, dims = 1:30, seed.use = WORKFLOW_SEED) seurat_singlets <- FindNeighbors(seurat_singlets, dims = 1:30) -seurat_singlets <- FindClusters(seurat_singlets) +seurat_singlets <- FindClusters(seurat_singlets, random.seed = WORKFLOW_SEED) seurat_singlets <- add_silhouette_to_metadata(seurat_singlets) saveRDS(seurat_singlets,file=output) write_cluster_metadata(seurat_singlets, nclusters_output, cluster_ids_output) diff --git a/workflow/scripts/silhouette_utils.R b/workflow/scripts/silhouette_utils.R index 747f105..9bd1a4a 100644 --- a/workflow/scripts/silhouette_utils.R +++ b/workflow/scripts/silhouette_utils.R @@ -1,3 +1,12 @@ +set_workflow_seed <- function(seed = Sys.getenv("SCRNASEQ_PREPROCESS_SEED", "12345")) { + seed <- suppressWarnings(as.integer(seed)) + if (length(seed) != 1 || is.na(seed)) { + stop("SCRNASEQ_PREPROCESS_SEED must be an integer") + } + set.seed(seed) + seed +} + add_silhouette_to_metadata <- function( seurat_obj, cluster_col = "seurat_clusters", diff --git a/workflow/scripts/soupx.R b/workflow/scripts/soupx.R index 59946a4..163fcdb 100644 --- a/workflow/scripts/soupx.R +++ b/workflow/scripts/soupx.R @@ -13,6 +13,7 @@ library("SoupX") options(future.globals.maxSize = 16 * 1024^3) source("workflow/scripts/silhouette_utils.R") +WORKFLOW_SEED <- set_workflow_seed() write_cluster_metadata <- function(seurat_obj, nclusters_output, cluster_ids_output) { cluster_ids <- levels(Idents(seurat_obj)) @@ -36,17 +37,18 @@ soup_channel <- SoupX::setClusters(soup_channel, clusters = as.factor(Idents(seurat_base))) soup_channel <- setDR(soup_channel, DR=Seurat::Embeddings(seurat_base, "umap")) +set.seed(WORKFLOW_SEED) soup_channel <- autoEstCont(soup_channel) corrected_counts <- adjustCounts(soup_channel,roundToInt=TRUE) seurat_soupx <- CreateSeuratObject(counts = corrected_counts) rm(filtered_matrix, raw_matrix, seurat_base, soup_channel, corrected_counts) gc() seurat_soupx[["percent.mt"]] <- PercentageFeatureSet(seurat_soupx, pattern = "(?i)^mt-") -seurat_soupx <- SCTransform(seurat_soupx, vars.to.regress = "percent.mt", verbose = FALSE) -seurat_soupx <- RunPCA(seurat_soupx, verbose = FALSE) -seurat_soupx <- RunUMAP(seurat_soupx, dims = 1:30) +seurat_soupx <- SCTransform(seurat_soupx, vars.to.regress = "percent.mt", seed.use = WORKFLOW_SEED, verbose = FALSE) +seurat_soupx <- RunPCA(seurat_soupx, seed.use = WORKFLOW_SEED, verbose = FALSE) +seurat_soupx <- RunUMAP(seurat_soupx, dims = 1:30, seed.use = WORKFLOW_SEED) seurat_soupx <- FindNeighbors(seurat_soupx, dims = 1:30) -seurat_soupx <- FindClusters(seurat_soupx) +seurat_soupx <- FindClusters(seurat_soupx, random.seed = WORKFLOW_SEED) seurat_soupx <- add_silhouette_to_metadata(seurat_soupx) saveRDS(seurat_soupx,file=output) write_cluster_metadata(seurat_soupx, nclusters_output, cluster_ids_output) diff --git a/workflow/scripts/tenx2seuratrds.R b/workflow/scripts/tenx2seuratrds.R index 78a3d00..747708b 100644 --- a/workflow/scripts/tenx2seuratrds.R +++ b/workflow/scripts/tenx2seuratrds.R @@ -10,6 +10,7 @@ library("glmGamPoi") options(future.globals.maxSize = 16 * 1024^3) source("workflow/scripts/silhouette_utils.R") +WORKFLOW_SEED <- set_workflow_seed() write_cluster_metadata <- function(seurat_obj, nclusters_output, cluster_ids_output) { cluster_ids <- levels(Idents(seurat_obj)) @@ -24,11 +25,11 @@ write_cluster_metadata <- function(seurat_obj, nclusters_output, cluster_ids_out filtered_loaded <- Seurat::Read10X(filtered) seurat_obj <- CreateSeuratObject(counts = filtered_loaded) seurat_obj[["percent.mt"]] <- PercentageFeatureSet(seurat_obj, pattern = "(?i)^mt-") -seurat_obj <- SCTransform(seurat_obj, vars.to.regress = "percent.mt", verbose = FALSE) -seurat_obj <- RunPCA(seurat_obj, verbose = FALSE) -seurat_obj <- RunUMAP(seurat_obj, dims = 1:30) +seurat_obj <- SCTransform(seurat_obj, vars.to.regress = "percent.mt", seed.use = WORKFLOW_SEED, verbose = FALSE) +seurat_obj <- RunPCA(seurat_obj, seed.use = WORKFLOW_SEED, verbose = FALSE) +seurat_obj <- RunUMAP(seurat_obj, dims = 1:30, seed.use = WORKFLOW_SEED) seurat_obj <- FindNeighbors(seurat_obj, dims = 1:30) -seurat_obj <- FindClusters(seurat_obj) +seurat_obj <- FindClusters(seurat_obj, random.seed = WORKFLOW_SEED) seurat_obj <- add_silhouette_to_metadata(seurat_obj) saveRDS(seurat_obj,file=output) write_cluster_metadata(seurat_obj, nclusters_output, cluster_ids_output) diff --git a/workflow/scripts/write_marker_manifest.py b/workflow/scripts/write_marker_manifest.py new file mode 100644 index 0000000..ed8cd19 --- /dev/null +++ b/workflow/scripts/write_marker_manifest.py @@ -0,0 +1,17 @@ +from pathlib import Path + + +def read_cluster_ids(cluster_ids_path): + with open(cluster_ids_path) as handle: + return [line.strip() for line in handle if line.strip()] + + +manifest_dir = Path(snakemake.output.manifest) +manifest_dir.mkdir(parents=True, exist_ok=True) + +for existing_path in manifest_dir.iterdir(): + if existing_path.is_file(): + existing_path.unlink() + +for cluster_id in read_cluster_ids(snakemake.input.cluster_ids): + (manifest_dir / f"{cluster_id}.txt").write_text(f"{cluster_id}\\n")