Upgrading to NextSeq Control Software v4

What is the upgrade?

• The NextSeq 500/550 system upgrade is accomplished by updating to NextSeq Control Software (NCS) v4. NCS v4 provides several enhancements:

  • Upgrade to Windows 10 operating system

  • Local Run Manager (LRM) for simplified run setup and onboard analysis

  • LRM replaces PrepTab

  • Improved BaseSpace Sequence Hub (BSSH) connection and run setup

  • Infinium MethylationEPIC array scanning ability (NextSeq 550 systems)

  • Compatibility with NextSeq v2.5 kits on all NextSeq systems including research mode of NextSeq 550Dx* - *For In Vitro Diagnostic Use

For more detailed information refer to:

• NextSeq Software v4.0.0 Release Notes

• NextSeq Software v4.0.1 Release Notes

Upgrade Process

• The upgrade must be performed by a qualified Illumina Field Service Engineer. To schedule an upgrade, contact Illumina Technical Support or your Illumina Field Representative. To ensure a successful upgrade process, consider:

  • Arrange for your local IT to be available during the upgrade to ensure smooth networking.

  • The NextSeq 500/550 system hard drives will be replaced: - Transfer all output files to a safe storage location in case a run was stored locally - Transfer all NextSeq 550 BeadChip product files (manifest, cluster files) to a safe storage location

  • Be prepared to create an LRM admin account. (Admin user provides name and temporary password to Illumina. After upgrading, admin user will enter the final password.) NCS v4 cannot be downgraded to previous versions of NCS.

Run setup with NCS v4

• NCS v4 has two options to set up a run: Manual and LRM. Both options can upload runs to BSSH for Run Monitoring Only or Monitoring and Storage. To use LRM mode, the run is set up in LRM with sample and analysis information. When the run completes, the specified analysis workflow will automatically start.

The output folder path is entered during run setup in NCS v4. As the default location, NCS v4 uses the output folder path from the previous run. NCS v4 does not have the option to save a default output folder location. Network paths must be entered as UNC path and the output folder must be at least two levels deep (eg, \server\level1\level2).

To set up a run in LRM, select Edit Runs from the NCS v4 home screen. LRM will open via Chromium Localhost.

To start a run, select Experiment and then select Sequence on the next screen

Option 1: Manual Mode 1. For BaseSpace Hub Settings, select Run Monitoring Only or Run Monitoring and Storage. Run Monitoring and Storage requires a sample sheet.

  ![](./000002755_files/image2.png)    

2. To start a run, enter the information for read length and index length. Sample and analysis information is not required in Manual Mode. In this scenario, the analysis will not start automatically when the run completes.    
    
  ![](./000002755_files/image3.png)    

3. When changing the output folder directory during run setup, do not accidentally activate the **Custom Primer** boxes. Make sure that the primer settings are correct before starting the run.  
4. To start the analysis after the run is completed, import the run to LRM or run bcl2fastq2 on your local server.    

Option 2: Local Run Manager: 1. To access LRM, select Edit Runs from the NCS v4 home screen or use the Chrome or Chromium Browser at: https://localhost 2. If LRM user management is enabled: Log in to LRM on your instrument. User management can be enabled/disabled under Tools > User Management by an Admin role user.

  ![](./000002755_files/image4.png)    

3. Select **Create Run** and select an analysis module.    
    
  ![](./000002755_files/image5.png)    

  
  * Analysis modules must be downloaded and installed individually from [Local Run Manager software downloads](https://www.illumina.com/content/illumina-support/language-master/en/sequencing/sequencing_software/local-run-manager/downloads.html). Make sure to use admin credentials when installing modules.  
  * In C:\Illumina\Genomes**,** check if genomes are present (genomes can be downloaded from [igenomes](https://support.illumina.com/sequencing/sequencing_software/igenome.html)).  
  * Under **Tools** > **Modules & Manifests,** upload manifests to LRM if required by the analysis workflow.  
4. Enter the sample and analysis information and **Save Run**.  
5. Saved runs are now available in NCS v4 for run start under LRM mode.  
  * Select the run and follow instructions on the screen.  
  * Select **Edit** to change the run settings before starting the run.    
  ![](./000002755_files/image6.png)    

Currently, only LRM accounts with admin-level permissions can start runs using LRM if user management is enabled. For more information, see NextSeq 500/550 Control Software v4.0.1 "Invalid Role" error with Local Run Manager v2.4.0.

Sample sheet i5 index sequence orientation

• On the NextSeq, MiniSeq, and iSeq, the second index (i5) is read in the reverse complement orientation compared to the MiSeq. The correct orientation to enter the i5 index depends on the instrument run mode:

  • In Local Run Manager mode, always enter the i5 sequence in the forward orientation, no matter which instrument type you are using. Local Run Manager generates the final sample sheet with the i5 sequence in the correct orientation depending on the instrument type. When creating a sample sheet with Illumina Experiment Manager for import into LRM, select MiSeq.

  • In Manual mode, the attached sample sheet is copied to the output location (including BaseSpace Sequence Hub) without validation or conversion of the indexes; therefore; the i5 sequence must be in the final orientation, which, for the NextSeq 500/550 and MiniSeq, is the reverse complement orientation.

Former BaseSpace PrepTab Users

• BaseSpace PrepTab is no longer supported in NCS v4. To directly upload a run to BaseSpace, use LRM or Manual Mode mode with Run Monitoring and Storage.

Option 1: Create Run in LRM with the GenerateFASTQ workflow. Select Library Prep Kit and enter read length, indexes, and sample information and save the run. If the required library kit is not available by default, create a custom library prep kit. For more information, see How to use a custom library prep kit in Local Run Manager v2.

Option 2: For Manual Mode, create a sample sheet with Illumina Experiment Manager (select NextSeq) or manually from a template sample sheet (email Illumina Technical Support). Save the sample sheet as .csv file. Use a text editor when making changes to the sample sheet. If using Excel, make sure that no changes are accidentally introduced due to language settings.

First run after upgrade

• During the first run after the upgrade, the flow cell registration step of the pre-run check can take longer (about 40 minutes). After the first run, the pre-run check time will go back to normal.

When the first run is completed, a prompt may appear asking to upload Operational Qualification results to BSSH. Until a selection is made, the run will still show as “Analyzing” in BSSH. Select Don’t Ask Again to keep this prompt from reappearing.

Networking considerations

• Setting the output folder to a network location has special considerations due to Windows 10 authentication requirements. To configure the network properly, work with your IT team and follow the instructions in the Knowledge Base article How to connect benchtop instruments and Local Run Manager to a network location.

Admin and Non-Admin Privileges in NCS v4

With NCS v4, non-admin users can run the instrument control software. There are several considerations for non-admin accounts:

• Accounts that are added to the NextSeq 500/550 OS will need to be managed through customer IT administration for permissions and access.

• NCS v4 allows only one user to be logged in at a time. For this reason, users should not log out during an active sequencing run, to prevent premature termination of the run.

• Remote logins (like RDP, SSH, Telnet) should be avoided and/or disabled to avoid premature run termination (same reasons as above).

• Admin account login will be needed for certain situations, such as installation of new software and LRM modules.

• Only admin users have read/write access to the C:\ drive, nonadmin users have read-only access.

Resources

• NextSeq resources

  • NextSeq Control Software v4
  • Local Run Manager Support Resources
  • Training video "how to start a run"
  • NextSeq 550 System Support Page
  • How to update Windows 10 account credentials on Benchtop Instruments LRM resources Local Run Manager Support Resources
  • Local Run Manager v4 Software Guide
  • LRM training video (general LRM overview) Currently available analysis options for LRM
  • DNA Amplicon Analysis Module

  • RNA Amplicon Analysis Module

  • Generate FASTQ Analysis Module

  • Assembly Analysis Module

  • Resequencing Analysis Module

  • RNA Fusion Analysis Module

  • Small RNA Analysis Module

  • DNA Enrichment Analysis Module

  • 16S Metagenomics Analysis Module

  • PCR Amplicon Analysis Module

  • TruSight Tumor 15

  • Amplicon DS

For any feedback or questions regarding this article (Illumina Knowledge Article #2755), contact Illumina Technical Support [e-mail].