Introduction

In this section we will use a software tool called Prokka to annotate a draft genome sequence. Prokka is a “wrapper”; it collects together several pieces of software (from various authors), and so avoids “re-inventing the wheel”.

Prokka finds and annotates features (both protein coding regions and RNA genes, i.e. tRNA, rRNA) present on on a sequence. Note, Prokka uses a two-step process for the annotation of protein coding regions: first, protein coding regions on the genome are identified using Prodigal; second, the function of the encoded protein is predicted by similarity to proteins in one of many protein or protein domain databases. Prokka is a software tool that can be used to annotate bacterial, archaeal and viral genomes quickly, generating standard output files in GenBank, EMBL and gff formats. More information about Prokka can be found here.

Agenda

In this tutorial, we will deal with:

1. Introduction
   1. Import the data
   2. Annotate the genome
   3. Examine the output
   4. View annotated features in JBrowse
   5. What’s Next

Import the data

Prokka requires assembled contigs.

Hands-on: Obtaining our data

1. Make sure you have an empty analysis history. Give it a name.

   Tip: Creating a new history

   Click the new-history icon at the top of the history panel.

   If the new-history is missing:

   1. Click on the galaxy-gear icon (History options) on the top of the history panel
   2. Select the option Create New from the menu

2. Import the following files from Zenodo or from the shared data library

   https://zenodo.org/record/1156405/files/contigs.fasta
   

   Tip: Importing via links

   • Copy the link location

   • Open the Galaxy Upload Manager (galaxy-upload on the top-right of the tool panel)

   • Select Paste/Fetch Data

   • Paste the link into the text field

   • Press Start

   • Close the window

   Tip: Importing data from a data library

   As an alternative to uploading the data from a URL or your computer, the files may also have been made available from a shared data library:

   • Go into Shared data (top panel) then Data libraries
   • Navigate to the correct folder as indicated by your instructor
   • Select the desired files
   • Click on the To History button near the top and select as Datasets from the dropdown menu
   • In the pop-up window, select the history you want to import the files to (or create a new one)
   • Click on Import

Annotate the genome

Now we will run the tool called Prokka.

Hands-on: Annotate genome

1. Prokka Tool: toolshed.g2.bx.psu.edu/repos/crs4/prokka/prokka/1.14.5+galaxy0 with the following parameters (leave everything else unchanged)
   • param-file “contigs to annotate”: contigs.fasta

Examine the output

Once Prokka has finished, examine each of its output files.

• The GFF and GBK files contain all of the information about the features annotated (in different formats.)
• The .txt file contains a summary of the number of features annotated.
• The .faa file contains the protein sequences of the genes annotated.
• The .ffn file contains the nucleotide sequences of the genes annotated.

View annotated features in JBrowse

Now that we have annotated the draft genome sequence, we would like to view the sequence in the JBrowse genome viewer. First, we have to make a JBrowse file. Then, we can view it within Galaxy.

Hands-on: Visualize the annotation

1. JBrowse Tool: toolshed.g2.bx.psu.edu/repos/iuc/jbrowse/jbrowse/1.16.9+galaxy0 with the following parameters
   • “Reference genome to display”: Use a genome from history
     • param-file “Select the reference genome”: fna output of Prokka Tool: toolshed.g2.bx.psu.edu/repos/crs4/prokka/prokka/1.14.5+galaxy0

     This sequence will be the reference against which annotations are displayed

   • “Produce Standalone Instance”: Yes
   • “Genetic Code”: 11: The Bacterial, Archaeal and Plant Plastid Code

   • Click on Insert Track Group

     We will now set up one track - each track is a dataset displayed underneath the reference sequence (which is displayed as nucleotides in FASTA format). We will choose to display the annotations (the Prokka.gff file).

     • In 1: Track Group
       • “Track Category”: gene annotations
       • Click on Insert Annotation Track and fill it with:
         • “Track Type”: GFF/GFF3/BED Features
         • param-file “GFF/GFF3/BED Track Data”: gff output of Prokka Tool: toolshed.g2.bx.psu.edu/repos/crs4/prokka/prokka/1.14.5+galaxy0

   A new file will be created in your history, this contains the JBrowse interactive visualisation. We will now view its contents and play with it

2. Inspect the JBrowse on data XX and data XX - Complete file by clicking on the galaxy-eye (eye) icon

   The JBrowse window will appear in the centre Galaxy panel.

3. Display all the tracks and practice maneuvering around
   1. Click on the tick boxes on the left to display the tracks
   2. Select contig 1 in the drop down box. You can only see one contig displayed at a time.
   3. Zoom by clicking on the plus and minus buttons.
   4. JBrowse displays the sequence and a 6-frame amino acid translation.
   5. Right click on a gene/feature annotation (the bars on the annotation track), then select View Details to see more information.
      • gene name
      • product name
      • you can download the FASTA sequence by clicking on the disk icon

What’s Next

After automatic annotation of prokaryotic genome, if inspection of predicted genes with JBrowse introduced mistakes, e.g. wrong exon/intron limits, splitted genes, or merged genes – or simply if you wish to rename genes or provide additional functional (e.g., Gene Ontology) data, setting up a manual curation project using Apollo helps a lot to manually fix these errors.

The Apollo training should provide additional guidance.

Key points

• Prokka is a useful tool to annotate a bacterial genome.

• JBrowse can be used to inspect the annotation of a genome.

Frequently Asked Questions

Have questions about this tutorial? Check out the tutorial FAQ page or the FAQ page for the Genome Annotation topic to see if your question is listed there. If not, please ask your question on the GTN Gitter Channel or the Galaxy Help Forum

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Citing this Tutorial

1. Anna Syme, Torsten Seemann, Simon Gladman, 2022 Genome annotation with Prokka (Galaxy Training Materials). https://training.galaxyproject.org/training-material/topics/genome-annotation/tutorials/annotation-with-prokka/tutorial.html Online; accessed TODAY
2. Batut et al., 2018 Community-Driven Data Analysis Training for Biology Cell Systems 10.1016/j.cels.2018.05.012

BibTeX

@misc{genome-annotation-annotation-with-prokka,
author = "Anna Syme and Torsten Seemann and Simon Gladman",
title = "Genome annotation with Prokka (Galaxy Training Materials)",
year = "2022",
month = "10",
day = "18"
url = "\url{https://training.galaxyproject.org/training-material/topics/genome-annotation/tutorials/annotation-with-prokka/tutorial.html}",
note = "[Online; accessed TODAY]"
}
@article{Batut_2018,
    doi = {10.1016/j.cels.2018.05.012},
    url = {https://doi.org/10.1016%2Fj.cels.2018.05.012},
    year = 2018,
    month = {jun},
    publisher = {Elsevier {BV}},
    volume = {6},
    number = {6},
    pages = {752--758.e1},
    author = {B{\'{e}}r{\'{e}}nice Batut and Saskia Hiltemann and Andrea Bagnacani and Dannon Baker and Vivek Bhardwaj and Clemens Blank and Anthony Bretaudeau and Loraine Brillet-Gu{\'{e}}guen and Martin {\v{C}}ech and John Chilton and Dave Clements and Olivia Doppelt-Azeroual and Anika Erxleben and Mallory Ann Freeberg and Simon Gladman and Youri Hoogstrate and Hans-Rudolf Hotz and Torsten Houwaart and Pratik Jagtap and Delphine Larivi{\`{e}}re and Gildas Le Corguill{\'{e}} and Thomas Manke and Fabien Mareuil and Fidel Ram{\'{\i}}rez and Devon Ryan and Florian Christoph Sigloch and Nicola Soranzo and Joachim Wolff and Pavankumar Videm and Markus Wolfien and Aisanjiang Wubuli and Dilmurat Yusuf and James Taylor and Rolf Backofen and Anton Nekrutenko and Björn Grüning},
    title = {Community-Driven Data Analysis Training for Biology},
    journal = {Cell Systems}
}
                   

Congratulations on successfully completing this tutorial!