16.1 Galaxy Workflows: Build a Workflow

16.1.1 Purpose

A workflow is a reusable pipeline that chains tools and dataset actions so you can repeat the analysis consistently. Galaxy Workflows let you:

  • Automate a series of analysis steps (tools + settings) so you don’t rebuild it each time.
  • Ensure reproducibility by keeping the same order of steps and parameters when rerunning on new data.
  • Scale and save time by running the same process across many samples consistently.
  • Share and collaborate by exporting or sharing workflows so others can run the same analysis.

16.1.2 Learning Objectives

  1. Build a Galaxy Workflow from Scratch
  2. Run a Workflow
  3. Edit a Workflow
  4. Share a Workflow

16.1.3 Activity 1 - Create a Galaxy Workflow from scratch

Estimated time: 10 min

16.1.3.1 Instructions

Follow steps below to:

  1. Plan a Workflow - 3-tool workflow using Flye, MetaBAT2 and GTDB-Tk tools
  2. Create a Workflow - Start a new workflow
  3. Build a Workflow - Configure tool parameters, connect inputs/outputs

16.1.3.2 Step 1 - Plan a Workflow

  1. Use a high-quality, Zymo Gut Standard D6331 subset2:
Tool Goal Specify Parameters Required input type
Flye De novo genome assembly Mode: PacBio HiFi & metagenomic assembly fastq
MetaBAT2 Metagenomic binning Default fasta
GTDB-Tk Classify genomes Change versions to: Galaxy Version 2.5.2+galaxy1 fasta (bins)

16.1.3.3 Step 2 - Create a Workflow

  1. In Galaxy’s menu on the Left, click Workflows
  2. Click Create new workflow
  3. Give it a name (e.g. pacbio-assemble-bin-classify)
  4. Click Save

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16.1.3.4 Step 3 - Build a Workflow

Note: While building your workflow you can save it at any point, but after saving, will need to click on Edit to go back and add.

In the Workflow Editor layout, on the canvas:

  1. Add Input Dataset (this is the workflow’s required input).
    1. For this activity, we are adding Single Dataset input as required input.
      • For future workflows that you may run using more than one input file, you can specify Input Dataset Collection instead.
    2. Exit out of Input Dataset box to move on to another action.

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  1. Add tool #1 - Flye
    1. Specify parameters appropriate for your input
      • Specify Mode: PacBio HiFi (–pacbio-hifi)
      • Under Perform metagenomic assembly: select Yes.
      • Under Generate a log file: select Yes.
      • Leave rest as default
      • Exit out of the Flye tool box
    2. Connect input/output
      • On Workflow Canvas, reposition Input Dataset box and Tool box so it’s nice and comfortable for you to connect inputs and outputs
      • Connect Input Dataset to Flye by dragging connector from Input Dataset to Flye.

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  1. Add tool #2 - MetaBAT2
    1. Specify parameters appropriate for your input
      • Leave as default
      • Exit out of the MetaBAT2 tool box
    2. Connect input/output
      • On Workflow Canvas, reposition Input Dataset box and Tool box so it’s nice and comfortable for you to connect inputs and outputs
      • Connect relevant output of Flye (consensus (fasta)), to MetaBAT2 by dragging connector from consensus (fasta) to MetaBAT2.
  2. Add tool #3 - GTDB-Tk
    1. Specify parameters appropriate for your input
      • Click on Versions button (next to the star) and Switch to 2.5.2+galaxy1 to be able to select an older GTDB-Tk database version
      • Under GTDB-Tk database, select Full database - release 220 (2024-10-19)
      • Leave rest as default
      • Exit out of the GTDB-Tk tool box
    2. Connect input/output
      • On Workflow Canvas, reposition Input Dataset box and Tool box so it’s nice and comfortable for you to connect inputs and outputs
      • Connect relevant output of MetaBAT2(Bin sequences, fasta), to GTDB-Tk by dragging connector from bins to GTDB-Tk
      • Click Save

16.1.3.5 Questions

1. Take a snapshot of your workflow and paste below:

16.1.4 Activity 2 - Run Workflow

16.1.4.1 Instructions

  1. Import the dataset PacBio-sequenced Zymo gut standard D6331 subset:
  2. Go to Workflows, click to select your workflow and click Run
    • Always double-check/select to ensure you are using the correct input file(s)
    • USEFUL: check ‘Attempt to re-use jobs with identical parameters’. This option will allow you to re-run select steps of workflow if a step fails, without effectively re-running successful steps.

Note: It may take a few minutes to get the Workflow running e.g. time for loading and queuing steps.

16.1.4.2 Questions

1. How many contigs did you obtain after Flye?

2. How many bins did you obtain after MetaBAT2?

3. How many classified genomes do you have after GTDB-Tk?

16.1.5 Activity 3 - Modify Workflow

16.1.5.1 Instructions

  1. Add another tool (tool #4) to your workflow - add fastp!
    1. Think about where this tool fits in - why do we use it, and what would you do with fastp output?
    2. Think of a fastp parameter you might want to change to improve sequence quality.
    3. Think about input/output connections, and possible re-connections!
  2. When running the workflow, ensure you check ‘Re-use jobs with identical parameters’ to avoid re-running your other tools.

16.1.5.2 Questions

1. Take a snapshot of your workflow and paste below:

2. Did you ‘Re-use jobs with identical parameters’ and avoided effective re-running of jobs you already ran before? (Flye, MetaBAT2, GTDB-Tk)? Yes or No?

16.1.6 Activity 4 - Share Workflow

16.1.6.1 Instructions

  1. To share your workflow, obtain a sharable URL
    1. Go to Workflows and click on Share
    2. Click to make Workflow accessible
    3. Copy URL

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16.1.6.2 Questions

1. Copy and paste your workflow URL below:

16.1.7 Grading Criteria

  • Download as Microsoft Word (.docx) and upload on Canvas

16.1.8 Footnotes

Resources

Contributions and Affiliations

  • Valeriya Gaysinskaya, Johns Hopkins University
  • Frederick Tan, Johns Hopkins University

Last Revised: March 2026

All materials CC-BY unless noted otherwise.