mRNAcal

mRNAcal is an R package designed to streamline the analysis of mRNA regions and RNA secondary structures. This package offers tools for organizing GenBank data, extracting FASTA sequences, generating mRNA regions, and processing RNA secondary structures using viennaRNA package.

Features

• GenBank Data Organization: Easily manage and extract information from GenBank files.
• FASTA Extraction: Extract coding sequences (CDS) or other genomic regions in FASTA format.
• Custom Downstream Sequences: Define a specific gene of interest (GOI) sequence to append to upstream regions.
• mRNA Region Generation: Automatically generate mRNA regions with user-defined upstream lengths and optional GOI.
• RNA Secondary Structure Prediction: Integrate RNAfold to calculate dot-bracket structures and minimum free energy (ΔG).
• Pipeline Automation: Use run_mRNAcal() to execute the full pipeline in one step with customizable parameters.

Installation

To install the mRNAcal package and its dependencies, please follow these steps: Check if devtools is installed, and install it if necessary

if (!requireNamespace("devtools", quietly = TRUE))
    install.packages("devtools")

Step 1: Install Bioconductor dependencies

First, install the necessary Bioconductor packages:

# Install Bioconductor packages
if (!requireNamespace("BiocManager", quietly = TRUE))
    install.packages("BiocManager")

BiocManager::install(c("Biostrings", "ComplexHeatmap", "Peptides"))

Step 2: Install CRAN dependencies

Next, install the required CRAN packages. Some packages may not be installed automatically, so you can install them manually: sometimes it can not installed automatically followed

Install CRAN packages

install.packages(c("qdap", "seqinr", "splitstackshape"))
#remotes::install_github("trinker/qdap")

Step 1: Install Java Development Kit (JDK)

The rJava package requires Java to be installed. Please install the Java Development Kit (JDK) from the following sources:

• Windows/Mac: Oracle JDK Downloads
• Linux: Use your package manager to install OpenJDK. For example:

  sudo apt install openjdk-11-jdk
  

After installation, ensure that the JAVA_HOME environment variable is set correctly.

Windows:

    1.    Go to System Properties -> Environment Variables -> System Variables -> New
    2.    Variable name: JAVA_HOME
    3.    Variable value: C:\Program Files\Java\jdk-11.0.1 (your JDK installation path)

Mac/Linux:
    Add the following to your .bash_profile or .zshrc:
    ```bash
    export JAVA_HOME=$(/usr/libexec/java_home)

Step 2: Install rJava

Once Java is installed and configured, you can install the rJava package: Windows: bash Sys.setenv(JAVA_HOME="C:/Program Files/Java/jdk-11.0.1") r install.packages("rJava") Mac/Linux: r install.packages("rJava")

Step 3: Install mRNAcal package

devtools::install_github("JAEYOONSUNG/mRNAcal")

Usage

1. Organizing GenBank Data

# Organize GenBank file into a structured table
Genbank_organizer("example.gbk")

2. Extract FASTA Sequences

genbank_fna_extractor()

3. Generate mRNA Regions

generate_mRNA_regions(upstream_values = seq(100, 500, 100), downstream = 100)
# Define upstream and downstream regions of coding sequences for mRNA.
# In cases where you have a specific gene of interest (GOI) sequence to append to upstream regions, you can use the custom_downstream_seq parameter.

locus_tag	seq_u100_dGOI	seq_u200_dGOI	...
gene1	ATCGG...ATGAAAAAAAAAAAAAAAAAAAAAAAAAAAA	GCTAG...ATGAAAAAAAAAAAAAAAAAAAAAAAAAAAA	...
gene2	CGTTA...ATGAAAAAAAAAAAAAAAAAAAAAAAAAAAA	TACGG...ATGAAAAAAAAAAAAAAAAAAAAAAAAAAAA	...

---

4. Process RNAfold Results The resulting genbank_table will include columns for each combination of upstream length and downstream region, as well as the custom sequence. Example:

Process RNA sequences using RNAfold

process_all_sequences(data = "genbank_table", rnafold_path = "/path/to/RNAfold")

Quick start

5. Automate the Entire Pipeline

native sequences

run_mRNAcal(
  genbank_file = "example.gbk",
  rnafold_path = "/path/to/RNAfold",
  upstream_values = seq(100, 500, 100),
  downstream = 100
)

specific sequence

run_mRNAcal(
  genbank_file = "example.gbk",
  rnafold_path = "/path/to/RNAfold",
  upstream_values = seq(100, 500, 100),
  custom_downstream_seq = "ATGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA"

Result

Sequence	Structure	Free Energy (ΔG)
seq_u100_d100	..((((((((((.(((((((.......)))))...))))))..	-51.3 kcal/mol
seq_u200_d100	....(((.(((.((((((((...).)))))))))))).)))..	-85.6 kcal/mol
seq_u300_d100	...((((...((((.((((....))))..))))..........	-102.4 kcal/mol
seq_u400_d100	((((((........((((((...)............)))))).	-138.4 kcal/mol
seq_u500_d100	....((..((((.(((((((...))).))).))))..))....	-39.3 kcal/mol

To be updated

• plot mRNA fold
• plot heatmap generation using free energy values
• delete duplicated mRNA sequences in excel file
• ciruclar form auto detection algorithm