RNake

RNake is a Snakemake pipeline designed for Bacterial RNA sequencing data analysis. This pipeline is built for convenience, allowing users to easily process both single and paired-end FASTQ inputs. It automates the steps of trimming, mapping, counting, and merging RNA-seq data, ensuring reproducibility and efficiency.

Features

• Automated Workflow: Automates the RNA-seq analysis process from raw FASTQ files to final count tables.
• Single & Paired-End Support: Handles both single-end and paired-end reads.
• Flexible Configuration: Easily configurable for different projects and input data.

Installation

Clone the repository and navigate to the project directory:

git clone https://github.com/bbagy/RNake.git
cd RNake

Make sure you have all necessary dependencies installed, including Snakemake, Python, and other tools specified in the YAML files.

Usage

To run the pipeline, use the following command:

pro=(your_project_name)
fastqs=(your_fastq_directory)
genome=(your_reference_genome_file.fna)
gff=(your_reference_gff_file.gff)

snakemake -s /your_path/Go_bacteriaRNake_paired_V3.smk --cores 16 --config project=$pro read_dir=$fastqs genome=$genome gff=$gff --rerun-incomplete --use-conda

Replace your_project_name, your_fastq_directory, your_genome_file, your_gff_file, and your_path with the appropriate paths or filenames for your specific analysis.

Handling Prokka Output GFF Files

If you are using a GFF file generated by Prokka, it may contain a FASTA section at the end, which can cause issues during processing. To clean the GFF file, removing the FASTA part, use the following command:

sed '/^##FASTA$/,$d' your_prokka_output.gff > your_cleaned_output.gff

Replace your_prokka_output.gff with the name of your Prokka output file, and your_cleaned_output.gff with the desired output filename.

Output

The final output of the pipeline is a CSV file named merged_counts_with_gene_names.csv. This file contains the merged count data with gene names mapped from the provided GFF file. It is located in the project’s output directory and can be used for downstream analysis.

Required Tools

The pipeline requires several tools, which are specified in the YAML files for Conda environments:

• Trimmomatic: For trimming the reads.
• Bowtie2: For mapping reads to the reference genome.
• SAMtools: For processing SAM/BAM files.
• HTSeq: For counting reads mapped to genes.

These tools are managed by Conda environments to ensure that the correct versions are used.

Contributing

We welcome contributions to improve this pipeline. Please fork the repository and create a pull request with your changes.

License

This project does not have a specific license. Please contact the repository owner for permissions regarding usage, distribution, or modification.

Contact

For any questions or issues, please open an issue on this repository or contact hp2523@cumc.columbia.edu.