NovaSeq 6000: BCL Convert Auto-launch Analysis in Cloud

This tutorial will walk through the end-to-end flow for running a NovaSeq 6000 sequencing run with secondary analysis auto-launch in ICA. The secondary analysis will consist of BCL Convert only.

This tutorial is intended to provide guidance for the cloud analysis-related functions. Specific settings and configurations for sequencing will need to be modified for compability.

The main steps in the tutorial consist of:

  1. Run Planning

  2. Prepare Sample Sheet

  3. Instrument Run Setup

  4. Run Monitoring

  5. Analysis Monitoring

  6. Review Analysis Output

Follow the BaseSpace Setup instructions to configure BaseSpace to upload run data to ICA.

Run Planning

To plan the sequencing run, navigate to BaseSpace Sequence Hub.

  1. Go to the "Runs" view using top menu navigation

  2. Click the "New Run" button

  3. Click "Run Planning"

Follow the Run Planning input form:

  1. Input the RunName (ie, NovaSeq6000Tutorial)

  2. Set the Instrument Platform to "NovaSeq"

  3. Ensure Secondary Analysis is set to "BaseSpace"

  4. Click "Next"

Complete the next input form:

  1. Set the Application to "Illumina BCL Convert for ICA - 4.1.5"

  2. Set the Library Prep Kit (ie, Illumina DNA Prep)

  3. Set the Index Adapter Kit (ie, Nextera DNA CD Indexes (24 Indexes, 24 Samples))

  4. Click Next

Complete the next input form to configure the settings for the secondary analysis, including settings used for demultiplexing.

  1. Select the Library Prep Kit (ie, Illumina DNA Prep)

  2. Set the Index Reads (ie, 2 Indexes)

  3. Set the Read Type (ie, Paired End)

  4. Set the Read Lengths

  5. Complete the Sample form, including Lane, Sample ID, and index information

  6. (Optional) Add analysis settings

  7. Click Next

Review the planned run on the final screen. Once the planned run is confirmed, click the "Export" button to download the Sample Sheet. The Sample Sheet location will need to be accessible from the instrument (ie, USB Drive, Network Location, etc).

Instrument Run Setup

Start the NovaSeq 6000 instrument. Refer to the NovaSeq 6000 documentation for details.

During the Login step, select "Illumina Cloud Options" and "Run Monitoring and Storage" to have the run monitoring and secondary analysis performed in BaseSpace and ICA. Login to the same account used to plan the run and click Next.

Refer to the NovaSeq 6000 documentation for guidance on the Run Setup screen.

Select to "Browse" for the Samplesheet and select the modified sample sheet from the last section. Click Next when complete

Review the run configuration on the final screen. Once confirmed, click to Start Run.

Run Monitoring

Once the run has been started on the instrument, the run will show in the BaseSpace Runs view after the connection has been established from the instrument to BaseSpace.

Navigate to BaseSpace and select "Runs" in the top menu navigation. Find the run name specified during Run Planning. Click to view the Run Summary view.

Run data will be uploaded to ICA. Navigate to ICA to verify the data is present.

Analysis Monitoring

Secondary analysis will start after the sequencing run data has been uploaded. BaseSpace will indicate the analysis starting on the Run Summary view in the "Pending Analyses" list.

Once the analysis has been launched, a workflow session is launched in ICA. This can be found in the BaseSpace externally-managed project where the sequencing run data is uploaded. See Sequencer Run Data in ICA for more information.

Navigate to the "Analyses" view within the project to find the workflow session. As the workflow session runs, the secondary analysis pipelines are launched and show in the "Analyses" view, or in the "Orchestrated Analyses" section of the workflow session details view. See Automated Secondary Analysis in ICA for more information.

Review Analysis Output

The results of the secondary analysis can be viewed in BaseSpace and ICA.

To view the analysis results in BaseSpace:

  1. Navigate to the Run Summary view

  2. Click the Analysis in the Latest Analysis or Prior Analyses sections

  3. Analysis results are shown in the Summary. Navigate to the Files tab to find the output files.

To view the analysis results in ICA:

  1. Navigate to the BaseSpace externally-managed ICA project corresponding to the workgroup/user the run was performed with

  2. Navigate to the Project Analyses

  3. Find the Analysis with the User Reference prefixed with the sequencing run name

  4. Click to view the Analysis details

  5. Copy the User Reference

  6. Navigate to the Project Data

  7. Search the Project Data using the copied User Reference value from step 5

  8. Navigate into the folder resulting from the filter to find the analysis output data

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