Functional Genomics
(MBG419)
Week 6 (03.04.2026)
‘Genome Annotation & Functional Genomics’
Dr Ömer Faruk Bay
Image credit: Laura Olivares Boldú / Wellcome Connecting Science
What is genome annotation?
• Genome annotation is the process of deriving the structural and
functional information of a protein or gene from a raw data set using
different analysis, comparison, estimation, precision, and other mining
techniques.
• Main objectives:
• Identify genes (protein-coding and non-coding)
• Locate regulatory elements (promoters, enhancers, terminators)
• Identify repetitive elements (transposons, microsatellites)
• Assign functions to genes (based on sequence similarity, structural features,
and experimental evidence)
Genome annotation
Structural annotation Functional annotation
• Structural annotation focuses on • Functional annotation assigns
identifying and labelling genomic biological meaning to genes by
features such as: predicting their:
• Genes
• Function
• Exons
• Biochemical role
• Introns
• Involvement in pathways
• Regulatory regions
• Non-coding elements
Structural annotation
• There are two types of gene prediction: ab initio (meaning ‘from the
beginning’ in Latin) and evidence based. The information taken from
these two prediction methods is then combined and lined up with the
sequenced genome.
• Ab initio gene prediction: Based solely on the DNA sequence, using statistical
models of gene structure (e.g., codon usage, splice site signals).
• Evidence-based gene prediction: Incorporates external evidence, such as:
• Transcriptome data (RNAseq)
• Protein homology (sequence similarity to known proteins)
• Expressed Sequence Tags (ESTs)
Prokaryotic genes - ab initio gene prediction
• Regulatory Region (Upstream Elements): • Coding Region (Open Reading Frame, ORF):
• Promoter • Start Codon
• Operator • Coding DNA Sequence (CDS)
• Ribosome Binding Site (RBS or Shine-Dalgarno • Stop Codon
Sequence)
• Termination Region (Downstream Elements):
• Terminator Sequence - Intrinsic termination and Rho-dependent termination
Prokaryotic genes - ab initio gene prediction
Regulatory region
• Promoter: The DNA sequence where RNA polymerase binds to initiate
transcription.
• Operator (if present in operon systems): A regulatory sequence where
repressor proteins can bind to control gene expression.
• Ribosome Binding Site (RBS or Shine-Dalgarno Sequence): A short sequence
upstream of the coding region that helps ribosomes initiate translation.
Prokaryotic genes - ab initio gene prediction
Coding region
Eukaryotic genes - ab initio gene prediction
Eukaryotic genes - ab initio gene prediction
Exon & introns
• Exons and Introns
• Splice sites (GT-AG rule for introns)
• Start (AUG) and stop codons (UAA, UGA, UAG)
Eukaryotic genes – evidence-based gene prediction
• Multiple pieces of evidence:
• Alignment of RNAseq reads
• Alignment of EST or transcripts (same species or closely related species
• Alignment of proteins (closely related species)
Structural annotation
• Evaluation of annotation:
• BUSCO – number of genes, fragmented genes, duplicated genes etc…
• rRNAs & tRNAs: Essential for translation
• MicroRNAs (miRNAs) & lncRNAs: Involved in gene regulation
• Manual curation:
• APOLLO (tool)
APOLLO
• Apollo software allows you to compare gene prediction models with evidence tracks to
determine if the prediction is correct.
• In this example the predicted gene (gene
18005) shown in dark blue matches the
RNA sequencing data (Illumina coverage,
Isoseq reads and intron data) quite well,
indicating this is a good gene model.
Dragging the gene to the yellow scratch
area at the top of the screen selects this
gene for further review.
Functional annotation
• Functional annotation is the process of assigning biological meaning to identified
features of genes. It's about figuring out what the gene does.
• Functional annotation answers following questions:
• What is the gene's most likely function? Is it an enzyme, a structural protein, a regulator?
• What biological pathways is it involved in? Does it participate in metabolism, signalling,
development, or some other process?
• Where is the gene expressed? Is it specific to certain tissues, developmental stages, or
environmental conditions?
• What other proteins does it interact with? Does it form complexes with other proteins to carry
out its function?
Functional annotation - methods
• Similarity search – homology
• BLAST (Basic Local Alignment Search Tool), DIAMOND (double index alignment of next-generation
sequencing data)
• Pattern search – domain-based annotation
• Pfam, InterProScan
• Gene ontology
• Blast2GO, EggNOG-mapper, InterProScan
• Pathway analysis
• DAVID (Database for Annotation, Visualization and Integrated Discovery), KEGG
Similarity search – homology
BLAST
• Genes with similar DNA or protein sequences often have similar
functions due to shared evolutionary ancestry. This is the single
most common starting point for functional annotation.
• If a gene in your newly sequenced organism has a high degree of
similarity to a well-characterized gene in a different organism, it's
likely to perform a similar function.
Pattern search – domain-based annotation
InterProScan
• Protein domains are evolutionarily conserved regions that have
specific functions.
• For example, a kinase domain is responsible for adding phosphate
groups to other proteins, while a DNA-binding domain allows a
protein to bind to DNA. By identifying these domains, we can gain
valuable insights into the protein's overall function.
• InterPro provides functional analysis of proteins by classifying
them into families and predicting domains and important sites. To
classify proteins in this way, InterPro uses predictive models,
known as signatures, provided by several different databases that
make up the InterPro consortium.
[Link]
Gene ontology
Blast2GO, EggNOG-mapper, InterProScan
[Link]
• GO is a structured vocabulary describing gene function in three
categories:
• Biological Process (BP) – What the gene does in the cell (e.g., DNA repair).
• Molecular Function (MF) – What activity the gene performs (e.g., ATP binding).
• Cellular Component (CC) – Where the gene product functions (e.g., nucleus).
Gene ontology
Blast2GO, EggNOG-mapper, InterProScan
• eggNOG-mapper v2 is a tool for
functional annotation of large sets
of sequences based on fast
orthology assignments using
precomputed eggNOG v5.0
clusters and phylogenies.
[Link]
Pathway analysis
DAVID
[Link]