AllTheBacteria​

Zamin Iqbal's Group, who brought us 661k bacterial assemblies, has now taken it a step further with AllTheBacteria. As someone once tasked with assembling "all the Staphylococcus aureus genomes" (although, it was only about 700 samples in 2010!), this is truly an impressive feat, and a valuable community resource! With the latest assemblies, the collection is now nearly 2,000,000 bacterial assemblies!

Similar to their previous methods, the latest version of AllTheBacteria uses Shovill for assembly. In addition, each assembly has basic metrics calculated, undergoes taxonomic abundance estimation, and has sketches made available. For more details about this project, please see:

• Preprint: AllTheBacteria - all bacterial genomes assembled, available and searchable
• GitHub: AllTheBacteria

Since Zamin revealed the latest updates on AllTheBacteria, I've been wondering: How could Bactopia users take advantage these assemblies? Especially, through available Bactopia Tools?

Why Bactopia Tools?​

The really nice thing about Bactopia Tools is they make it super easy to run 60 additional analyses on your genomes. It's really as simple as adding --wf <tool> to your Bactopia command, then Bactopia will then handle the rest for you, including container selection and audit trails.

Obviously, I'm a bit biased here, but utilizing Bactopia Tools in this situation would greatly streamline a lot of downstream analyses of AllTheBacteria assemblies. I think this would allow researchers to quickly get to the science behind these assemblies.

To give you an idea, there are currently 38 Bactopia Tools that use assemblies as inputs. In other words, each of these tools would be easy to run on the 2,000,000 AllTheBacteria assemblies.

bactopia atb-formatter​

Bactopia already allows assemblies as inputs, but I didn't want users to have to go through the full Bactopia pipeline to use the Bactopia Tools. Instead, I wanted to make a quick and easy way for users to go directly to using Bactopia Tools. To accomplish this, I created a new Bactopia command called atb-formatter (AllTheBacteria Formatter). With atb-formatter, the necessary Bactopia output directory structure will be created from a directory of _AllTheBacteria assemblies.

That's cool and all, but let's actually demonstrate the usage of atb-formatter on some Legionella pneumophila assemblies from AllTheBacteria.

Example Usage for Legionella pneumophila​

To demonstrate the usage of bactopia atb-formatter, I will use assemblies for Legionella pneumophila from AllTheBacteria and run legsta, a typing tool for L. pneumophila assemblies, written by Torsten Seeman, To be specific, I will run legsta from the available Bactopia Tool.

Getting Setup​

Before we get started, you'll need to have Bactopia installed. If you haven't done this yet, please see the installation instructions.

You will also want to make sure you are using at least version 3.0.1 of Bactopia, as this is the first release to have the atb-formatter command.

bactopia --version
bactopia 3.0.1

Download the Assemblies​

First I will download the L. pneumophila assemblies from AllTheBacteria, then extract them into a folder called legionella-assemblies. Easy enough!

mkdir atb-legionella
cd atb-legionella

# Download the assemblies
wget https://ftp.ebi.ac.uk/pub/databases/AllTheBacteria/Releases/0.1/assembly/legionella_pneumophila__01.asm.tar.xz
wget https://ftp.ebi.ac.uk/pub/databases/AllTheBacteria/Releases/0.1/assembly/legionella_pneumophila__02.asm.tar.xz

# Extract the assemblies
mkdir legionella-assemblies
tar -C legionella-assemblies -xJf legionella_pneumophila__01.asm.tar.xz
tar -C legionella-assemblies -xJf legionella_pneumophila__02.asm.tar.xz

At the time of writing this, there were 5,393 L. pneumophila assemblies available from AllTheBacteria. While its not Salmonella enterica with it's hundreds of thousands of assemblies, it's a great number to demonstrate the usage of bactopia atb-formatter.

Create the Bactopia Directory Structure​

With the assemblies extracted, now I need to create the required Bactopia directory to make use of Bactopia Tools. For this, I used bactopia atb-formatter, which creates a sample folder for each assembly that matches the BioSample accession.

# Create the Bactopia directory structure
bactopia atb-formatter --path legionella-assemblies --recursive

After running the above command, you should see something like the following:

2024-03-22 14:30:07:root:INFO - Setting up Bactopia directory structure (use --verbose to see more details)
2024-03-22 14:30:08:root:INFO - Bactopia directory structure created at bactopia
2024-03-22 14:30:08:root:INFO - Total assemblies processed: 5393

Use Bactopia to run Legsta​

Fancy! Now we have all the assemblies sym-linked into a Bactopia directory structure. It's time to let Bactopia Tools shine! To do this, I will run the legsta Bactopia Tool and demonstrate how seamless it is to type 5,393 assemblies.

With a simple addition of --wf legsta and pointing to the Bactopia directory, legsta will be executed on all 5,393 assemblies! It really is that simple!

# Run legsta
bactopia --wf legsta -profile singularity

After some time, the legsta tool will complete for all 5,393 assemblies, and you should be met with something like the following:

[5d/d04297] process > BACTOPIATOOLS:LEGSTA:LEGSTA_MODULE (SAMN29911258) [100%] 5393 of 5393 ✔
[71/c63bf7] process > BACTOPIATOOLS:LEGSTA:CSVTK_CONCAT (legsta)        [100%] 1 of 1 ✔
[16/833262] process > BACTOPIATOOLS:CUSTOM_DUMPSOFTWAREVERSIONS (1)     [100%] 1 of 1 ✔

    Bactopia Tools: `legsta Execution Summary
    ---------------------------
    Bactopia Version : 3.0.1
    Nextflow Version : 23.10.1
    Command Line     : nextflow run /home/rpetit3/bactopia/main.nf --wf legsta \
                                    --bactopia bactopia/ -profile singularity
    Resumed          : false
    Completed At     : 2024-03-22T15:09:54.959834620-06:00
    Duration         : 32m 51s
    Success          : true
    Exit Code        : 0
    Error Report     : -
    Launch Dir       : /home/rpetit3/test-legsta

This took about 30 minutes on my laptop, but it was incredibly simple to run legsta on all 5,393 L. pneumophila assemblies.

Results of Typing​

Here's the fun part, typing results for all 5,393 L. pneumophila assemblies! Another nice thing about the Bactopia Tools in that in most cases it will merge all the results at the end leaving you with just a single file to review.

To share the results of this analysis, I've uploaded the results to Google Drive and made them available from this link: Bactopia - Legsta Results from AllTheBacteria

From this Google Sheet, you can make a copy or export the results as a CSV file.

Conclusion​

In this post, I demonstrated how in just a few steps you can make use of Bactopia Tools to rapidly and seamlessly start analyzing the 2,000,000 bacterial assemblies from AllTheBacteria. If you are planning to do your own downstream analyses on these assemblies, I hope this post has convinced you that Bactopia can make this process much easier.

If you have any questions or ideas for additional Bactopia Tools, please feel free to reach out to me!

Also! This the first ever blog post for Bactopia!