homerun

This repository is the SLURM-oriented wrapper around the Python package in csrnaseq. It is meant to help you launch a complete csRNA-seq analysis workflow from a project directory on a cluster.

What this repo contains

• submit_array.sh: submits the full 7-phase workflow
• prepare.sbatch: prepares the project and runs the initial setup
• align_array.sbatch: runs trim + align for one leaf run per array task
• tagdir_array.sbatch: builds leaf-level TagDirs in parallel, one array task per leaf
• tagdirs_combo_array.sbatch: merges replicates into combo TagDirs in parallel, one array task per Species/Sample
• tss_array.sbatch: calls TSRs with findcsRNATSS.pl in parallel, one array task per Species/Sample
• bedgraphs_array.sbatch: builds bedGraphs in parallel, one array task per Species/Sample
• collect.sbatch: runs ritrie, QC, stability, and reporting after every array phase finishes
• csrnaseq: the Python package that actually performs the pipeline steps

Project structure

Projects use a compact Species/<category> layout. Species and sample names are parsed from FASTQ filenames; sample identity remains in filenames and TagDir names, so multiple samples can safely share each species-level category directory.

For example, homo_sapiens_K562_csRNA-r1_DB422_S1_R1_001.fastq.gz becomes species homo_sapiens, sample K562, and leaf csRNA_r1.

This produces a tree like:

homerun_test/
├── config.txt                        # auto-generated summary of every run's config + samples
└── homo_sapiens/
    ├── RawData/                       # all staged FASTQs for the species
    ├── Trimmed/                       # sample-prefixed trimmed reads
    ├── Aligned/                       # sample-prefixed SAMs and mapping logs
    ├── TagDirs/                       # K562_csRNA_r1, K562_csRNA-combo, ...
    ├── bedGraphs/                     # names match their source TagDirs
    ├── TSS/                           # K562.tss.txt, IMR90.tss.txt, ...
    ├── RITRIE/                        # sample-prefixed working directories
    └── QC/
        ├── csRNA_nucleotide_divergence_heatmap.png
        ├── sRNA_nucleotide_divergence_heatmap.png
        ├── K562/                     # detailed sample report and plots
        │   └── qc_report.html
        └── IMR90/
            └── qc_report.html

• Raw, trimmed, and aligned files share species-level category folders and remain self-identifying through their filenames.
• Leaf and combo TagDirs/bedGraphs are sample-prefixed to prevent collisions.
• TSS outputs share Species/TSS/ and use sample-prefixed filenames.
• QC provides compact cross-sample A/C/G/T divergence heatmaps in Species/QC/, plus detailed reports in Species/QC/<sample>/.
• config.txt at the project root records every config value used for the run (genome, aligner, thresholds, --gtf, etc.) and every sample/RawData file discovered — see Run configuration (config.txt) below.

Typical workflow

1. Create a project directory and place your FASTQ files in it — they are parsed and staged into Species/RawData/ automatically (for example, homo_sapiens/RawData/).
2. Run the submission script with the required paths and cluster settings.
3. Let the prepare, align-array, tagdir/tagdirs-combo-array, tss/bedgraphs-array, and collect stages run in sequence (see dependency graph below).
4. Inspect the species-level QC heatmaps and each detailed sample report (for example, homo_sapiens/QC/K562/qc_report.html), then check config.txt to confirm the detected configuration and samples.

Job-array dependency graph

prepare
  └─afterok─> align_array[0..N-1]
                ├─afterok─> tagdir_array[0..N-1]          (leaf TagDirs)
                └─afterok─> tagdirs_combo_array[0..S-1]   (combo TagDirs)
                              ├─afterok─> tss_array[0..S-1]
                              └─(+ tagdir_array)─afterok─> bedgraphs_array[0..S-1]
                                                              └─afterok─(+tss_array)─> collect

• N = number of leaf runs (R1 files in RawData) — align_array/tagdir_array indexing, one task per leaf run.
• S = number of Species/Sample groups — tagdirs_combo_array/bedgraphs_array/tss_array indexing, one task per Species/Sample (usually smaller than N, since a sample typically has several leaf runs).

tagdirs_combo_array only needs align_array to finish (not tagdir_array), so it runs in parallel with the leaf TagDir build rather than after it — combo TagDirs are built straight from the aligned SAM files, not from the leaf TagDirs. tss_array needs the combo TagDirs. bedgraphs_array needs both tagdir_array and tagdirs_combo_array, since it builds bedGraphs for every TagDir (leaf and combo alike) under a sample. collect then runs ritrie, QC, stability, and reporting over everything the array phases built.

TagDir generation

• Leaf TagDirs — built in parallel via tagdir_array.sbatch, one array task per leaf (e.g. one task each for K562/csRNA_r1, K562/csRNA_r2, K562/sRNA_r1, K562/sRNA_r2, K562/RNA_r1).
• Combined TagDirs — built in parallel via tagdirs_combo_array.sbatch, one array task per Species/Sample, merging that sample's replicate leaves into a <assay>-combo/ directory (e.g. K562/csRNA-combo/, K562/sRNA-combo/).

RITRIE (RIT/RIE QC metric)

ritrie computes RIT/RIE — reads at called TSRs vs. reads at exons (excluding TSR overlap) — a RIN-free proxy for RNA degradation. Higher values generally indicate stronger enrichment of transcription-initiation signal relative to exonic RNA; lower values may indicate more degraded RNA. Thresholds haven't yet been formally established.

This step requires a genome annotation GTF, passed via --gtf (or CSRNA_GTF). If it's unset, ritrie is simply skipped — that's not an error. If it is set but points at a missing/unreadable file, prepare fails immediately with a clear error rather than letting the pipeline run for hours and fail on ritrie at the very end.

Stability handling

The stability step is automatically skipped for samples without matching total RNA data. For example, if a sample has csRNA_r1/csRNA_r2 and sRNA_r1/sRNA_r2 leaves but no RNA_r* leaf, stability analysis is skipped for that sample. When stability analysis isn't performed, stability-specific columns are omitted from the report.

Run configuration (config.txt)

Every prepare run writes (or refreshes) <project>/config.txt — a plain-text summary of the run, meant to make a project directory self-documenting without digging through config.env, CLI history, or logs:

# HomeRun csRNA-seq pipeline — run configuration
# Generated: 2026-07-14T12:01:49

[Config]
project = /path/to/homerun_test
aligner = star
genome_index = /path/to/STARIndex
genome = hg38
gtf = /path/to/gencode.v44.annotation.gtf
threads = 8
...

[Samples]
count = 2
homo_sapiens/HepG2
homo_sapiens/K562

[RawData]
count = 3
homo_sapiens/HepG2/sRNA_r1/RawData/homo_sapiens_HepG2_sRNA-r1_DB477_S7_R1_001.fastq.gz
homo_sapiens/K562/csRNA_r1/RawData/homo_sapiens_K562_csRNA-r1_DB422_S1_R1_001.fastq.gz
homo_sapiens/K562/csRNA_r2/RawData/homo_sapiens_K562_csRNA-r2_DB423_S2_R1_001.fastq.gz

[Config] lists every setting the run actually used — whatever was passed via CLI flag or CSRNA_* env var, or its built-in default if neither was set. [Samples] and [RawData] list every Species/Sample discovered and every staged FASTQ, so you can confirm what the pipeline saw before it spends hours processing it. It's safe to re-run prepare on a project that already has staged data or an old config.txt sitting in it — the file is simply overwritten with current information each time, and any leftovers from an older/different layout are ignored.

Quick start (w/o copying files and having fastqs in the project dir)

path/to/homerun/submit_array.sh \
  --project /PATH/TO/PROJECT \
  --partition kamiak \
  --conda-env CONDA_ENV_NAME \
  --genome-index /path/to/STARIndex \
  --genome hg38

Quick start (with copying files via copy-src)

path/to/homerun/submit_array.sh \
  --project /PATH/TO/PROJECT \
  --partition kamiak \
  --conda-env CONDA_ENV_NAME \
  --genome-index /path/to/STARIndex \
  --genome hg38 \
  --copy-src '/PATH/TO/*.FASTQ.GZ'

Quick start (with RITRIE enabled)

path/to/homerun/submit_array.sh \
  --project /PATH/TO/PROJECT \
  --partition kamiak \
  --conda-env CONDA_ENV_NAME \
  --genome-index /path/to/STARIndex \
  --genome hg38 \
  -- --gtf /path/to/genome.gtf

The controller will:

• parse and stage raw FASTQs into the compact Species/RawData/ layout (e.g. homo_sapiens_K562_csRNA-r1_DB422_S1_R1_001.fastq.gz → homo_sapiens/RawData/)
• write/refresh config.txt at the project root
• submit and wait for the prepare job (which stages all --copy-src files, validates --gtf, and finalizes array sizes)
• submit a sample-array job for trim + align
• submit a tagdir-array job (leaf TagDirs) and a tagdirs-combo-array job (combo TagDirs) in parallel
• submit a tss-array job and a bedgraphs-array job, each depending on the combo TagDirs (bedgraphs also needs the leaf TagDirs)
• submit a collect job that runs ritrie, QC, stability, and reporting

All flags

Required:

Flag	Description
--project	Path to your project directory
--partition	SLURM partition name
--conda-env	Conda environment with pipeline dependencies
--genome-index	Path to STAR --genomeDir or HISAT2 -x index prefix
--genome	HOMER genome name (e.g. hg38)

Optional:

Flag	Default	Description
--conda-module	anaconda3	Cluster module that provides Conda
--aligner	star	Aligner to use: star or hisat2
--throttle	16	Max array tasks running at once
--tss-throttle	1	Max concurrent TSS tasks; the safe default serializes writes to shared species-level TSS/ directories
--email		Email address for SLURM notifications
--copy-src		Quoted glob path to FASTQs copied into Species/RawData/ before array sizes are calculated
--starindex-url		URL to auto-download a STAR index tarball if none is found
--ntag-threshold	7	Minimum tags to call a TSS cluster
--trim-min	20	Discard reads shorter than this after trimming
--trim-max	58	Discard reads longer than this after trimming
--trim-adapter	AGATCGGAAGAGCACACGTCT	3′ adapter sequence
--skip-chr	chrEBV	Chromosome to exclude from bedGraphs
--gtf		Genome annotation GTF; enables the ritrie step. Skipped (not an error) if unset

To pass extra flags directly to the Python pipeline (e.g. STAR tuning, or --gtf):

path/to/homerun/submit_array.sh \
  --project /PATH/TO/PROJECT \
  --partition kamiak \
  --conda-env CONDA_ENV_NAME \
  --genome-index /path/to/STARIndex \
  --genome hg38 \
  -- --star-filter-multimap 5000 --gtf /path/to/genome.gtf

To see all the flags do:

path/to/homerun/submit_array.sh \
    --h (or --help)

Or call the Python package directly for the same output:

$ python -m csrnaseq --help
usage: csrnaseq [-h] [--project PROJECT] [--log-path LOG_PATH]
                 [--steps {trim,align,tagdirs,tagdirs-combo,bedgraphs,tss,ritrie,qc,stability,report} [{trim,align,tagdirs,tagdirs-combo,bedgraphs,tss,ritrie,qc,stability,report} ...]]
                 [--sample-index SAMPLE_INDEX] [--group-index GROUP_INDEX] [--skip-prepare] [--only-prepare] [--count-samples] [--count-groups]
                 [--stage-raw] [--aligner {star,hisat2}] [--genome-index GENOME_INDEX] [--genome GENOME] [--gtf GTF] [--copy-src COPY_SRC]
                 [--threads THREADS] [--trim-adapter TRIM_ADAPTER] [--trim-min TRIM_MIN] [--trim-max TRIM_MAX] [--ntag-threshold NTAG_THRESHOLD]
                 [--skip-chr SKIP_CHR] [--star-filter-multimap STAR_FILTER_MULTIMAP] [--star-multimap-out STAR_MULTIMAP_OUT]
                 [--star-multimap-order {Random,Old_2.4}] [--hisat2-strandness {F,R,FR,RF}]

    __  __                                    
   / / / /___  ____ ___  ___  _______  ______ 
  / /_/ / __ \/ __ `__ \/ _ \/ ___/ / / / __ \
 / __  / /_/ / / / / / /  __/ /  / /_/ / / / /
/_/ /_/\____/_/ /_/ /_/\___/_/   \__,_/_/ /_/ 

RNA-seq analysis pipeline for HPC clusters.
Version: 1.0.0

optional arguments:
  -h, --help            show this help message and exit
  --project PROJECT     Project root (default: $CSRNA_PROJECT or CWD).
  --log-path LOG_PATH   Pipeline log file path (overrides CSRNA_LOG; else timestamped file under <project>/logs/).
  --steps {trim,align,tagdirs,tagdirs-combo,bedgraphs,tss,ritrie,qc,stability,report} [{trim,align,tagdirs,tagdirs-combo,bedgraphs,tss,ritrie,qc,stability,report} ...]
                        Run only these steps (still executed in canonical order).
  --sample-index SAMPLE_INDEX
                        0-based index into RawData R1 files. Restricts trim/align/tagdirs to one leaf run. Used by SLURM_ARRAY_TASK_ID.
  --group-index GROUP_INDEX
                        0-based index into list_samples(cfg). Restricts tagdirs-combo/bedgraphs/tss to one Species/Sample group. Used by
                        SLURM_ARRAY_TASK_ID.
  --skip-prepare        Skip folder creation/raw copy/STARIndex setup.
  --only-prepare        Run prepare and exit.
  --count-samples       Print number of leaf runs (R1 files in RawData) and exit.
  --count-groups        Print number of Species/Sample groups and exit.
  --stage-raw           Move loose *_R1*/*_R2* FASTQs into RawData/ and exit.

config overrides (override config.env when given):
  --aligner {star,hisat2}
                        Aligner (overrides CSRNA_ALIGNER).
  --genome-index GENOME_INDEX
                        STAR genomeDir or HISAT2 prefix (overrides CSRNA_GENOME_INDEX).
  --genome GENOME       HOMER genome (overrides CSRNA_GENOME).
  --gtf GTF             GTF annotation file for RIT/RIE metric (overrides CSRNA_GTF).
  --copy-src COPY_SRC   FASTQ copy source (overrides CSRNA_COPY_SRC).
  --threads THREADS     Threads (overrides CSRNA_THREADS / SLURM_CPUS_PER_TASK).
  --trim-adapter TRIM_ADAPTER
                        3' adapter sequence.
  --trim-min TRIM_MIN   Minimum read length after trimming.
  --trim-max TRIM_MAX   Maximum read length after trimming.
  --ntag-threshold NTAG_THRESHOLD
                        Minimum tags for TSS calling.
  --skip-chr SKIP_CHR   Chromosome excluded from bedGraphs.

alignment overrides:
  --star-filter-multimap STAR_FILTER_MULTIMAP
                        STAR outFilterMultimapNmax.
  --star-multimap-out STAR_MULTIMAP_OUT
                        STAR outSAMmultNmax.
  --star-multimap-order {Random,Old_2.4}
                        STAR outMultimapperOrder.
  --hisat2-strandness {F,R,FR,RF}
                        HISAT2 RNA strandness.

Requirements

The conda environment passed via --conda-env must provide:

Tool	Used for	Notes
HOMER	Tag directories, bedGraphs, TSS calling/annotation, RITRIE	Must include homerTools on PATH; genome data for --genome (e.g. hg38) must be installed via perl configureHomer.pl -install hg38
Python ≥3.9	Runs the csrnaseq package	See csrnaseq/README.md for Python package dependencies
STAR or HISAT2	Read alignment	Select via --aligner; a matching pre-built genome index is required (--genome-index)
SLURM	Job scheduling	Only required if using submit_array.sh. Tested on partitions with sbatch/squeue; --partition must be a valid partition on your cluster

Not on a SLURM cluster? The same HOMER/Python/STAR-or-HISAT2 requirements apply — SLURM is only used by submit_array.sh to schedule the jobs. The underlying csrnaseq Python package can be run directly (python -m csrnaseq ...) on any machine that meets the requirements above, without SLURM at all. See csrnaseq/README.md for running it standalone.

Quick check that required tools are available in your environment:

conda activate CONDA_ENV_NAME
for t in homerTools STAR hisat2; do
    command -v "$t" >/dev/null && echo "OK: $t" || echo "MISSING: $t"
done

Note: only one of STAR/hisat2 is required, depending on --aligner.

Notes

• The repo is built around the csrnaseq CLI, so most of the actual processing logic lives in the package under csrnaseq.
• Outputs live in category directories under each species; detailed sample reports live at Species/QC/<sample>/qc_report.html, with cross-sample heatmaps directly in Species/QC/.
• Combined TagDirs/bedGraphs are per-assay <assay>-combo/ directories (e.g. csRNA-combo, sRNA-combo), not a single catch-all folder — this keeps replicate merges scoped to their own assay.
• tagdirs-combo, bedgraphs, and tss each run as their own parallel SLURM array (indexed by --group-index, one task per Species/Sample) rather than looping serially inside collect — see the job-array dependency graph above.
• config.txt at the project root is regenerated at the end of every prepare run — check it first if a run behaves unexpectedly, to confirm the pipeline actually saw the config/samples you intended.
• For package-specific installation details, module descriptions, and CLI internals, see csrnaseq/README.md.