End-to-End

This tutorial demonstrates major functions of the ICA platform, beginning with setting up a project with instrument run data to prepare to use pipelines, and concluding with viewing pipeline outputs in preparation for eventually ingesting outputs into available modules.

In the following example, we start from an existing ICA project with demultiplexed instrument run data (fastq), use the DRAGEN Germline pipeline, and view output data.

Set-up

This tutorial assumes you already have an existing project in ICA. To create a new project, please see instructions in the Projects page.

Additionally, you will need the DRAGEN Demo Bundle linked to your existing ICA project. The DRAGEN Demo Bundle is an an entitled bundle provided by Illumina with all standard ICA subscriptions and includes DRAGEN pipelines, references, and demo data.

Linking an Entitled Bundle

For general steps on creating and linking bundles to your project, see the Bundles page. This tutorial explores the DRAGEN Germline Published Pipeline, so we will need to link the DRAGEN Demo Bundle to our existing project.

Steps:

• Go to your project's Details page

• Click the Edit button

• Click the + button, under LINKED BUNDLES

• Click on the DRAGEN Demo Bundle, then click the Link Bundles button

  • There may be multiple versions of DRAGEN Demo Bundles. This tutorial details steps for DRAGEN Demo Bundle 3.9.5. Steps for other versions after 3.9.5 should be similar.

• Click the Save button

DRAGEN Demo Bundle assets should be available now in your projects Data and Pipelines pages.

Pipelines

After setting up the project in ICA and linking a bundle, we can run various pipelines.

DRAGEN Germline Published Pipeline

This example demonstrates how to run the DRAGEN Germline Published Pipeline (version 3.9.5) in your ICA project using the demo data from the linked DRAGEN Demo Bundle.

The required pipeline input assets for this tutorial include:

• Under Data page

  • Illumina DRAGEN Germline Demo Data folder

  • Illumina DRAGEN Enrichment Demo Data folder

  • Illumina References folder

• Under Pipelines page

  • DRAGEN Germline

Launching the DRAGEN Germline Published Pipeline with Demo Data

From the Pipelines page, select DRAGEN Germline 3.9.5, and then click Start New Analysis. Initial set-up details require a User Reference (pipeline run name meaningful to the user) and an Entitlement Bundle from the drop-down menu under Pricing.

Running the DRAGEN Germline pipeline uses the following inputs:

• FASTQ files

  • Select the FASTQ files in the Illumina DRAGEN Enrichment Demo Data folder

• Reference:

  • Select a reference genome from the Illumina References folder (do not select a methyl-converted reference genome for this tutorial)

    • Ie: hg38_altaware_nohla-cnv-anchored.v8.tar (suggested, if enabling CNV analysis)

The DRAGEN Germline Settings to be selected are:

• Enable germline small variant calling: Set to true

• Enable SV (structural variant) calling: Set to true

  • If true, Enable map align output must also be set to true

• Enable repeat genotyping: Set to true

• Enable map align: Set to true

  • When using FASTQ files as input, as in this example, set this to true as default.

  • When using BAM files as input, set to true to realign reads in input BAMs; set to false to keep alignments in input BAM files.

• Enable CNV calling: Set to true

  • Enabling Copy Number Variant calling requires one of the following:

    • Enable CNV self normalization is set to true

    • A panel of normals (PON) is provided in the Input Files

• Output format: Set to CRAM

  • Other available options for alignments output are BAM and SAM format.

• Enable CNV self-normalization: Set to true

  • Required if Enable CNV calling is set to true and no panel of normals (PON) is provided in the Input Files.

• Enable duplicate marking: Set to true

• Emit Ref Confidence: Set to GVCF to enable banded gVCF generation for this example

  • To enable base pair resolution in the gVCF, set to BP_RESOLUTION

• Additional DRAGEN args: None

  • Users can provide additional DRAGEN arguments here (see the DRAGEN user guide for examples), but we will leave this blank for this example run.

• Sample sex: Leave blank

  • Users may specific the sex of the sample here if known, but the user will omit this setting for this example run.

• Enable HLA: Set to true to enable HLA typing

• Enable map align output: Set to true

  • The format for alignment output was selected previously in the "Output format setting" above

• Resources

  • Use the default resources settings:

    • Storage size: Set to small

    • FPGA Medium Tier: Set to Standard

    • FPGA Medium Resources: Set to FPGA Medium

Once all parameters have been set, click Start Run

Monitoring analysis run status

You can monitor the status of analysis pipeline runs from the Runs page in your project.

Possible statuses include:

• Requested

• In Progress

• Succeeded

• Failed

Click the refresh button in upper left corner of the ICA environment page to update the status.

Click on the run to view more information about it. The various tabs (Result, Details, Logs) under a given run provide additonal context regarding the status of the completed run. (Note: Details are only available in ICA for graphical CWL pipelines, of which DRAGEN Germline is one.)

If you encounter a failed run, an error message can be found by expanding the "Error" reporting in the Result tab.

Analysis run logs can be found on the Logs tab; check the boxes next to the log files to view them in the text viewer. Check the box next to "Show technical steps" to view additional log files.

Viewing DRAGEN analysis output

DRAGEN analysis output folders are found on the project's Data page, along with all other data loaded to the project (such as assets from a linked entitled bundle). Output analysis will be grouped into folders, so users can click through the directory structure to explore outputs.

Related Resources

• DRAGEN Support Site: https://support.illumina.com/sequencing/sequencing_software/dragen-bio-it-platform.html

• ICA Pricing: https://help.ica.illumina.com/reference/r-pricing