Prokka Genome Annotation

Prokka Genome Annotation

Overview

Prokka is a command-line pipeline for rapid annotation of prokaryotic genomes (bacteria, archaea, and viruses). It uses a tiered search strategy: protein-coding genes (CDS) are predicted with Prodigal and searched first against a genus-specific database, then RefSeq proteins, then Pfam/TIGRFAMs HMMs. Non-coding RNA genes (rRNA, tRNA, tmRNA) are identified with Barrnap, Aragorn, and Infernal. Prokka processes a single FASTA assembly in minutes and outputs a comprehensive annotation in GFF3, GenBank, FASTA, and tabular formats.

When to Use

• Annotating a newly assembled bacterial or archaeal genome from Illumina, PacBio, or Nanopore assemblies
• Getting functional protein annotations (CDS with product names, EC numbers, GO terms) from a draft or complete genome
• Preparing annotation files for downstream comparative genomics (Roary pan-genome, OrthoFinder)
• Annotating viral or phage genomes when kingdom-specific databases are important
• Performing metagenome-assembled genome (MAG) annotation with the --metagenome flag
• Parsing annotated outputs in Python with BioPython for downstream sequence or feature analysis
• Use PGAP (NCBI Prokaryotic Genome Annotation Pipeline) instead when the goal is NCBI GenBank submission with standards compliance
• Use Bakta instead for faster annotation with built-in NCBI-compatible outputs and a more regularly updated database

Prerequisites

• Software: Prokka ≥ 1.14, Perl 5, Prodigal, Barrnap, HMMER3, BLAST+, Aragorn, Infernal, tbl2asn
• Python packages (for output parsing): biopython, pandas, matplotlib
• Input: assembled genome in FASTA format (complete or draft with multiple contigs)
• Environment: conda strongly recommended to handle the Perl and C dependency stack

Check before installing: The tool may already be available in the current environment (e.g., inside a pixi / conda env). Run command -v prokka first and skip the install commands below if it returns a path. When running inside a pixi project, invoke the tool via pixi run prokka rather than bare prokka.

# Install Prokka via conda/mamba (recommended)
conda install -c conda-forge -c bioconda prokka

# Or with mamba (faster)
mamba install -c conda-forge -c bioconda prokka

# Verify installation and database setup
prokka --version
# prokka 1.14.6

# Check that required tools are on PATH
prokka --depends
# prokka needs: awk, sed, grep, makeblastdb, blastp, hmmscan, ...