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NextSeq 550/550Dx: TSO 500 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 NextSeq 550 or NextSeq 500Dx (RUO Mode) with automated launch of secondary and tertiary analysis in Illumina Connected Insights.

Prerequisites

  • The latest v4.2.0 version of NextSeq 550 Control Software (NCS)

  • 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 NextSeq Control Software

  1. From the NextSeq Control Software home page, navigate to Manage Instrument.

  2. Select System Configuration.

  3. Select BaseSpace Sequence Hub Configuration.

  4. Change the Hosting Location to the appropriate region and check the box for Private Domain. Enter your domain name in the text box to designate the Enterprise Account Domain. Then select Save.

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

  1. Click on the workgroup name from the BaseSpace header, select Settings from the expanded menu.

  2. Make sure ICA Run Storage is turned on.

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.

  1. Navigate to Configuration -> Test Definition and create a test definition with GRCh37 human reference genome. Turn on Report Automation if you configured report automation settings in the previous step.

  1. Navigate to Configuration -> Data Upload -> From Illumina Connected Analytics, add the DRAGEN TruSight Oncology 500 Analysis Software v2.5.2 (with HRD) pipeline, and select the test definition created in the previous step.

Plan the Sequencing Run in BaseSpace

  1. Log in to BaseSpace and make sure the same workgroup is selected.

  2. Navigate to the Runs tab, select New Run, and then select Run Planning.

  1. Select Instrument Platform as "NextSeq 500/550".

  2. Set the Secondary Analysis to "BaseSpace/Illumina Connected Analytics".

The configuration input forms vary depending on the instrument platform, secondary analysis pipeline, and other selections (see Sequencer Auto-launch Analyses Compatibility for details). The following images demonstrate screens using a NextSeq 550 instrument with RAGEN TruSight Oncology 500 Analysis Software v2.5.2 (with HRD) analysis.

  1. Select "DRAGEN TruSight Oncology 500 Analysis Software - 2.5.2 (with HRD)" from the Application dropdown menu.

  2. Select "TruSight Oncology 500" from the Library Prep Kit dropdown menu.

  3. Select "TruSight Oncology 500 (NovaSeq6000Dx with SP Flow Cell, NovaSeq6000, NextSeq)" from the Index Adapter Kit dropdown menu.

  4. Click Next to continue.

  1. Use default values for Index Reads, Read Type, and Read Length or change as needed.

  2. Input Sample IDs and appropriate Index IDs for each sample. Alternatively, download a sample template and use it to import the sample information.

  3. In Per Sample Configuration, add Sample Type, Pair ID and Sample Feature. 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.

  4. Upon completion, export sequencing run settings as a SampleSheet.csv file.

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.

  1. Find [Cloud_TSO500S_Data] section.

  2. Add ,Tumor_Type at the end of the next line after [Cloud_TSO500S_Data].

  3. Add SNOMED CT code for each sample following a ,. Note: samples sharing the same Pair ID should have the same Tumor Type.

  4. Save the file.

Set Up Sequencing Run with Cloud Analysis Sample Sheet

  1. From the NextSeq Control Software Home screen, select Experiment.

  2. On the Select Assay screen, select Sequence.

  3. On the Run Setup screen, select Manual as run mode.

  4. Select Use BaseSpace Sequence Hub Setting and then select "Run Monitoring and Storage". Enter your BaseSpace Sequencing Hub username and password. If prompted, select a workgroup to upload run data to. You are prompted only if you belong to multiple workgroups.

  5. Select Next.

  6. Enter a run name of your preference.

  7. Optional: Enter a library ID of your preference.

  8. From the Recipe drop-down list, select a recipe. Only compatible recipes are listed.

  9. Select Paired-End as read type.

  10. Enter the number of cycles for each read in the sequencing run using the same value as the sample sheet generated from previous step.

  11. Set the output folder location for the current run. Select Browse to navigate to a network location.

  12. Select Browse to navigate to a sample sheet.

  13. Select Purge consumables for this run. The setting purges consumables automatically after the current run.

  14. Select Next.

  15. Select Next to start the sequencing.

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.

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.

  1. Click the Edit Case button, then input the tumor type in the Disease field.

  2. 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.

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.

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

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

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

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