CODA setup and use

Dear user,
CODA is a workflow developed for the preprocessing of Combo-Seq data as described in the manuscript "CODA: a combo-Seq data analysis workflow" (https://doi.org/10.1093/bib/bbac582).

Important!

After the first use, every time you re-run the CODA_preprocess.sh script, you only need to perform step 6.
System requirements: since CODA uses STAR as its aligner, it requires at least 10 GB of RAM to work.


Before you start using CODA, some initial setup is required:

1. Install conda on your machine (https://conda.io/projects/conda/en/latest/user-guide/install/index.html)
2. Download the GTF and FASTA files of your reference genome of interest (you can skip this step if you already have the indexed files for RSEM).
   Some examples of possible sources for these files are Gencode and Ensembl for mRNA and several other RNA biotypes, or piRNAclusterDB for piRNA.
3. Download the miRge3.0 library for the organism(s) of interest (https://mirge3.readthedocs.io/en/latest/quick_start.html#mirge3-0-libraries)
4. Clone this GitHub repository by typing in the terminal

git clone https://github.com/marta-nazzari/CODA

The following files will be downloaded:

• CODA_preprocess.sh: this is main script for processing the Combo-Seq data
• merge_mirge_files.R: this script is called by the CODA_preprocess.sh script and is necessary to group together all files output by miRge3.0 (note: skipping this step causes the CODA_preprocess.sh script to crash)
• CODA and R_CODA: with these files, you can create a Conda environment (explained at step 5)

5. On you machine, you can create the necessary Conda environments by running the following commands in the terminal:
   
   • conda create -n CODA --file CODA
     
   • conda create -n R_CODA --file R_CODA
     (Note: the environments you create must be named CODA and R_CODA for the script to properly work)
     

Alternatively, you can manually install the required tools on your machine (in which case, the calls to conda environments activation and deactivation need to be commented out). CODA uses:

• Cutadapt v3.7
• FastQC v0.11.9
• miRge3.0 v0.0.9
• MultiQC v1.12
• R v3.6.3
• RSEM v1.3.3
• STAR v2.7.10a

6. Make sure that the CODA_preprocess.sh script has execute and write permissions (you can do so by typing in the command line chmod 777 /path/to/script/CODA_preprocess.sh)
7. Once the files have been downloaded and the Conda environments created, you can open the CODA_preprocess.sh script on a text editor and edit with the necessary parameters that are user-specific (e.g. location of raw files, output directory, genome files location, etc)

You can then launch the CODA_preprocess.sh script from the command line.


Have fun!