Genome-assembly: Genome assembly with `hifiasm` on Galaxy Australia

Galaxy Australia is capable of de novo assembling genomes based on PacBio high fidelity reads built from circular consensus sequence HiFi reads.

This How-to-Guide will describe the steps required to assemble your genome on the Galaxy Australia platform, using multiple workflows (see Fig 1) developed in consultations between the Bioplatforms Australia Threatened Species Initiative, Galaxy Australia, and the Australian BioCommons.

Note: If you need help, the Galaxy community is both approachable and helpful. Ask them questions!

Quick start guide

Login to Galaxy Australia
Create a new history
Upload your HiFi ccs.bam data files to your Galaxy history
Load and execute workflows (links included below), using required options
- FILE CONVERSION workflow: BAM to FASTQ + QC v1.0 optional
- ASSEMBLY workflow: PacBio HiFi genome assembly workflow v2.1
- PURGE DUPLICATES workflow: Purge duplicates from hifiasm assembly v1.0 optional
Review workflow report and perform additional QC as needed
Re-run workflows, or individual tools, as needed

How to cite the workflows

Price, G. (2022). BAM to FASTQ + QC v1.0. WorkflowHub. https://doi.org/10.48546/WORKFLOWHUB.WORKFLOW.220.2

Price, G., & Farquharson, K. (2022). PacBio HiFi genome assembly using hifiasm v2.1. WorkflowHub. https://doi.org/10.48546/WORKFLOWHUB.WORKFLOW.221.3

Price, G. (2022). Purge duplicates from hifiasm assembly v1.0. WorkflowHub. https://doi.org/10.48546/WORKFLOWHUB.WORKFLOW.237.2

The overall workflow

Fig 1. The approach described in this How-to-Guide, including Quick Start guide steps 1) registration, 2) upload of input BAM file, 3) BAM to FASTQ conversion workflow, 4) assembly workflow, 5) purge duplicates workflow and 6) reviewing the assembly report and FASTA metrics. Required workflow steps are blue, and optional steps are red.

Further to this, a summary of the different elements of this assembly approach are detailed below:

Process name	Workflow name	Description	Inputs	Outputs
UPLOAD FILES	Not applicable	See the different upload options.	BAM (start at step 3 in Fig 1), or FASTQ (start at step 4 in Fig 1)	Uploaded data!
FILE CONVERSION	BAM to FASTQ + QC	Conversion of files from BAM to FASTQ, including a FASTQC quality control (QC) step.		FASTQ file, FASTQC report HTML file
ASSEMBLY	PacBio HiFi genome assembly using hifiasm	Assembly using the hifiasm tool, including Bandage visualisation and QC.		Assembly file in FASTA format, FASTA metrics, Assembly report file
PURGE DUPLICATES	Purge duplicates from hifiasm assembly	Optional workflow to purge duplicates from the contig assembly.		Purged primary sequences from draft assembly (FASTA), Purged haplotig sequences from draft assembly (FASTA)

The WorkflowHub entries are all available in this collection.

In-depth workflow guide

To register for Galaxy Australia, visit the login page.
Click the Register here link, as shown in Fig 2.
Complete the registration wizard and click Create.
Login to your account!

Fig 2. Log-in / registration menu for Galaxy Australia.

Upload data file(s)

Note: FAQ: can data be seamlessly transferred from the Data Portal to Galaxy Australia? The Australian BioCommons is working with the Bioplatforms Australia data portal team to develop a direct transfer mechanism from the portal to Galaxy Australia histories. This solution will be made available as soon as possible.

Select your data using the Bioplatforms Data Portal
- The files you need for the assembly are .css.bam format
- Fig 3 shows a HiFi data set selected in the data portal browser interface.
Fig 3. The appearance of a HiFi data set in the Bioplatforms Australia data portal browser interface.
Click Access and select Copy Download URL in the drop down menu (see Fig. 3).
- Note: This will copy a download URL to your clipboard.
- The URL is time sensitive and will expire after 10 minutes.
- Please note you only need to instigate the download within this 10 minute window. The import itself can take longer than 10 minutes.
In Galaxy Australia, create a new history

Now, perform the steps outlined in Fig. 4
- Step 1: Click on Upload Data
- Step 2: Select Paste/Fetch data
- Step 3: Paste the URL you obtained from the data portal into the content box.
- Step 4: Select Start

Fig 4. Process required for download of data based on a URL provided to Galaxy Australia.

Note: you can paste multiple URLs in this box. If you have more than 1 SMRT cell of data, you will need to copy the URLs for each of the ccs.bam files and paste them in here.

Other data transfer options are also available

Self-managed (download & upload)

Download required HiFi data to your personal computer, then
Upload / transfer to Galaxy Australia (see Fig 5)
- Note: the following Galaxy Training Network tutorial provides guidance on how to upload files via URL. The same mechanism can be used to upload local files, by selecting Choose local files (see Fig 5).

Fig 5. Choosing local files to upload to Galaxy Australia.

Supported

Contact the Galaxy Australia Support team for data chaperoning.

OPTIONAL STEP: convert BAM files to FASTQ

Important: please note the following important points

This workflow is not needed if files are already in FASTQ format

You must do this step if your files are in ccs.bam format

You will need to complete this workflow for each BAM file

Make sure you are logged into Galaxy Australia
Visit this link to:
- retrieve the workflow for file conversion,
- add it to your Galaxy Australia workflows list, and
- open your workflows list (which can also be reached by clicking the Workflow tab, highlighted by a red box in Fig 6, in the Galaxy interface)

Tip: Did you know? You can view the workflow diagram by clicking on the workflow name in the list and selecting Edit.

Once you have reached the workflow screen, select the play button (highlighted by a red box in Fig 7) for the BAM to FASTQ + QC workflow.

Fig 7. The workflows page of the Galaxy Australia service is where your workflows appear. The blue box is a zoom view of the workflow table, and the `play` button to launch your workflow is highlighted by a red box.

The workflow invocation window will open. Select the BAM file that you previously loaded into your Galaxy history using the drop-down menu (step 1 in Fig 8).

Warning: Depending on the contents of your BAM file, the parameters (options) chosen will impact the file produced.

Click Run workflow (step 2 in Fig 8).

Fig 8. The workflow invocation menu for the BAM to FASTQ + QC workflow. Step 1 is to select the BAM file for conversion using the drop-down menu, and Step 2 is to select Run workflow.

The workflow will produce
- A FASTQ file that will be the input for the assembly workflow covered in the next section (Fig 9a), and
- A FastQC report which you can view in the Galaxy user interface (Fig 9b).
If you only have a single BAM file, stop here! If you have multiple BAM files, repeat this entire section of the tutorial for each BAM file:

Important: if you have multiple BAM files to convert, you will need to join the multiple FASTQ files produced by the workflow. Another tool called Concatenate datasets tail-to-head (Galaxy Version 0.1.1) can be used for this purpose.

Tip: Did you know? You don’t need to re-run entire workflows! You can re-run individual tools in Galaxy Australia by selecting the previous tool run in your history and pushing the button indicated below.

OPTIONAL STEP: joining multiple FASTQ files (e.g. from multiple flow cells)

Note: If you need to concatenate multiple FASTQ files, for example from multiple flow cells, you can use the Concatenate datasets tail-to-head tool in Galaxy Australia (see below). Note that you can insert multiple data sets (i.e. FASTQs), and should only concatenate files with identical formats.

Search for the Concatenate datasets tail-to-head tool in the Galaxy Australia browser interface (step 1 in Fig 10).
Select the tool from the search results (step 2 in Fig 10).

Fig 10. Search for and open a tool using the Galaxy Australia interface.
In the tool menu, select the first FASTQ data set (step 3 in Fig 11).
Insert additional FASTQ data sets by selecting the + Insert Dataset button (step 4 in Fig 11).
When all data sets are selected, press Execute (step 5 in Fig 11).

Fig 11. Select data sets and execute the Concatenate datasets tool.

Assembly workflow

Important: This workflow includes an adapter filtering step that makes use of HiFiAdapterFilt. Adapter contamination in the reads could cause misassemblies, and will result in NCBI rejecting the upload of affected genome assemblies. HiFiAdapterFilt identifies .ccs reads containing adapter sequences using the same method as NCBI and removes the entire read prior to genome assembly to avoid such misassemblies.

Make sure you are logged into Galaxy Australia
Visit this link to:
- retrieve v2.1 of the assembly workflow,
- add it to your Galaxy Australia workflows list, and
- open your workflows list (which can also be reached by clicking the Workflow tab [highlighted by a red box in Fig 6] in the Galaxy interface)

Tip: Did you know? version 2.1 of the workflow uses hifiasm v0.16.1.

Select the play button (highlighted by a red box in Fig 7) for the PacBio HiFi genome assembly using hifiasm v2.1 workflow (the workflow is shown in Fig 12).

Fig 12. PacBio HiFi genome assembly using hifiasm. The blue boxes indicate the main steps in the workflow, which produce the primary hifiasm assembly. The grey boxes indicate the other outputs of hifiasm, which are hidden but still accessible after the workflow has been run.

The workflow invocation window will open.
Select the FASTQ file that was produced by the BAM to FASTQ + QC workflow using the drop-down menu.
Select correct workflow input parameters
Click the Run workflow button (as in step 2 of Fig 8).
The PacBio HiFi genome assembly using hifiasm workflow produces the following outputs (Fig 13):
- Bandage info and images for:
  - Primary assembly contig graph
  - Alternate assembly contig graph
  - Processed unitig graph
  - Haplotype resolved raw unitig graph
- FASTA file for the primary assembly contig GFA file
- Fasta statistics for primary assembly contig FASTA file

Fig 13a. Example Bandage info file for primary assembly contig graph.

Fig 13b. Example Bandage image file for primary assembly contig graph.

Fig 13c. Example Fasta statistics for primary assembly contig FASTA file.

Tip: Did you know? You can access the other assembly output types for hifiasm (haplotype resolved, unitig graph and alternate assembly). They are just hidden so that the workflow highlights the provision of the primary assembly contig graph. For instructions on how to reveal hidden files, see this link.

OPTIONAL STEP: Purge duplicates from hifiasm assembly

Make sure you are logged into Galaxy Australia
Visit this link to:
1. retrieve the purge duplicates workflow,
2. add it to your Galaxy Australia workflows list, and
3. open your workflows list (which can also be reached by clicking the Workflow tab [highlighted by a red box in Fig 6] in the Galaxy interface)
Select the play button (highlighted by a red box in Fig 7) for the Purge duplicates from hifiasm assembly workflow (the workflow is shown in Fig 14).

Fig 14. Purge duplicates from hifiasm assembly.

The workflow invocation window will open.
Select the raw reads in FASTQ format, and hifiasm primary contig assembly file (FASTA format) using the drop-down menu.

Note: These inputs are both produced by the PacBio HiFi genome assembly using hifiasm workflow.

Select correct workflow input parameters
Click the Run workflow button (as in step 2 of Fig 8).
The Purge duplicates from hifiasm assembly workflow produces the following outputs:
- Purged primary sequences from draft assembly (FASTA)
- Purged haplotig sequences from draft assembly (FASTA)

Review Workflow Report

Note: A workflow report is generated automatically following completion of the workflow run (see Fig 15).

Review quality control contents of the workflow report (Fig 15).
Inspect bandage image (Fig 16).
Review fasta statistics (Fig 17).

Fig 16. Bandage image for the primary hifiasm assembly.

Fig 17. Basic summary statistics are included in the fasta statistics output. This includes: `number of contigs (num_seq)`, `assembly size (num_bp)`, `contig N50 (len_N50)`, `L50`.

OPTIONAL STEP: Post-assembly quality control workflow

A genome assembly quality control workflow guide is available.

Appendices

Revealing hidden files

Note: By default, the workflow hides 3 of the outputs of hifiasm. The only one that is visible is the primary assembly contig graph. To reveal the hidden outputs, follow these steps:

In your history panel, click on hidden files (as shown by the red box in Fig S1).
If you would like to unhide the data set, click on Unhide it in the expanded history panel that appears following step 1 (as shown by the red box in Fig S2).

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