RNAEdgeFlow

RNAEdgeFlow is a modular pipeline for processing RNA 5′/3′ end-tag and internal reads.
It supports end-tag classification (5P, 3P, internal), UMI handling, adapter trimming, alignment, expression quantification, coverage profiling, and metagene analysis.

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Features

• Automatic separation of 5P, 3P, and internal reads from paired-end FASTQ.
• UMI extraction and deduplication (via umi_tools).
• Adapter trimming and quality control (via cutadapt and fastp).
• Alignment with STAR and expression quantification with StringTie.
• Coverage profiling and base content statistics.
• Two major functions:
  1. pipeline → full end-to-end pipeline for a sample, from FASTQ to processed BAMs, QC, expression matrices, and coverage.
  2. metagene → metagene coverage plots (e.g., TSS/TES) from BAMs or directories of BAM files.

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Installation

Step 1. Clone the repository

git clone https://github.com/Gaoyang-Wang/RNAEdgeFlow.git
cd RNAEdgeFlow

Step 2. Create the Conda environment

bash install.sh

This will create a Conda environment named rnaedgeflow and install all required dependencies (Python, R, Bioconductor packages, bedtools, samtools, STAR, fastp, cutadapt, umi_tools, etc.).

Step 3. Activate the environment

conda activate rnaedgeflow

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Usage

1. Run the full pipeline

./rnaedgeflow pipeline --profile path/to/sample.profile

--profile points to a .profile file describing all required parameters (sample name, input FASTQ prefix, STAR genome index, barcode/UMI patterns, etc.).

Outputs will be created under:

OutputDir/SampleName/
  ├── process/           # intermediate FASTQs and BAMs
  ├── result/            # final outputs
  │   ├── stat/          # QC and read statistics
  │   ├── internal_expr/ # StringTie expression
  │   └── terminal_bed/  # coverage profiles
  └── log/               # pipeline logs

Example:

./rnaedgeflow pipeline --profile example_data/example.profile

2. Metagene profiling

(a) Scan all BAMs in a directory

./rnaedgeflow metagene --inputDir path/to/bam_dir --outputDir results/metagene_out [--bins 100] [--up 1000] [--down 1000]

(b) Explicitly provide BAM files + sample names

./rnaedgeflow metagene \
  --inputBam path/to/sample1.bam --sampleName Sample1 \
  --inputBam2 path/to/sample2.bam --sampleName2 Sample2 \
  --inputBam3 path/to/sample3.bam --sampleName3 Sample3 \
  --outputDir results/metagene_out [--bins 100] [--up 1000] [--down 1000]

Options:
--upstream number of bases upstream of TSS (default: 1000)
--downstream number of bases downstream of TES (default: 1000)
--bins number of bins per region (default: 100)

Example:

./rnaedgeflow metagene \
  --inputDir example_data/test_001/process \
  --upstream 500 --downstream 500 --bins 100 \
  --outputDir example_data/test_001/result/metagene

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Example Data

A small test dataset is included under example_data/test_001.
You can test the pipeline with:

./rnaedgeflow pipeline --profile example_data/example.profile
./rnaedgeflow metagene --inputDir example_data/test_001/process --bins 100

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