Genome annotation with Prokka

Overview

question Questions
  • How to annotate a bacterial genome?
  • How to visualize annoted genomic features?
objectives Objectives
  • Load genome into Galaxy
  • Annotate genome with Prokka
  • View annotations in JBrowse
requirements Requirements

time Time estimation: 1 hour

Supporting Materials

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. Import the data
  2. Annotate the genome
  3. Examine the output
  4. View annotated features in JBrowse

Import the data

Prokka requires assembled contigs.

hands_on Hands-on: Obtaining our data

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

    tip 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 Sample Data.

    • Obtain data directly from Zenodo: DOI
    • Download contigs.fasta
    • Upload the file to your history.

Annotate the genome

Now we will run the tool called Prokka.

hands_on Hands-on: Annotate genome

  1. Prokka tool with the following parameters (leave everything else unchanged)
    • param-file “contigs to annotate”: contigs.fasta
    • “Locus tag prefix (–locustag)”: P
    • “Force GenBank/ENA/DDJB compliance (–compliant)”: No
    • “Sequencing Centre ID (–centre)”: V
    • “Genus Name (–genus)”: Staphylococcus
    • “Species Name (–species)”: aureus
    • “Use genus-specific BLAST database”: No

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 Hands-on: Visualize the annotation

  1. JBrowse tool with the following parameters
    • “Reference genome to display”: Use a genome from history
      • param-file “Select the reference genome”: fna output of Prokka tool

      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
    • “JBrowse-in-Galaxy Action”: New JBrowse Instance
    • 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 Cateogry”: gene annotations
        • Click on Insert Annotation Track and fill it with:
          • “Track Type”: GFF/GFF3/BED/GBK Features
          • param-file “GFF/GFF3/BED Track Data”: gff output of Prokka tool
          • “JBrowse Track Type [Advanced]”: Canvas Features
          • Click on “JBrowse Styling Options [Advanced]”
            • “JBrowse style.label”: product,name,id
          • “Track Visibility”: On for new users

    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 out by clicking on the minus button to see sequence reads and their coverage (the grey graph)
    4. Zoom in by clicking on the plus button to see annotations.
    5. JBrowse displays the sequence and a 6-frame amino acid translation.
    6. 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. JBrowse

keypoints Key points

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

congratulations Congratulations on successfully completing this tutorial!