Module 2: Genome Annotation
Genome annotation is the process of identifying and labeling functional elements in a genome assembly — including genes, repetitive elements, regulatory regions, and non-coding RNAs. A raw assembly is just a string of nucleotides; annotation transforms it into a biologically interpretable resource.
Types of Annotation
| Type | What it finds | Examples |
|---|---|---|
| Structural | Gene boundaries, exons, introns, UTRs | BRAKER3, Helixer |
| Functional | Gene function, protein domains | BLAST, InterProScan |
| Repeat | Transposable elements, repeat families | RepeatMasker |
| Quality | Annotation completeness | BUSCO, OMArk |
Learning Objectives
- Mask repetitive elements before gene prediction
- Predict gene structures using evidence-based and ab initio methods
- Assess annotation completeness and quality
- Visualize annotated features in a genome browser
Hands-on Tutorials
Hands-on 1 — Getting Started
Introduction to genome annotation tools and workflows in Galaxy.
Galaxy Training Network tutorials: Genome Annotation Tutorials
Hands-on 2 — Basic Gene Structure Annotation
Gene structure annotation for sequences not yet at contig/scaffold level.
Key tools used:
- RepeatMasker — identify and soft-mask repetitive elements
- RNA-STAR — align RNA-seq reads to the genome for splice site evidence
- BRAKER3 — ab initio gene prediction with RNA-seq and protein evidence
- GFFread — process and convert GFF/GTF annotation files
Hands-on 3 & 4 — Annotation Using Pipelines
Running complete annotation pipelines in Galaxy and assessing outputs.
Key tools used:
- Helixer — deep learning–based gene structure prediction
- BUSCO — benchmarking annotation completeness using conserved gene sets
- OMArk — proteome quality assessment
Hands-on 4 — Visualization with WebIGV
Exploring annotated features interactively in the browser.
Key tools used:
- JBrowse / WebIGV — view GFF annotation tracks alongside the assembly sequence
Key Concepts
GFF3 / GTF — Standard file formats for storing genome annotation. GFF3 (Generic Feature Format version 3) and GTF (Gene Transfer Format) describe genomic features with their chromosomal coordinates.
Evidence-based vs Ab Initio — Evidence-based annotation uses experimental data (RNA-seq, protein homology) to guide gene finding. Ab initio prediction relies solely on statistical models trained on known gene structures.
Repeat masking — Before gene prediction, repetitive sequences are masked (replaced with lowercase letters or Ns) to prevent them from confusing gene finders.
BUSCO score — Measures the percentage of expected conserved genes found in an assembly or annotation. A high complete BUSCO score indicates a high-quality, near-complete result.