Project 3

Project 3: Assembly and annotation of bacterial genomes

You will be working with PacBio sequencing data of eight different bacterial species. Divide the species over the members of the group and generate an assembly and annotation. After that, guess the species.

Project aim

Generate and evaluate an assembly of a bacterial genome out of PacBio reads.

There are eight different species: sample_[1-8].fastq.gz

Each species has a fastq file available. You can download all fastq files like this:

wget https://ngs-longreads-training.s3.eu-central-1.amazonaws.com/project3.tar.gz
tar -xvf project3.tar.gz
rm project3.tar.gz

Note

Download the data file package in your shared working directory, i.e. : /group_work/<group name> or ~/<group name>. Only one group member has to do this.

This will create a directory project3 with the following structure:

project3
|-- sample_1.fastq.gz
|-- sample_2.fastq.gz
|-- sample_3.fastq.gz
|-- sample_4.fastq.gz
|-- sample_5.fastq.gz
|-- sample_6.fastq.gz
|-- sample_7.fastq.gz
`-- sample_8.fastq.gz

0 directories, 8 files

Before you start

You can start this project with dividing the species over the different group members. In principle, each group member will go through all the steps of assembly and annotation:

1. Quality control with NanoPlot
2. Assembly with flye
3. Assembly QC with BUSCO
4. Annotation with prokka

Tasks and questions

Note

You have four cores available. Use them! For most tools you can specificy the number of cores/cpus as an argument.

Note

All require software can be found in the conda environment assembly. Load it like this:

conda activate assembly

• Perform a quality control with NanoPlot.
  • How is the read quality? Is this quality expected?
  • How is the read length?
• Perform an assembly with flye.
  • Have a look at the helper first with flye --help. Make sure you pick the correct mode (i.e. --pacbio-??).
  • Check out the output. Where is the assembly? How is the quality? For that, check out assembly_info.txt.
  • What species did you assemble? Choose from this list:

    Acinetobacter baumannii
    Bacillus cereus
    Bacillus subtilis
    Burkholderia cepacia
    Burkholderia multivorans
    Enterococcus faecalis
    Escherichia coli
    Helicobacter pylori
    Klebsiella pneumoniae
    Listeria monocytogenes
    Methanocorpusculum labreanum
    Neisseria meningitidis
    Rhodopseudomonas palustris
    Salmonella enterica
    Staphylococcus aureus
    Streptococcus pyogenes
    Thermanaerovibrio acidaminovorans
    Treponema denticola
    Vibrio parahaemolyticus
    

  • Did flye assemble any plasmid sequences?
• Check the completeness with BUSCO. Have a good look at the manual first. You can use automated lineage selecton by specifying --auto-lineage-prok. After you have run BUSCO, you can generate a nice completeness plot with generate_plot.py. You can check its usage with generate_plot.py --help.
  • How is the completeness? Is this expected?
• Perform an annotation with prokka. Again, check the manual first. After the run, have a look at for example the statistics in PROKKA_[date].txt. For a nice table of annotated genes have a look in PROKKA_[date].tsv.
• Compare the assemblies of the different species. Are assembly qualities similar? Can you think of reasons why?

This tutorial is based on data provided by Pacific Biosciences at https://downloads.pacbcloud.com/public/dataset/2021-11-Microbial-96plex/