Nov 12, 2021
Running the Titan_Illumina_PE Workflow on Terra.bio
- Jill V Hagey1,
- Kevin Libuit2,
- Frank J Ambrosio2,
- Technical Outreach and Assistance for States Team1
- 1Centers for Disease Control and Prevention;
- 2Theiagen Genomics
- Theiagen
Protocol Citation: Jill V Hagey, Kevin Libuit, Frank J Ambrosio, Technical Outreach and Assistance for States Team 2021. Running the Titan_Illumina_PE Workflow on Terra.bio. protocols.io https://dx.doi.org/10.17504/protocols.io.bzykp7uw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: November 10, 2021
Last Modified: November 15, 2021
Protocol Integer ID: 55020
Keywords: Illumina, Sequencing, MiSeq, iSeq, MiniSeq, NextSeq, NovaSeq, Paired-End, Next Generation Sequencing, NGS, SARS-CoV-2, Covid, Pangolin, Short-Read, Coronavirus, Genomics, Genetics, Virology, Molecular Biology, input data for titan illumina pe, public health viral genomics titan series, additional technical documentation for the titan-illumina-pe workflow, titan-illumina-pe data, titan-illumina-pe workflow, consensus genome assembly, bed format along with the raw illumina, primer sequence coordinates of the pcr scheme, theiagen genomic, generated consensus genome, consensus genome, titan illumina pe, sequencing, pcr scheme, artic v3 protocol, bed file format, primer sequence coordinate, clade designation, titan-illumina-pe job, titan-illumina-pe, quality of both raw read data, qiaseq primer panel, bed format, common read data, input primer scheme coordinate, pcr, alternative primer scheme, raw read data, artic v3, fastq files per sample
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Abstract
The Titan_Illumina_PE workflow is a part of the Public Health Viral Genomics Titan series for SARS-CoV-2 genomic characterization. Titan_Illumina_PE was written specifically to process Illumina paired-end (PE) read data. Input reads are assumed to be the product of sequencing tiled PCR-amplicons designed for the SARS-CoV-2 genome. The most common read data analyzed by the Titan_Illumina_PE workflow are generated with the ARTIC V3 protocol. However, alternative primer schemes such as the QIAseq Primer Panel are also suitable for this workflow. The primer sequence coordinates of the PCR scheme utilized must be provided in BED format along with the raw Illumina read data. Upon initiating a Titan_Illumina_PE job, the input primer scheme coordinates and raw paired-end Illumina read data provided for each sample will be processed to perform consensus genome assembly, infer the quality of both raw read data and the generated consensus genome, and assign lineage or clade designations as outlined in the Titan_Illumina_PE data workflow diagram below.
Additional technical documentation for the Titan_Illumina_PE workflow is available at:
Required input data for Titan Illumina PE:
Illumina paired-end read data (forward and reverse FASTQ files per sample)
Primer sequence coordinates of the PCR scheme utilized in BED file format
Video Instruction:
Theiagen Genomics: Titan Genomic Characterization
Theiagen Genomics: Titan Outputs QC
For technical assistance please contact us at: [email protected]
Attachments
Troubleshooting
Running the Titan Illumina PE Workflow
To run the Titan workflow, click on the 'Workflows' panel in the newly created workspace. It should bring you to the following page. if you do not see the Titan_Illumina_PE workflow in your workspace, please see our video on importing a workflow to Terra.bio: https://youtu.be/ZRpQylIDMzo).
The workflows panel in the newly created Terra workspace
Click on the 'Titan_Illumina_PE' tile and it will take you to a new page (if you do not see the Titan_ClearLabs workflow in your workspace, please see our video on importing a workflow to Terra.bio: https://youtu.be/ZRpQylIDMzo). Double check that you are using the latest version of the workflow. Alternately, you may specify another version, but should only pick a stable version (with numbers) NOT a 'main' or 'dev' version.
The Titan Illumina PE workflow page. Check version you are using.
Click the 'Run workflow(s) with inputs defined by data table' option button and then in the 'Select root entity type' pull-down menu, select 'sample' or whichever entity type you specified in the sample table. The first four rows in the 'Inputs' tab should each have a yellow caution icon. These four rows represent variables that have to be provided by the user. This is the information that we populated the sample data table with in the previous steps.
Make sure "Use call caching" is checked and then select the root entity type for the data you wish to analyze (here it's sample).
NOTE: Call caching allows Terra to identify and skip jobs that have been run previously; this option is by default enabled to avoid unnecessary compute costs. More information on Terra call caching, including examples of when you may want to disable this feature, is available through the Terra Support Documentation.
The Titan Illumina PE workflow input panel after defining inputs by the sample data table
For the first row, click on the 'Attribute' text box and then click 'workspace.Artic_V3_primers' (or whatever you named the primer bed file). In the second row we input our primer_bed file of choice, which should be saved as a workspace element in Terra (if you do not have this saved as a workspace element please see our video on uploading a workspace element: https://youtu.be/Qrbz7uRtwmQ). For the next row, the 'read1_raw' variable, click on the 'Attribute' text box and then click 'this.rea1'. In the third row, the 'read2_raw' variable, click on the 'Attribute' text box and then click 'this.read2'. Finally, in the third row, the 'samplename' variable, click on the 'Attribute' text box and then click option that displays your root entity in the notiation: 'this.root_entity'. Click the 'Save' button on the top right-hand side of the page. The yellow caution icons should disappear.
Note
VERSION UPDATE:
Beginning with v1.4.4, you have the option to specify either UShER or pangoLEARN methods for PANGO lineage assignment. The default is UShER unless "pangolearn" is entered into the "inference_engine" row.
Module 3.3: Real-time phylogenetics with UShER
Next, click the "output" tab and select "use defaults" to autofill in the names of the output files according to the sample names. If you forget to select this option, you will have trouble accessing the results! Save changes by clicking the 'Save' button.
Setting output names by clicking "use defaults"
You are now ready to run the Titan_Illumina_PE workflow! Click the blue 'select data' button and select the samples you want to run or click the check box at the very top left to select all of them to be run.
Check the box to have all samples run through the Titan_Illumina_PE workflow.
Click on the 'Run Analysis' button to the right of the 'Outputs' tab. A popup window should appear titled 'Confirm launch'. If the 'Run Analysis' button is greyed out, you need to save your recent changes by clicking the 'Save' button.
The 'Confirm launch' popup window
Click the 'Launch' button to access the 'Job History' panel where each sample will be queued for the Titan_Illumina_PE analysis.
The 'Job History' page showing each sample queued for the Titan_Illumina_PE run
The status will change from 'Queued' to 'Submitted' to 'Running.' After the workflow finishes, the status column will indicate either 'successful' or 'failed.'
If you run into any trouble at all please feel free to check out our video on Running the Titan_Illumina_PE Workflow, or reach out to our support email: [email protected]