Genome annotation with Prokka

Contributors

Anna Syme Torsten Seemann Simon Gladman

Questions

• How to annotate a bacterial genome?

• How to visualize annoted genomic features?

Objectives

• Load genome into Galaxy

• Annotate genome with Prokka

• View annotations in JBrowse

Overview

• What is genome annotation?

• Tools for genome annotation

• The tool “Prokka”

Speaker Notes

• In these slides, we will learn what is genome annotation, and which tools can be used for genome annotation.
• We will describe in detail a tool called Prokka.

What is Annotation?

• Classifying and describing parts of the genome sequence

• Annotations are biological or other features on a genome, e.g.

  • a ribosome binding site: a biological feature

  • a sequence of TTTTTT: may/may not be biological but could be interesting

• We can name features by type and location, e.g. gene, pseudogene, repeat

• We can hypothesise functions, e.g. antioxidant activity

Speaker Notes

• Annotating a genome means positioning features along the sequence of a genome.
• Those features can be anything one can find in a genome sequence: genes, but also binding sites for example.
• When a feature, like a gene for example, is positioned, you can add information about its function.
• This operation is named “functional annotation”.

First: assemble the genome

Speaker Notes

• Before annotating a genome, you need to assemble it.
• If you get a high quality assembly, it will be easier to perform a good quality annotation.

Then: annotate

Speaker Notes

• Once you have a good genome sequence, you can annotate it.
• In this example, there a gene coding for a delta toxin.
• There is a ribosome binding site in red, and the coding sequence of this gene is in green.

Annotation

Speaker Notes

• For each feature annotated on a genome, you can get its position, its type, and some information about its function or how it is expressed.

How do we annotate? many different ways:

• sequence: does it match known sequences in databases?

• sequence structure: e.g. does it look like an exon (start and stop codons)?

• use other data: e.g. do lab experiments to investigate biological function

Speaker Notes

• You can annotate features by looking at similarities with known sequences from international databases.
• Some tools annotate features on a genome by seeking motifs corresponding to known structure (for example gene or exon start or stop).
• Some lab experiments can help annotate specific regions of a genome, even though it is often much more expensive than an automatic annotation.
• The lab experiments can provide certainty about function, where automatic annotation is more of a guess.

Prokka

Speaker Notes

• Prokka is a pipeline that runs several other tools to annotate prokaryotic genomes.
• The input is the assembly of the genome in Fasta format.
• Prokka runs Aragorn to annotate transfer RNAs.
• Ribosomal RNAs are annotated with RNAmmer.
• Infernal uses the Rfam database to annotate non coding RNAs.
• Finally Prodigal annotated coding genes.
• Each coding sequence is then compared to the SwissProt sequence database using Blast, and to TIGR and Pfam motif datases using Hmmer3.
• SignalP is also run to detect signal peptides in each predicted coding sequence.
• The final result of the whole Prokka pipeline is a set of GFF3, GBK ans ASN1 files.

More information

Galaxy Training Network Slides: Introduction to Genome Annotation

Speaker Notes

• More information is available in the “Introduction to Genome Annotation” slides.

Key Points

• Prokka is a useful tool to annotate a bacterial genome.
• JBrowse can be used to inspect the annotation of a genome.

Thank you!