Step-by-Step Manual: Converting GFF3 to GTF

Converting GFF3 (General Feature Format version 3) to GTF (Gene Transfer Format) is a common task in bioinformatics, especially for downstream analyses like RNA-seq. Below is a detailed guide on how to perform this conversion using popular tools.

1. Understand the Formats

• GFF3: A flexible format for describing genomic features. It has 9 columns: seqid, source, type, start, end, score, strand, phase, and attributes.
• GTF: A stricter format derived from GFF2, commonly used for gene annotation. It also has 9 columns but requires specific attributes like gene_id and transcript_id.

2. Use gffread from Cufflinks

gffread is a widely used tool for converting GFF3 to GTF.

Step 2.1: Install Cufflinks

If you don’t have gffread, install Cufflinks:

# Download Cufflinks
wget http://cole-trapnell-lab.github.io/cufflinks/assets/downloads/cufflinks-2.2.1.Linux_x86_64.tar.gz

# Extract the tarball
tar -xzvf cufflinks-2.2.1.Linux_x86_64.tar.gz

# Add to PATH
export PATH=$PATH:/path/to/cufflinks-2.2.1.Linux_x86_64

Step 2.2: Convert GFF3 to GTF

Run gffread to convert your GFF3 file:

gffread input.gff3 -T -o output.gtf

• input.gff3: Your input GFF3 file.
• -T: Specifies output format as GTF.
• output.gtf: The output GTF file.

3. Use AGAT (Another GFF Analysis Toolkit)

AGAT is a powerful toolkit for working with GFF/GTF files.

Step 3.1: Install AGAT

Install AGAT using Conda:

conda install -c bioconda agat

Step 3.2: Convert GFF3 to GTF

Run the following command:

agat_convert_sp_gff2gtf.pl --gff input.gff3 -o output.gtf

• input.gff3: Your input GFF3 file.
• output.gtf: The output GTF file.

4. Use rtracklayer in R

If you prefer working in R, you can use the rtracklayer package from Bioconductor.

Step 4.1: Install rtracklayer

if (!require("BiocManager", quietly = TRUE))
    install.packages("BiocManager")
BiocManager::install("rtracklayer")

Step 4.2: Convert GFF3 to GTF

Run the following R script:

library(rtracklayer)

# Import GFF3 file
gff3_file <- "input.gff3"
gff3_data <- import(gff3_file)

# Export as GTF
gtf_file <- "output.gtf"
export(gff3_data, gtf_file, format = "gtf")

5. Use GenomeTools

GenomeTools is another tool for working with GFF/GTF files.

Step 5.1: Install GenomeTools

# On Ubuntu/Debian
sudo apt-get install genometools

# On macOS
brew install genometools

Step 5.2: Convert GFF3 to GTF

Run the following command:

gt gff3_to_gtf input.gff3 > output.gtf

6. Validate the Output

After conversion, validate the GTF file to ensure it meets the required format:

• Check for mandatory attributes like gene_id and transcript_id.
• Use tools like gtf2bed or IGV to visualize the GTF file.

7. Automate the Workflow

If you frequently convert GFF3 to GTF, consider automating the process using a script or workflow manager like Snakemake or Nextflow.

Example Snakemake Workflow

rule all:
    input:
        "output.gtf"

rule convert_gff3_to_gtf:
    input:
        "input.gff3"
    output:
        "output.gtf"
    shell:
        "gffread {input} -T -o {output}"

Recent Tools and Tips

1. AGAT: A comprehensive toolkit for GFF/GTF manipulation.
2. gffread: Fast and reliable for GFF3-to-GTF conversion.
3. rtracklayer: Ideal for R users working with genomic data.
4. GenomeTools: A versatile tool for GFF/GTF manipulation.

Tips for Conversion

• Check Attribute Consistency: Ensure mandatory attributes like gene_id and transcript_id are present in the GTF file.
• Handle Large Files: Use tools like AGAT or gffread for efficient processing of large GFF3 files.
• Validate Output: Always validate the converted GTF file to ensure it meets the required format.

By following this guide, you can efficiently convert GFF3 files to GTF format using the latest tools and best practices.