# MiSeq: DRAGEN Amplicon and Connected Insights Auto-launch Analysis in Cloud

This tutorial provides a guided example of the end-to-end workflow for a sequencing run on MiSeq with automated launch of secondary and tertiary analysis in Illumina Connected Insights.

## Prerequisites

* The latest v4.1.0 version of MiSeq Control Software (MCS)
* Illumina Connected Analytics (ICA) subscription (comes with included BaseSpace subscription)
* Illumina Connected Insights subscription
* Positive balance of Genome Equivalent Samples and iCredits
* At least one workgroup

  > Personal context is not supported in Connected Insights

## Configure Cloud Connectivity in MiSeq Control Software

1. From the **Main Menu**, select **System Settings**.
2. Select the **Run Settings** tab.
3. Select the **BaseSpace** tab.
4. *Optional*: Select **Turn on Illumina Proactive Support** to enable the Illumina Proactive monitoring service.
5. Select the region the instrument should connect to. This setting is used to ensure proper data storage is sent to Illumina Proactive Support and BaseSpace Sequence Hub.
6. Select **Private Domain** and enter the private domain of your BaseSpace Sequence Hub account.

## Configure BaseSpace

1. Log in to BaseSpace as the Workgroup Owner and make sure the desired workgroup is selected.

> Workgroup Administrators can change the workgroup owner in IAM Console

2. Click on the workgroup name from the BaseSpace header, select **Settings** from the expanded menu.
3. Make sure **ICA Run Storage** is turned on.

![](https://3223063594-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2F2YNCzdEu5yaFoMebBG1p%2Fuploads%2Fgit-blob-b11493765b43e36adf45fd44b3136efcaa2f9592%2Fbasespace_settings_account.png?alt=media)

## Configure Connected Insights

1. Log in and navigate to Connected Insights, making sure the same workgroup is selected as in the previous steps.
2. *Optional*: Navigate to **Configuration -> Report Automation** and configure report automation with preferred settings. This feature allows for the automatic generation of a draft report based on user settings.

![](https://3223063594-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2F2YNCzdEu5yaFoMebBG1p%2Fuploads%2Fgit-blob-8d53159a8df1fd432285cba57de75616d5d1009b%2Fici_configuration_general_sop-automation_edit_full.png?alt=media)

3. Navigate to **Configuration -> Test Definition** and create a test definition with preferred settings. **Turn on Report Automation** if you configured report automation settings in the previous step.

![](https://3223063594-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2F2YNCzdEu5yaFoMebBG1p%2Fuploads%2Fgit-blob-e4572c8592c3eec3a41a63cb2a1acd881c2c6665%2Fici_configuration_test-definitions_edit.png?alt=media)

4. Navigate to **Configuration -> Data Upload -> From Illumina Connected Analytics**, add the **DRAGEN Amplicon v4.2** pipeline, and select the test definition created in the previous step.

![](https://3223063594-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2F2YNCzdEu5yaFoMebBG1p%2Fuploads%2Fgit-blob-0b0bb76afb5dd5aa86a2d4d80870c3dff601def0%2Fici_configuration_general_data-upload_cloud.png?alt=media)

## Plan the Sequencing Run in BaseSpace

1. Log in to [BaseSpace](https://basespace.illumina.com/) and make sure the same workgroup is selected.
2. Navigate to the **Runs** tab, select **New Run**, and then select **Run Planning**.

![](https://3223063594-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2F2YNCzdEu5yaFoMebBG1p%2Fuploads%2Fgit-blob-2c8c893e7c0c79856ab3830f222ab3f029d0d626%2Fbasespace_runs_planned.png?alt=media)

3. Select **Instrument Platform** as "MiSeq".
4. Set the **Secondary Analysis** to "BaseSpace/Illumina Connected Analytics".

![](https://3223063594-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2F2YNCzdEu5yaFoMebBG1p%2Fuploads%2Fgit-blob-9c496412b6a9783121f1604a389ab8c1913ed4d6%2Fbasespace_run-planning_run-settings_miseq.png?alt=media)

The configuration input forms vary depending on the instrument platform, secondary analysis pipeline, and other selections (see [Sequencer Auto-launch Analyses Compatibility](https://help.connected.illumina.com/analysis/sequencer-reference) for details). The following images demonstrate screens using a MiSeq instrument with DRAGEN Amplicon analysis.

![](https://3223063594-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2F2YNCzdEu5yaFoMebBG1p%2Fuploads%2Fgit-blob-fbebd3f3d44daf15b7762147f40c9f4b7d31a377%2Fbasespace_run-planning_config_1_config-setup.png?alt=media)

5. Select "DRAGEN Amplicon for ICA - 4.2.7" from the **Application** dropdown menu.
6. Select the desired library prep kit from the **Library Prep Kit** dropdown menu.
7. Select **Index Adapter Kit**.
8. Click **Next** to continue.

![](https://3223063594-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2F2YNCzdEu5yaFoMebBG1p%2Fuploads%2Fgit-blob-0eff3cb08993b48fa8f80ef5b1feaff02d0c6a2a%2Fbasespace_run-planning_config_1_sample-analysis-setup.png?alt=media)

9. Use default values for **Index Reads**, **Read Type**, and **Read Length** or change as needed.
10. Input Sample IDs and appropriate I7 and I5 indices for each sample. Alternatively, download a sample template and use it to import the sample information.

Under the **Analysis Settings** section:

11. Make sure **BAM** is selected in the **Map/Align Output Format** field. Note: Connected Insights doesn't support CRAM yet.
12. Select the **DNA BED file** matching the Library Prep Kit if DNA samples are included in the run.
13. Select **Somatic** from the **DNA Caller Variant** dropdown menu.
14. *Optional*: Select or upload a new **CNV Panel of Normal** file if DNA samples are included in the run and you want to enable the CNV caller.
15. *Optional*: Select Yes for **DNA Enable SV Calling** if DNA samples are included in the run and you want to enable the SV caller.
16. Select the **RNA BED file** matching the Library Prep Kit if RNA samples are included in the run.
17. Select No for **RNA Enable Differential Expression**.
18. In **Per Sample Configuration**, add **DNA or RNA** and **Pair ID**. One DNA sample and one RNA sample can be paired and analyzed in Connected Insights in a single case if they share the same Pair ID.
19. Upon completion, export sequencing run settings as a SampleSheet.csv file.

![](https://3223063594-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2F2YNCzdEu5yaFoMebBG1p%2Fuploads%2Fgit-blob-34da9e69fc1cd25ed091d4298c9633704e5f25a8%2Fbasespace_run-planning_summary.png?alt=media)

### Optional: Update the Sample Sheet to Provide Disease Information

Connected Insights requires sample's disease information to start the analysis. Users can provide disease information in several ways, incorporating disease information into the sample sheet is one of them.

1. Use a text editor to open the SampleSheet.csv file generated in the previous step.

![](https://3223063594-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2F2YNCzdEu5yaFoMebBG1p%2Fuploads%2Fgit-blob-c1e1b9612f1b67db9c62ee6168ca57879df257a0%2Fsamplesheet_before_edit.png?alt=media)

2. Find `[Cloud_DragenAmplicon_ICA_Data]` or `[Cloud_DragenAmplicon_Data]` section.
3. Add `,Tumor_Type` at the end of the next line after `[Cloud_DragenAmplicon_ICA_Data]`.
4. Add [SNOMED CT](https://browser.ihtsdotools.org/) code for each sample following a `,`. Note: samples sharing the same Pair ID should have the same Tumor Type.
5. Save the file.

![](https://3223063594-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2F2YNCzdEu5yaFoMebBG1p%2Fuploads%2Fgit-blob-b494de9131301359f8a5416c8710796208b73b0c%2Fsamplesheet_after_edit.png?alt=media)

## Set Up Sequencing Run with Cloud Analysis Sample Sheet

1. From the Home screen, select **Sequence**.
2. From the Run Setup Option screen, select **Manual**.
3. From the BaseSpace Options screen, select **Use BaseSpace™ Sequencing Hub for this run**.
   1. Select the **Run analysis, collaboration, and storage** option to send all run data to BaseSpace Sequence Hub.
   2. Enter your BaseSpace Sequence Hub account credentials.
   3. Select **Sign In**.
   4. Select the Workgroup.
4. Select **Next**.
5. Set the Read Type, Read Length, and Custom Primers for all of your cycles.
6. Browse to select the sample sheet CSV file generated and updated in the previous step.
7. Select **Next**.
8. Continue to set up the sequencing run as per the [MiSeq System Product Documentation](https://support.illumina.com/downloads/miseq-product-documentation.html).

## Monitoring Sequencing Run

Sequencing run results will be uploaded to ICA. Users can monitor the sequencing run status from the instrument or BaseSpace.

## Secondary Analysis Auto-launch

Secondary analysis will automatically start upon the sequencing run completion. Users can monitor the secondary analysis status from BaseSpace or ICA.

![](https://3223063594-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2F2YNCzdEu5yaFoMebBG1p%2Fuploads%2Fgit-blob-9a0749e7fb0f980847205e1e9243e66da44ee0f3%2Fbasespace_run_details.png?alt=media)

## Tertiary Analysis Auto-launch in Connected Insights

Cases are automatically created in Connected Insights upon the successful completion of secondary analysis. The tertiary analysis automatically starts if disease information is provided in the sample sheet.

If disease information was not provided in the sample sheet, the case will be created with the "Missing Required Data" status. To start the analysis, follow these steps:

1. Log in to Connected Insights and make sure the right workgroup is selected.
2. Open a case with the "Missing Required Data" status.

![](https://3223063594-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2F2YNCzdEu5yaFoMebBG1p%2Fuploads%2Fgit-blob-652203e8b55795ca865a9418388ebf5919afefbb%2Fici_case_detail_panel_mrd.png?alt=media)

3. Click the **Edit Case** button, then input the tumor type in the **Disease** field.
4. Click the **Save** button. The case will start processing after a few seconds.

For additional options to provide case disease information with the Case Metadata file, access \[DATA UPLOAD -> Custom Case Data Upload] in the Connected Insights user guide accessible in the software.

## View Tertiary Analysis Results

Users can open the case to review the tertiary analysis results after the case finishes processing and the status changes to "Ready for Interpretation". A draft report will be ready for review if the Report Automation feature is turned on.

![](https://3223063594-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2F2YNCzdEu5yaFoMebBG1p%2Fuploads%2Fgit-blob-2cd43d967f1cf23a9e3bdcb08b9f337de01c96a8%2Fici_amplicon_case_overview.png?alt=media)

## Requeue Secondary Analysis

Use the following steps to requeue secondary analysis in case of its failure:

1. Delete failed cases in Connected Insights if needed.
2. Log in to BaseSpace and open the run.
3. Click the hourglass icon, then select **Requeue -> Planned Run**.

![](https://3223063594-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2F2YNCzdEu5yaFoMebBG1p%2Fuploads%2Fgit-blob-406d348f0075b814eb2e3aed8f3642a5ca7460b6%2Fbasespace_run_requeue.png?alt=media)

4. Choose either the Sample Sheet from the run or upload a new Sample Sheet to requeue the secondary analysis.

![](https://3223063594-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2F2YNCzdEu5yaFoMebBG1p%2Fuploads%2Fgit-blob-2bbacbc92cfdf2d0a3a056433c3005e59d2cf99d%2Fbasespace_requeue_option.png?alt=media)

5. New cases will be created in Connected Insights after the secondary analysis completes.