Creating Automated Workflows Tutorial

Exercise 1: Automate Sanger data analysis with a workflow

In this example we are going to automate the following steps for analyzing Sanger data into a Workflow:
– Trim Sanger sequences ends with error probability > 1%
– De novo assemble Sanger data of overlapping regions sequenced with forward and reverse primers based on the file naming and save the contigs
– Extract consensus sequences
– Multiple Align Consensus sequences
– Build a Phylogenetic tree

The input sequences are from a barcoding project of two shark species : the Longnose spurdog (Squalus blainville) and the Shortnose spurdog (Squalus megalops), very difficult to tell apart based on morphology, but easily barcoded by Sanger sequencing. The chromatogram data has been downloaded from the BOLD database (Accession BOLD:AAA1550). Each sample has been sequenced with forward and reverse primers.

Let’s build a workflow for the analysis of these sequences.

Create a new workflow

Press Workflow (in the Toolbar), select Manage Workflows and New Workflow from the dialog.

Name it Sanger analysis and add a description. i.e. “Sanger sequences trimming, de novo assembly, multiple alignment and tree building” and select the Chromatogram as Icon.

Now we are ready to add the steps to our workflow.

Add trimming operation to the workflow

Add the first operation by pressing Add step → Add operation.

The first step we want to run is to trim the sequences at 3’ and 5’ for low quality. Start writing the name of the operation you would like to run for automatically filter the available operations. Select Trim ends and OK.

The Trim Ends operation will be added to the workflow.

Double click on the operation for checking/modifying the options. In this example we are not going to expose any option to the user and we leave all the options as default, except for decreasing the error probability limit to 0.01, as shown below. Then click OK.

Add de novo assembly operation to the workflow

Now we can add a second step, following the same procedure as above: Add step → Add operation and select Align/Assemble → De novo assembly.

Also for this operation, we are not going to expose any of the options. Double click on the new step and modify only the options highlighted in the following screenshot:

The assembly will take into account the file naming and assemble all the chromatograms that have the same name prior to the dash (hyphen) into one contig. In this example we want to assemble forward and reverse reads for each of the sample sequenced. As result of this operation we would like to save the contigs.

Note that if you intend to return more than one output at this stage (e.g. contigs and consensus sequences) and then add another step, you will need to specify which output you would like to use for the next step using for example a filtering option (Add step → Filter documents).

Add more operations to the workflow

The following two operations to be added, in this order, are:

– Generate Consensus Sequence
– Alignment → MUSCLE Alignment

Both with default options. The Workflow at this stage should look like the following:

The next and last step we would like to add to the workflow is:

– TreeBuilding → Geneious Tree Builder (nucleotide)

But in this case we would like to expose the option to change the Tree building method (Neighbor-Joining or UPGMA). For that, select Expose some options and then find the option corresponding to the Tree building method as shown below:

Finalize the workflow

Workflows return only the result of the last step . If you would like to save intermediate steps, you just need to add the step called Save Documents / Branch from the Add Step menu.

In this example, we would like to save the contigs generated by the de novo assembly step. Add the step called Save Document / Branch with the following options:

And drag and drop this step right after the de novo assembly step:

Congratulations! Your workflow it’s now set up. You can click OK now and in the next step you will test it on the example data.

Run the workflow

Now select all the example chromatograms provided:

And then run the Workflow you just designed Sanger analysis via the Tool menu or the Toolbar.

Select Neighbor-Joining as Tree Build method.

As a result you will get an assembly document per sample as well as a phylogenetic tree including all the samples.

Under the tree view option Show Tip Labels you can select the Species or the Common name of each sample (as in the screenshot). Notice how it is possible to tell apart the two morphologically similar species with one single barcoding PCR.

Congratulations! You’ve successfully designed and run your first workflow!

Debugging and Quick access

As a debugging option, we advise to test each step on your data before adding new steps, to make sure everything runs as expected.

If you want to easily access your newly built workflow, you can add a direct access to the Toolbar by right clicking on the Toolbar, select customize and then tick your workflow on the list.

Notice that the buttons can also be added to the Toolbar using the option Show Toolbar Shortcut that you find by pressing the gear button in the lower left corner in any Geneious operation.