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README.md

README.md

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A bioinformatics toolkit for mapping rNMPs embedded in DNA

© 2017 Alli Gombolay, Fredrik Vannberg, and Francesca Storici
School of Biological Sciences, Georgia Institute of Technology

If you use Ribose-Map, please use the following citation:

Gombolay, AL, FO Vannberg, and F Storici. Ribose-Map: a bioinformatics toolkit to map ribonucleotides embedded in genomic DNA. Nucleic Acids Research 2018. DOI: 10.1093/nar/gky874.

Modules

Process rNMP sequencing data independent of the rNMP sequencing technique:

  • Alignment: Align reads to the reference, de-depulicate based on UMIs, and de-multiplex by barcode
  • Coordinate: Locate genomic coordinates of rNMPs for ribose-seq, Pu-seq, emRibo-seq, or HydEn-seq

Analyze sequence context of embedded rNMPs and their genome-wide distribution:

  • Sequence: Calculate and visualize frequencies of nucleotides at and flanking sites of embedded rNMPs
  • Distribution: Visualize coverage of rNMPs across genome and create bedgraph files for genome browser

 

Required Data

Ribose-Map requires the following files:

  1. FASTQ files of sequencing data generated using NGS
  2. Bowtie2 indexes (extension .bt2) for reference genome
  3. FASTA file of nucleotide sequence of reference genome
  4. Configuration file (example is provided in ribose-map/lib)

Note: If you have a list of single nucleotide coordinates that were not created using the Coordinate Module and would like to use the Sequence and/or Distribution Modules, you will need to first create a directory with the path shown below:

"$repository/results/$sample/coordinate" ($repository and $sample variables should be those provided in the config file)

 

Software Installation

  1. Clone Ribose-Map GitHub repository:

    • Click here for information on installing Git on Linux
    git clone https://github.com/agombolay/ribose-map/
    

    Ribose-Map uses Bowtie 2, BEDtools, SAMtools, cutadapt, UMI-tools, R, and Python to analyze and visualize data.
    To ensure easy installation and versioning of these software, we recommend using the MiniConda package manager.

  2. Install pre-requisite packages for conda:

    python3 -m pip install pycosat pyyaml requests --user
    
  3. Install MiniConda and source your .bashrc file:

    • Follow prompts to install MiniConda in home directory
    • Run these commands from within ribose-map/lib directory
    sh Miniconda3-latest-Linux-x86_64.sh && source ~/.bashrc
    
  4. Create environment in which to run Ribose-Map:

    • Run these commands from within ribose-map/lib directory
    conda update conda
    conda install anaconda-client anaconda-build conda-build
    conda env create --name ribosemap_env --file ribosemap.yaml
    

 

How to run Ribose-Map

Before proceeding, close current terminal window and open a new window to refresh the settings

  • If SAMtools gives an error, then install this dependency: conda install -c conda-forge ncurses
  1. Activate environment to access software:
source activate ribosemap_env
  1. Run scripts with configuration_file as input:

    1. Process the data
    ribose-map alignment config
    ribose-map coordinate config
    
    1. Analyze the data
    ribose-map sequence config
    ribose-map distribution config
    
  2. Once the analysis is complete, exit environment:

source deactivate ribosemap_env
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