DRAGEN Array Cloud Analysis

DRAGEN Array Cloud Analysis Overview

DRAGEN Array Cloud utilizes the user-friendly graphical interface of BaseSpace Sequence Hub to simplify DRAGEN Array analysis setup and kickoff. Optional integration with the iScan System allows data to be streamed directly from the instrument to the cloud platform. Analysis data is stored on the Illumina Connected Platform providing secure storage for both microarray and sequencing data.

Getting Started

The following prerequisites are needed to get started with DRAGEN Array Cloud:

• Illumina Connected Analytics subscription: An ICA Basic, Professional or Enterprise subscription can be used which include access to BaseSpace Sequence Hub. Follow the Illumina Software Registration Guide to register the software.

• Workgroup setup: Workgroups must be created before login. Using a workgroup allows all members of the workgroup to share access to resources, analyses, and data. Learn more about managing a Workgroup.

  • Designating a workgroup as ‘Collaborative’ allows projects to be shared with collaborators or Illumina Tech Support to assist with troubleshooting. To create a collaborative workgroup, select the Enable collaborators outside of this domain checkbox during workgroup creation.

• Software consumables: iCredits can be purchased for storage on the cloud platform and analysis pipelines with a compute charge. Per sample analysis can be purchased for relevant pipelines as listed in section Applications. Follow the Illumina Software Registration Guide (found under Example 3: Configuring the Software Consumables) to register the software consumables.

• [Optional] iScan integration: The iScan System is integrated with Illumina Connected Platform and can send IDATs for further analysis. The iScan System must be running iScan Control Software version 4.2.1 or later.

  • Instructions to Use Illumina Connect Analytics (ICA) with the iScan System

  • Troubleshooting iScan integration

• EULA acceptance: Accept all necessary End User License Agreements in BaseSpace Sequence Hub before scanning begins.

• Internet connection: For uploading product files or IDATs, a network connection 1 GbE or faster is recommended.

Note: Accessioning BeadChips before scanning and starting analysis is no longer a required step and has been automated within the system.

Running Analysis

Before beginning analysis, ensure workgroup context is being used so analysis can be viewed by all members of your workgroup. The name of your workgroup should appear in the top right corner.

Use the following steps to run the Microarray Analysis Setup on BaseSpace Sequence Hub:

1. Select the Runs tab

2. Select New Run

3. Select Microarray Analysis Setup

4. Enter the Analysis Name (Figure 1)
5. Use the Select Project link to choose the project for your output files To select an existing project, click the radio button next to the desired project name. You can also create a project by clicking the New button in the project selection window.

6. Select the Type of Analysis Further detail of each Type of Analysis is available in section Applications. Note: For PGx CNV calling, it is recommended that 96 or more samples passing LogRDev <= 0.2 are included in the analysis. For PGx star allele calling, it is recommended to QC the samples and review the samples that have Log R Dev > 0.2, call rate < 0.99, or TGA Control probe < 1.0 to assess the reliability of the analysis. These metrics are provided in the genotyping sample summary file (gt_sample_summary.csv).

7. (Optional) Create a custom configuration via the "Add Custom Configuration" option in Configuration Settings. Custom configurations must be assigned a name and product files can be uploaded or selected (Figure 2). Custom configuration options vary by Type of Analysis including:

• DRAGEN Array - Genotyping provides flexibility for turning off/on specific output files and adjusting GenCall score cutoff. Its recommended to turn off VCF output for non-human species and Final Report output for large sample numbers.

• DRAGEN Array - Methylation - QC provides options to adjust thresholds as detailed in section DRAGEN Array Methylation QC Threshold Adjustment.

1. Select your preferred option in the Configuration Settings drop-down menu Configuration setup will vary based on the Type of Analysis selected. More details are available in section Applications.

2. Select Next

3. Select either Import Sample Sheet, Select BeadChips, or Import IDAT Files (Figure 3)

• Import Sample Sheet presents a link to upload sample sheet. Users may download a template sample sheet by selecting the Download Template link.

• Select BeadChips allows users to select BeadChips from the displayed list of available BeadChips. If selecting specific samples within the BeadChip is desired the Import Sample Sheet option should be used.

• Import IDAT Files allows users to upload the IDAT files from a local folder to the cloud platform for use with the current and future analyses by users within the same workgroup.

1. Select Launch Analysis

View Outputs

1. On the Analyses tab, view the analysis status, e.g., initializing or complete.

2. After the analysis is complete, select the analysis and select the Files tab.

3. From the Files tab, select the Output folder.

Manage Data

The data management tab allows you to view and manage all your scanned IDAT files in the cloud. Before viewing, ensure workgroup context is being used so all data available your workgroup can be seen. The name of your workgroup should appear in the top right corner. For more information, see BaseSpace Data Management

DRAGEN Array Methylation QC

Threshold Adjustment

When using DRAGEN Array – Methylation – QC cloud analysis type, additional customization options will appear after product files are selected within Configuration Settings. Adjustments to these thresholds will be saved as part of the Configuration Setting. Thresholds can be adjusted based on study objectives. Adjusting thresholds will impact the pass or fail status of samples in the output files.

Illumina recommends thresholds for MethylationEPIC v1 & v2 and Methylation Screening Array (MSA). Users may use these thresholds as a starting point when defining thresholds for their custom or semi-custom BeadChip or other Infinium Methylation arrays. Further tuning may be required based on BeadChip used, laboratory conditions, iScan settings, bisulfite conversion methods, FPPE sample type, etc. A dataset deemed acceptable to the user based on proportion probes passing can be used for these additional threshold adjustments.

To customize thresholds, use the toggle to allow additional thresholds to be displayed and adjust as desired by typing in a numeric value or using the arrows to adjust up or down. Further detail of these thresholds including calculation method can be found in the Methylation Sample QC Summary Files section.

The recommended thresholds are pre-set within the software for MethylationEPIC and Methylation Screening Array with the following values:

The first 21 rows in the tables correspond to the 21 control metrics used in the methylation sample QC. See section Methylation Sample QC Summary Files for details.

DRAGEN Array Methylation QC and GenomeStudio Methylation Module Differences

DRAGEN Array Methylation QC software provides automated methylation sample QC using assay control probes on the Infinium Methylation Arrays. Unlike the manual visual QC in GenomeStudio, DRAGEN Array ultilizes 21 numerical metrics defined based on the control probes and uses standard thresholds to determine pass/fail status of a sample. Unlike GenomeStuio, probe detection rate (proportion of probes passing at a given p-value threshold) is not utilized to determine sample pass/fail status in DRAGEN Array. For more information, see High-throughput Infinium methylation array QC using DRAGEN Array Methylation QC software tech note.

DRAGEN Array Methylation QC performs background normalization, dye bias correction, and detection p-value calculation differently in comparison to the GenomeStudio Methylation module, leading to differences in probe detection p-values and detection rates. For the GenomeStudio Methylation Module, non-cancer samples at standard DNA input typically have detection rate > 96%. The detection rates from DRAGEN Array Methylation QC are typically lower compared to GenomeStudio, because the detection p-value from DRAGEN Array is more stringent than that from the GenomeStudio Methylation Module. The table below shows example detection rates from the DRAGEN Array Methylation QC software from MSA (Methylation Screening Array) datasets.

Note that only samples passing QC are included and all samples are at or above 50ng DNA input. Detection p-value threshold 0.05.

Troubleshooting and Additional Support

Troubleshooting iScan integration

The firewall protects the iScan control computer by filtering incoming traffic to remove potential threats. The firewall is enabled by default to block all inbound connections. Keep the firewall enabled and allow outbound connections.

For the instrument to connect to BaseSpace Sequence Hub, you will need to add regional platform endpoints and instrument specific endpoints to the allow list on your firewall. Regional endpoints and further detail can be found in Security and Networking for Illumina instrument control computers.

The following table shows the applicable endpoints for the iScan.

Some notes on IDAT fail status: iScan will mark certain samples with a FAIL status if the registration quality is too poor for that particular section. Selected samples that are marked with FAIL status will be excluded from analysis and there would be no results for that sample, even though IDATs are generated. The registration quality can be found in the metrics.txt file.

Sharing a project

Project sharing allows a user to share files with users outside the workgroup for collaboration or with Illumina Tech Support for troubleshooting. To share a project on BaseSpace Sequence Hub, first set the Workgroup type as ‘Collaborative’ during Workgroup setup, and then use the following steps to obtain a link to your project. The project can then be accessed by anyone with the link. All files in the project are shared.

1. Navigate to the Projects tab

2. Click the button next to the desired project

3. Select the Share button above to list (Figure 3)

4. Select the Get Link Option to Activate a link for the project

5. Copy the link and send it to the desired recipient(s)

Additional Notes:

• The project owner maintains ownership and write access. If project owner deletes the data, the collaborators lose access to it.

• Either sending or receiving domain must be collaborative

  • https://help.basespace.illumina.com/microarray/getting-started -> Workgroup Setup

• Must be in the same AWS regional instance

  • https://help.basespace.illumina.com/manage-your-account/regions -> "Data cannot be transferred directly between instances, however you can download and share data separately."

• For Enterprise domains, use this same method (share-by-link, not share-by-transfer)

  • https://help.basespace.illumina.com/collaborate/share-with-collaborators/share-by-link