The documents in this section support the Illumina NextSeq 1000/2000 Integrations. These integrations are compatible with Illumina cloud hosted deployments only.

NextSeq 1000/2000 Integration v2.5.0 supports future ICA services and includes new custom fields and modifications to existing automations. The integration package requires the NextSeq 1000/2000 Sequencing v2.4 workflow. You can install this workflow through the Illumina Preset Protocols (IPP). If you do not want to upgrade through the IPP, you can manually upgrade the workflow configuration.

Modify the Existing NextSeq 1000/2000 Workflow

To modify the existing NextSeq 1000/2000 Sequencing v2.3 workflow, you must make the following changes:

• Add the Measurement global field.

• Modify the Master Step field.

• Modify the Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) master and protocol steps.

• Update the Validate Reagent Cartridge Barcode step automation.

Modify NextSeq 1000/2000 Sequencing v2.5.0 Integration Package Configuration

When the /opt/gls/clarity/config/configure_nextseq1k2k.sh script is running as part of the installation and configuration process, do not use the default names for the automated sequencing and demultiplexing steps. Instead, use the names in the modified workflow.

To make modifications after configuration, use the omxProps-ConfigTool utility to update the following properties:

This section explains how to validate the installation of the Illumina NextSeq 1000/2000 Integration Package v2.5.0.

The validation process involves the following items:

• Running samples through the Library Prep Validation v2.3.3 workflow.

  • The workflow contains a single-step protocol that models the library prep workflow required to produce libraries tagged with index sequences. At the end of the step, these libraries are routed to NextSeq 1000/2000 Sequencing v2.4 workflow.

• Running the libraries through the NextSeq 1000/2000 Sequencing v2.4 workflow. This process validates the following information:

  • Successful sequential step advancement of samples in the following steps:

    1. Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4)

    2. Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.4)

    3. AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.4)

    4. Demultiplexing (NextSeq 1000/2000 Sequencing v2.4)

  • Automatic validation of run setup information before sample sheet generation or planned run creation.

  • Automated generation of a sample sheet. The sample sheet is used to start the sequencing run on NextSeq 1000/2000 Control Software (NCS) via Local run mode.

  • Automated creation of a planned run on Illumina Connected Analytics. The NextSeq 1000/2000 Control Software retrieves the planned run to start the run via Hybrid or Cloud run mode.

  • Automated tracking of the NextSeq 1000/2000 sequencing run and parsing of run statistics from BaseSpace Sequence Hub into Clarity LIMS using the integration service.

  • Automated tracking of the secondary analysis using DRAGEN (up to BCL Convert only).

  • Parsing of demultiplexing metrics from BaseSpace Sequence Hub into Clarity LIMS using the integration service.

Before running the validation steps below, these steps assume that the NextSeq 1000/2000 Integration Package v2.5.0 is installed, and that you have imported the default Clarity LIMS configuration.

Activate Workflow, Create Project, Add and Assign Samples

The following steps set up Clarity LIMS in preparation for running samples through the Library Prep Validation v2.3.3 and NextSeq 1000/2000 Sequencing v2.4 workflows.

1. On the Configuration tab, under Workflows, activate both the Library Prep Validation v2.3.3 and NextSeq 1000/2000 Sequencing v2.4 workflows.

2. On the Projects and Samples screen, create a project and add samples to it.

3. Assign the samples to the Library Prep Validation v2.3.3 workflow.

   • Label Group = TruSeq HT Adapters v2 (D7-D5)

   • Sequencing Instrument = NextSeq 1000/2000

   On exit from the step, the Routing Script automation is triggered. This automation assigns samples to the first step of the NextSeq 1000/2000 Sequencing v2.4 workflow, which is Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4) step.

NextSeq 1000/2000 Sequencing v2.4 Protocol

Step 1: Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4)

1. In Lab View, locate the NextSeq 1000/2000 Sequencing v2.4 protocol. The samples are queued for the Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4) step.

2. Add the samples to the Ice Bucket and select View Ice Bucket.

3. On the Ice Bucket screen, select Begin Work.

4. On the Pooling screen, perform the following actions:

   1. Create a pool by dragging samples into the Pool Creator.
   2. Type a name for the pool or accept the default name (Pool #1).

   1. Select Record Details.
5. In the Reagent Lot Tracking section, select from the active lots displayed in the drop-down list.

   The Step Details area contains two required fields:
6. Select Calculate Volumes.

   This selection triggers the Calculate Volumes automation. This automation calculates the volume required for each library to form a pool that has the concentration and volume specified in the step details fields.

   The automation also generates the Calculation File (CSV) and attaches it to the step. This file contains volume information of each of the samples and RSB buffer to add to the pool. Select the file to download it, then open it in Excel.

7. In the Sample Details table, select the pool icon to view details on the pool composition.
8. Select Next Steps.

   This selection triggers the Set Next Step automation, which sets the next step for samples to ADVANCE, advancing them to the next step in the protocol. The next step is Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.4).

   On the Assign Next Steps screen, the next step for samples is already set to the next step in the workflow. The next step is Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.4).

9. Select Finish Step.

At the end of this step, the pool of samples automatically advances to Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.4) step.

Step 2: Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.4)

1. In Lab View, locate the NextSeq 1000/2000 Sequencing v2.4 protocol. The pool of samples is queued for the Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.4) step.

2. Add the samples to the Ice Bucket and select View Ice Bucket.

3. On the Ice Bucket screen, select Begin Work.

   The Validate Single Input automation is triggered. This automation checks that there is only one container input to the step.

4. On the Placement screen, perform the following actions:

   1. Drag the pool into the NextSeq 1000/2000 Reagent Cartridge field in the Placed Samples area.
   2. Scan or type the barcode of the reagent cartridge into the NextSeq 1000/2000 Reagent Cartridge field.
   3. Select Record Details.

   On exit of the Placement screen, the Validate Reagent Cartridge Barcode automation checks that the reagent cartridge barcode conforms to the barcode mask [A-Z]{2}[0-9]{7}-[A-Z0-9]{4}. If not, an error message displays.

   On entry to the Record Details screen, the Retrieve Analysis Workflow Versions automation fetches the available analysis workflow versions from ICA. It then updates the preset values of both Local Analysis Workflow Versions & Cloud Analysis Workflow Versions field.

5. On the Record Details screen, the Reagent Lot Tracking section tracks the NextSeq 1000/2000 Reagent Cartridge lot information used in the step. Follow the steps in Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4) step if you must add a lot.

6. In the Reagent Lot Tracking section, select from the active lots displayed in the drop-down list.

7. The fields displayed on the Record Details screen are used to create planned run and generate the sample sheet file.

   • Run Name — Enter the experiment name. Only alphanumeric characters, dashes, and underscores are permitted. Spaces are not permitted.

   • Instrument Type — Select from preset options (NextSeq 1000 or NextSeq 2000).

   • Paired End — Select from preset options (True or False).

   • Read 1 Cycles — Select from preset options (301, 151, 101, 51) or type a custom value.

   • Read 2 Cycles — Select from preset options (301, 151, 101, 51) or type a custom value.

   • Index Reads — Select from preset options (No Index, Single Index, Dual Index).

   • Index Read 1 — Select from preset options (0, 6, 8) or type a custom value.

   • Index Read 2 — Select from preset options (0, 6, 8) or type a custom value.

   • Analysis Workflow — Read only. This field is set to GenerateFASTQ.

   • [Optional] Local Analysis Workflow Versions — Default is None. Only mandatory when Run Mode = Local/Hybrid and Analysis Workflow = GenerateFASTQ. Select according to the DRAGEN version installed on the instrument.

   • [Optional] Cloud Analysis Workflow Versions — Default is None. Only mandatory when Run Mode = Cloud and Analysis Workflow = GenerateFASTQ. Select the cloud DRAGEN version to be used.

   • FASTQ Compression Format:

     • If Run Mode = Local/Hybrid, Analysis Workflow = GenerateFASTQ, and Local Analysis Workflow Version >= 3.7.4, select gzip or DRAGEN.

     • If Run Mode = Cloud, Analysis Workflow = GenerateFASTQ, and Local Analysis Workflow Version >= 3.7.4, field is set to gzip automatically.

     • Otherwise, select None.

   • [Optional] Adapter Sequence Read 1 — Enter the Read 1 adapter sequence of the index adapter kit.

   • [Optional] Adapter Sequence Read 2 — Enter the Read 2 adapter sequence of the index adapter kit.

   • [Optional] Barcode Mismatches Index 1 — Select from preset options (0, 1, 2). Leave it blank if you are unsure.

   • [Optional] Barcode Mismatches Index 2 — Select from preset options (0, 1, 2). Leave it blank if you are unsure.

   • [Optional] Override Cycles — String used to specify UMI cycles and mask out cycles of a read (for example, N1Y150;I8;I7N1;Y141U10). Leave it blank if you are unsure.

   • Instructions — Read only.
8. On the Record Details screen, select Validate Run Setup and Create Planned Run.

   This selection triggers the automation script, which does the following actions:

   • Validates the parameters entered on the Record Details screen.

   • If Run Mode is Hybrid or Cloud, create the planned run.

   • Generates the sample sheet and attaches it to the placeholder in the Files area of the Record Details screen for all Run Mode types.

9. Select Next Steps.

   On the Assign Next Steps screen, the next step for samples is set to the next step in the workflow. The next step is AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.4).

10. Select Finish Step.

Step 3: AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.4)

This step is fully automated.

The integration starts the step automatically and data from the run is parsed back to Clarity LIMS. No user interaction is required. However, you can open and review the various stages of the step in Clarity LIMS. Do not perform any action when reviewing the data.

Read summary metrics are recorded for the library pool in the Step Details section and the Sample Details table.

Values are populated in the following master step fields:

• Run Name

• Current Read

• Flow Cell ID

• Reagent Cartridge Lot Number

• Instrument ID

• Run Status

• Current Cycle

• Flow Cell Lot Number

• Instrument Platform

• Instrument Control Software Version

• Output Folder

• Secondary Analysis Workflow

• Reagent Cartridge ID

• Instrument Type

• RTA Version

• Sequencing Log

The summary metrics (per run level) populate in the following global fields.

• % Bases >=Q30 R1

• % Bases >=Q30 R2

• % Error Rate R1

• % Error Rate R2

• Yield (Gb) R1

• Yield (Gb) R2

• Reads PF R1

• Reads PF R2

• %PF R1

• %PF R2

• % Aligned R1

• % Aligned R2

• % Phasing R1

• % Phasing R2

• % Prephasing R1

• % Prephasing R2

• Intensity Cycle 1 R1

• Intensity Cycle 1 R2

• Cluster Density R1

• Cluster Density R2

At the end of this step, the pool of samples automatically advances to (and queues for) the Demultiplexing (NextSeq 1000/2000 Sequencing v2.4) step.

Step 4: Demultiplexing (NextSeq 1000/2000 Sequencing v2.4)

This step is semi-automated.

The integration starts the step automatically and demultiplexing data from the GenerateFASTQ secondary analysis is parsed back to Clarity LIMS. Review the data parsed and assign QC, depending on the criteria set, and complete the step.

In the Record Details screen, perform the following actions:

1. Review demultiplexing data.

   • Demultiplexing metrics are recorded for the library pool in the Sample Details table. Metrics include columns for # Reads, # Perfect Index Reads, and # One Mismatch Index Reads.
   • The following metric files generated by the DRAGEN onboard or Cloud analysis module are stored under the Files section as Run_Metrics.zip:

     • Adapter_Metrics.csv

     • Demultiplex_Stats.csv

     • Index_Hopping_Counts.csv

     • Quality_Metrics.csv

     • Top_Unknown_Barcodes.csv

   • The Analysis Status step field is updated with the analysis status.

2. Assign QC flags to all the individual sample. There are two ways of doing this step.

   1. Manually assign QC flags through the QC column in Sample Details table.
   2. Automatically assign QC flag by running Assign QC flags automation. This option is for scenarios where a huge number of libraries are involved.

   In the Step Details section, the following step fields are visible. (N is the number of criteria. You can use one or more criteria.)

   • Criteria N - Source Data Field — Select from preset options (for example, # Reads).

   • Criteria N - Operator — Select from preset options (for example, >= (greater than or equal to)).

   • Criteria N - Threshold Value — Enter the desired threshold value.
3. After filling up the criteria fields, select Assign QC flags. This selection triggers the automation script, which loops through each library in the pool and apply QC flag base on the criteria set previously.

   This automation also generates an AssignQC Result file under Files section.
4. Select Next Steps.

   On the Assign Next Steps screen, the Next Step field for all samples is prepopulated with Mark protocol as complete.
5. Select Finish Step.

At this point, the whole NextSeq 1000/2000 Integration workflow is fully validated.

Troubleshooting

If the AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.4) step starts but does not finish, complete the following steps:

1. Log in to Clarity LIMS using the default user account and use one of the following methods to open the step in Clarity LIMS:

   • Method 1: In Lab View, find the step in the Recent Activities pane.

   • Method 2: Search for the step in Clarity LIMS using reagent cartridge barcode as the search term.

2. On the Record Details screen, locate the Sequencing Log field. The multiline text field contains logging information.

If you cannot reach the Record Details screen, or if the Sequencing Log field does not contain enough information to resolve the issue, contact the Clarity LIMS support team. Supply the relevant information from the troubleshooting steps already performed.

The integration includes the following features:

• Preconfigured NextSeq 1000/2000 Sequencing v2.4 workflow that maps to lab protocols and instrument runs.

• The following preconfigured protocols:

  • Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4)

  • Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.4)

  • AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.4)

  • Demultiplexing (NextSeq 1000/2000 Sequencing v2.4)

• Automatic validation of run setup information before sample sheet generation or planned run creation.

• Automated generation of a sample sheet. The sample sheet is used to start the sequencing run on NextSeq 1000/2000 Control Software (NCS) via Local run mode.

• Automated creation of a planned run. The NCS retrieves and starts the planned run via Hybrid or Cloud run mode.

• Automated tracking of the NextSeq 1000/2000 sequencing run and parsing of run statistics from BaseSpace Sequence Hub into Clarity LIMS.

• Automated tracking of the secondary DRAGEN analysis (up to BCL Convert only) and parsing of demultiplexing metrics from BaseSpace Sequence Hub into Clarity LIMS.

• The following Cloud analysis supported applications:

  • BCL Convert v4.2.7 or earlier under BaseSpace Sequence Hub DRAGEN Analysis

  • BCL Convert v4.2.7 under ICA Workflow Session Tracking

• Local analysis supported BCL Convert v4.2.7 and earlier.

• [Optional] Preconfigured Library Prep Validation v2.3.3 workflow used for validation purposes only. The workflow contains a single-step protocol that models the library prep workflow required to produce libraries tagged with index sequences. At the end of the step, these libraries are routed to NextSeq 1000/2000 Sequencing v2.4 workflow. For more information, refer to NextSeq 1000/2000 Integration v2.5.0 User Interaction, Validation and Troubleshooting.

• [Optional] An integration of Clarity LIMS Product Analytics (CLPA) that make Clarity LIMS data available to the ICA and CLPA. This integration also requires installation and configuration. For more information, refer to Clarity LIMS Product Analytics Integration v1.3.0 Configuration.

The integration includes the following features:

• Preconfigured NextSeq 1000/2000 Sequencing v2.3 workflow that maps to lab protocols and instrument runs.

• The following preconfigured protocols:

  • Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3)

  • Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3)

  • AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3)

  • Demultiplexing (NextSeq 1000/2000 Sequencing v2.3)

• Automatic validation of run setup information before sample sheet generation or planned run creation.

• Automated generation of a sample sheet. The sample sheet is used to start the sequencing run on NextSeq 1000/2000 Control Software (NCS) via Local run mode.

• Automated creation of a planned run on Illumina Connected Analytics (ICA). The NCS retrieves and starts the planned run via Hybrid or Cloud run mode.

• Automated tracking of the NextSeq 1000/2000 sequencing run and parsing of run statistics from BaseSpace Sequence Hub into Clarity LIMS using SIS integration service.

• Automated tracking of the secondary analysis using DRAGEN (up to BCL Convert only) and parsing of demultiplexing metrics from BSSH into Clarity LIMS using SIS integration service.

• Support for parsing the unicode Parameter.xml file generated by NCS v1.5.

• [Optional] Preconfigured Library Prep Validation v2.3.1 workflow used for validation purposes only. The workflow contains a single-step protocol that models the library prep workflow required to produce libraries tagged with index sequences. At the end of the step, these libraries are routed to NextSeq 1000/2000 Sequencing v2.3 workflow. For more information, refer to NextSeq 1000/2000 Integration v2.4 User Interaction, Validation and Troubleshooting.

• [Optional] An integration of Clarity LIMS Product Analytics (CLPA) that make Clarity LIMS data available to the ICA and CLPA. This integration also requires installation and configuration. For more information, refer to Clarity LIMS Product Analytics Integration v1.2 Configuration.

The integration includes the following features:

• Preconfigured NextSeq 1000/2000 Sequencing v2.4 workflow that maps to lab protocols and instrument runs.

• The following preconfigured protocols:

  • Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4)

  • Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.4)

  • AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.4)

  • Demultiplexing (NextSeq 1000/2000 Sequencing v2.4)

• Automatic validation of run setup information before sample sheet generation or planned run creation.

• Automated generation of a sample sheet. The sample sheet is used to start the sequencing run on NextSeq 1000/2000 Control Software (NCS) via Local run mode.

• Automated creation of a planned run. The NCS retrieves and starts the planned run via Hybrid or Cloud run mode.

• Automated tracking of the NextSeq 1000/2000 sequencing run and parsing of run statistics from BaseSpace Sequence Hub into Clarity LIMS.

• Automated tracking of the secondary DRAGEN analysis (up to BCL Convert only) and parsing of demultiplexing metrics from BaseSpace Sequence Hub into Clarity LIMS.

• The following Cloud analysis supported applications:

  • BCL Convert v4.2.7 or earlier under BaseSpace Sequence Hub DRAGEN Analysis

  • BCL Convert v4.2.7 under ICA Workflow Session Tracking

• Local analysis supported BCL Convert v4.2.7 and earlier.

• [Optional] Preconfigured Library Prep Validation workflow used for validation purposes only. The workflow contains a single-step protocol that models the library prep workflow required to produce libraries tagged with index sequences. At the end of the step, these libraries are routed to NextSeq 1000/2000 Sequencing v2.4 workflow.

• [Optional] An integration of Clarity LIMS Product Analytics (CLPA) that make Clarity LIMS data available to the ICA and CLPA. This integration also requires installation and configuration. For more information, refer to Clarity LIMS Product Analytics Integration v1.3.1 Configuration.

Last Updated: November 2024

Release Date: July 2024

Document Version: 2

These release notes describe the key changes to software components for the Clarity LIMS NextSeq 1000/2000 Integration Package v2.5.1.

Compatibility

Refer to Compatibility under Instruments & Integrations.

Defect Repairs

• Fixed the issue of unable to generate sample sheet and planned run when any sample in the pool has been assigned QC flag prior to entering the Load to Reagent Cartridge step.

Known Issues

• Sequencing run error triggered by disk space full on instrument causes RunParameters.xml to be empty and the run event cannot be processed on Clarity LIMS. This error is unlikely to occur as pre-run checks on the instrument check for sufficient disk space before run is started.

Documentations

The NextSeq 1000/2000 Integration v2.5.1 guides follow exactly that of the following NextSeq 1000/2000 Integration v2.5.0 with the following modifications:

• NextSeq 1000/2000 Integration v2.5.0 Configuration

  • NextSeq 1000/2000 Integration Package version has been updated from v2.5.0 to v2.5.1.

• NextSeq 1000/2000 Integration v2.5.0 User Interaction, Validation and Troubleshooting

  • NextSeq 1000/2000 Integration Package version has been updated from v2.5.0 to v2.5.1.

Revision History

Last Updated: November 2024

Release Date: June 2024

Document Version: 2

These release notes describe the key changes to software components for the Clarity LIMS NextSeq 1000/2000 Integration Package v2.5.0.

Compatibility

Refer to Compatibility under Instruments & Integrations.

New Features

• Updates the Demultiplexing step in NextSeq 1000/2000 Sequencing workflow with the following changes:

  • The Sample Details table displays the index sequence.

  • The Sample Details table only displays the following demultiplexing stats due to changes in Demultiplex_Stats.csv:

    • # Reads

    • # Perfect Index Reads

    • # One Mismatch Index Reads

  • The demultiplexing stats displayed are aggregated for multi-lane flow cells (for example, P3).

  • The following run metrics files are included in Run_Metrics.zip and attached to the step:

    • Adapter_Metrics.csv

    • Demultiplex_Stats.csv

    • Index_Hopping_Counts.csv

    • Quality_Metrics.csv

    • Top_Unknown_Barcodes.csv

  • Analysis Status is now visible on the step.

• The integration service and integration-related properties have been moved from configuration files to database.

• Supports DRAGEN BCL Convert v4.2.7 analysis, including:

  • Cloud BCL Convert v4.2.7 running under the ICA Workflow Session Tracking application

  • Local BCL Convert v4.2.7

Defect Repairs

• Demultiplexing stats are now correctly displayed in the Sample Details table in Demultiplexing step when the same samples with different indexes are in the run. For multi-lane flow cells, the demultiplexing stats are aggregated.

• The Validate Reagent Cartridge Barcode automation in the Load to Reagent Cartridge step now only accepts the [A-Z]{2}[0-9]{7}-[A-Z0-9]{4} barcode format.

Known Issues

• Sequencing run error triggered by disk space full on instrument causes RunParameters.xml to be empty and the run event cannot be processed on Clarity LIMS. This error is unlikely to occur as pre-run checks on the instrument check for sufficient disk space before run is started.

• Sample sheet and planned run generation will fail if any of the samples in the pools has been assigned QC flag in prior steps before entering the Load to Reagent Cartridge step.

Revision History

This section explains how to validate the installation of the Illumina NextSeq 1000/2000 Integration Package v2.4.0.

The validation process involves the following items:

• Running samples through the Library Prep Validation v2.3.1 workflow.

  • The workflow contains a single-step protocol that models the library prep workflow required to produce libraries tagged with index sequences. At the end of the step, these libraries are routed to NextSeq 1000/2000 Sequencing v2.3 workflow.

• Running the libraries through the NextSeq 1000/2000 Sequencing v2.3 workflow. This process validates the following information:

  • Successful sequential step advancement of samples in the following steps:

    1. Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3)

    2. Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3)

    3. AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3)

    4. Demultiplexing (NextSeq 1000/2000 Sequencing v2.3)

  • Automatic validation of run setup information before sample sheet generation or planned run creation.

  • Automated generation of a sample sheet. The sample sheet is used to start the sequencing run on NextSeq 1000/2000 Control Software (NCS) via Local run mode.

  • Automated creation of a planned run on Illumina Connected Analytics. The NextSeq 1000/2000 Control Software retrieves the planned run to start the run via Hybrid or Cloud run mode.

  • Automated tracking of the NextSeq 1000/2000 sequencing run and parsing of run statistics from BaseSpace Sequence Hub into Clarity LIMS using the SIS Integration Service.

  • Automated tracking of the secondary analysis using DRAGEN (up to BCL Convert only).

  • Parsing of demultiplexing metrics from BaseSpace Sequence Hub into Clarity LIMS using SIS integration service.

Before running the validation steps below, these steps assume that the NextSeq 1000/2000 Integration Package v2.4.0 is installed, and that you have imported the default Clarity LIMS configuration.

For information on how the integration works, refer to NextSeq 1000/2000 Integration v2.4.0 Configuration.

Activate Workflow, Create Project, Add and Assign Samples

The following steps set up Clarity LIMS in preparation for running samples through the Library Prep Validation v2.3.1 and NextSeq 1000/2000 Sequencing v2.3 workflows.

1. On the Configuration tab, under Workflows, activate both the Library Prep Validation v2.3.1 and NextSeq 1000/2000 Sequencing v2.3 workflows.

2. On the Projects and Samples screen, create a project and add samples to it.

3. Assign the samples to the Library Prep Validation v2.3.1 workflow.

   Follow the steps in Library Prep Validation Protocol to run the Library Prep Validation workflow with the following:

   • Label Group = TruSeq HT Adapters v2 (D7-D5)

   • Sequencing Instrument = NextSeq 1000/2000

   On exit from the step, the Routing Script automation is triggered. This automation assigns samples to the first step of the NextSeq 1000/2000 Sequencing v2.3 workflow, which is Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) step.

NextSeq 1000/2000 Sequencing v2.3 Protocol

Step 1: Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3)

1. In Lab View, locate the NextSeq 1000/2000 Sequencing v2.3 protocol. The samples are queued for the Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) step.

2. Add the samples to the Ice Bucket and select View Ice Bucket.

3. On the Ice Bucket screen, select Begin Work.

4. On the Pooling screen, perform the following actions:

   1. Create a pool by dragging samples into the Pool Creator.
   2. Type a name for the pool or accept the default name (Pool #1).
   3. Select Record Details.

5. On the Record Details screen, the Reagent Lot Tracking section tracks the Resuspension Buffer (RSB) lot information used in the step. If required, create new lots by referencing Add and Configure Reagent Kits and Lots in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.

6. In the Reagent Lot Tracking section, select from the active lots displayed in the drop-down list.
7. The Step Details area contains two required fields:

   • Final Loading Concentration (pM) — The value entered in this field is the recommended final loading concentration specified in the Illumina NextSeq 1000/2000 Sequencing System Guide at support.illumina.com. The value depends on the library type. The default drop-down list contains the values 650, 750, 1000, and 2000. A custom value is acceptable.

   • Final Loading Volume (ul) — The value in this field is the final loading volume of the pool into the reagent cartridge. The field is prepopulated with the configured default value, 24 µl, specified in the Illumina NextSeq 1000/2000 Sequencing System Guide at support.illumina.com. The value is editable when more volume is necessary.

8. Select Calculate Volumes.

   This selection triggers the Calculate Volumes automation. This automation calculates the volume required for each library to form a pool that has the concentration and volume specified in the step details fields.

   The automation also generates the Calculation File (CSV) and attaches it to the step. This file contains volume information of each of the samples and RSB buffer to add to the pool. Select the file to download it, then open it in Excel.

9. In the Sample Details table, select the pool icon to view details on the pool composition.
10. Select Next Steps.

    This selection triggers the Set Next Step automation, which sets the next step for samples to ADVANCE, advancing them to the next step in the protocol. The next step is Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3).

    On the Assign Next Steps screen, the next step for samples is already set to the next step in the workflow. The next step is Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3).

11. Select Finish Step.

At the end of this step, the pool of samples automatically advances to Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3) step.

Step 2: Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3)

1. In Lab View, locate the NextSeq 1000/2000 Sequencing v2.3 protocol. The pool of samples is queued for the Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3) step.

2. Add the samples to the Ice Bucket and select View Ice Bucket.

3. On the Ice Bucket screen, select Begin Work.

   The Validate Single Input automation is triggered. This automation checks that there is only one container input to the step.

4. On the Placement screen, perform the following actions:

   1. Drag the pool into the NextSeq 1000/2000 Reagent Cartridge field in the Placed Samples area.
   2. Scan or type the barcode of the reagent cartridge into the NextSeq 1000/2000 Reagent Cartridge field.
   3. Select Record Details.

   On exit of the Placement screen, the Validate Reagent Cartridge Barcode automation checks that the reagent cartridge barcode conforms to the barcode mask [A-Za-z]{2}[0–9]{7}-[A-Za-z0-9]{4}. If not, an error message displays.

   On entry to the Record Details screen, the Retrieve Analysis Workflow Versions automation fetches the available analysis workflow versions from ICA. It then updates the preset values of both Local Analysis Workflow Versions & Cloud Analysis Workflow Versions field.

5. On the Record Details screen, the Reagent Lot Tracking section tracks the NextSeq 1000/2000 Reagent Cartridge lot information used in the step. Follow the steps in Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) step if you must add a lot.

6. In the Reagent Lot Tracking section, select from the active lots displayed in the drop-down list.

7. The fields displayed on the Record Details screen are used to create planned run and generate the sample sheet file.

   • Run Name — Enter the experiment name. Only alphanumeric characters, dashes, and underscores are permitted. Spaces are not permitted.

   • Instrument Type — Select from preset options (NextSeq 1000 or NextSeq 2000).

   • Run Mode — Select from preset options: Local, Hybrid, Cloud. For more information on the different run modes, refer to Run Mode Details.

   • Paired End — Select from preset options (True or False).

   • Read 1 Cycles — Select from preset options (301, 151, 101, 51) or type a custom value.

   • Read 2 Cycles — Select from preset options (301, 151, 101, 51) or type a custom value.

   • Index Reads — Select from preset options (No Index, Single Index, Dual Index).

   • Index Read 1 — Select from preset options (0, 6, 8) or type a custom value.

   • Index Read 2 — Select from preset options (0, 6, 8) or type a custom value.

   • Analysis Workflow — Read only. This field is set to GenerateFASTQ.

   • Local Analysis Workflow Versions — [Optional] (default is None). Only mandatory when Run Mode = Local/Hybrid and Analysis Workflow = GenerateFASTQ. Select according to the DRAGEN version installed on the instrument.

   • Cloud Analysis Workflow Versions — [Optional] (default is None). Only mandatory when Run Mode = Cloud and Analysis Workflow = GenerateFASTQ. Select the cloud DRAGEN version to be used.

   • FASTQ Compression Format:

     • If Run Mode = Local/Hybrid, Analysis Workflow = GenerateFASTQ, and Local Analysis Workflow Version >= 3.7.4, select gzip or DRAGEN.

     • If Run Mode = Cloud, Analysis Workflow = GenerateFASTQ, and Local Analysis Workflow Version >= 3.7.4, field is set to gzip automatically.

     • Otherwise, select None.

   • Adapter Sequence Read 1 — [Optional] Enter the Read 1 adapter sequence of the index adapter kit.

   • Adapter Sequence Read 2 — [Optional] Enter the Read 2 adapter sequence of the index adapter kit.

   • Barcode Mismatches Index 1 — [Optional] Select from preset options (0, 1, 2). Leave it blank if you are unsure.

   • Barcode Mismatches Index 2 — [Optional] Select from preset options (0, 1, 2). Leave it blank if you are unsure.

   • Override Cycles — [Optional] String used to specify UMI cycles and mask out cycles of a read (eg, N1Y150;I8;I7N1;Y141U10). Leave it blank if you are unsure.

   • Instructions — Read only.
8. On the Record Details screen, select Validate Run Setup and Create Planned Run.

   This selection triggers the automation script, which does the following actions:

   • Validates the parameters entered on the Record Details screen.

   • Create the planned run and send it to ICA if Run Mode is Hybrid or Cloud.

   • Generates the sample sheet and attaches it to the placeholder in the Files area of the Record Details screen for all Run Mode types.

9. Select Next Steps.

   On the Assign Next Steps screen, the next step for samples is set to the next step in the workflow. The next step is AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3).

10. Select Finish Step.

At the end of this step, the pool of samples automatically advances to and is queued for the AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3) step. The sequencing run is ready to be started. For details on how to start the sequencing run for different run modes, refer to NextSeq 1000/2000 Integration v2.4.0 Configuration.

Step 3: AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3)

This step is fully automated.

The integration starts the step automatically and data from the run is parsed back to Clarity LIMS. No user interaction is required. However, you can open and review the various stages of the step in Clarity LIMS. Do not perform any action when reviewing the data.

Read summary metrics are recorded for the library pool in the Step Details section and the Sample Details table.

Values are populated in the following master step fields:

• Run Name

• Current Read

• Flow Cell ID

• Reagent Cartridge Lot Number

• Instrument ID

• Run Status

• Current Cycle

• Flow Cell Lot Number

• Instrument Platform

• Instrument Control Software Version

• Output Folder

• Secondary Analysis Workflow

• Reagent Cartridge ID

• Instrument Type

• RTA Version

• Sequencing Log

The summary metrics (per run level) populate in the following global fields.

• % Bases >=Q30 R1

• % Bases >=Q30 R2

• % Error Rate R1

• % Error Rate R2

• Yield (Gb) R1

• Yield (Gb) R2

• Reads PF R1

• Reads PF R2

• %PF R1

• %PF R2

• % Aligned R1

• % Aligned R2

• % Phasing R1

• % Phasing R2

• % Prephasing R1

• % Prephasing R2

• Intensity Cycle 1 R1

• Intensity Cycle 1 R2

• Cluster Density R1

• Cluster Density R2

At the end of this step, the pool of samples automatically advances to (and queues for) the Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) step.

Step 4: Demultiplexing (NextSeq 1000/2000 Sequencing v2.3)

This step is semi-automated.

• The DRAGEN version must be earlier than v3.9.3.

• The samples in the run must not repeat.

• The run must be for the single lane flow cell (P1 or P2).

The integration starts the step automatically and demultiplexing data from the GenerateFASTQ secondary analysis is parsed back to Clarity LIMS. Review the data parsed and assign QC, depending on the criteria set, and complete the step.

In the Record Details screen, perform the following actions:

1. Review demultiplexing data.

   • Demultiplexing metrics are recorded for the library pool in the Sample Details table. Metrics include columns for # Reads, # Perfect Index Reads, # One Mismatch Index Reads, and # of >= Q30 Bases (PF).
   • The Demultiplex_stats.csv file generated by the DRAGEN onboard or Cloud analysis module is stored under the Files section.
2. Assign QC flags to all the individual sample. There are two ways of doing this step.

   1. Manually assign QC flags through the QC column in Sample Details table.
   2. Automatically assign QC flag by running Assign QC flags automation. This option is for scenarios where a huge number of libraries are involved.

   In the Step Details section, the following step fields are visible. (N is the number of criteria. You can use one or more criteria.)

   • Criteria N - Source Data Field — Select from preset options eg # Reads.

   • Criteria N - Operator — Select from preset options, for example, >= (greater than or equal to).

   • Criteria N - Threshold Value — Enter the desired threshold value.
3. After filling up the criteria fields, select Assign QC flags. This selection triggers the automation script, which loops through each library in the pool and apply QC flag base on the criteria set previously.

   This automation also generates an AssignQC Result file under Files section.
4. Select Next Steps.

   On the Assign Next Steps screen, the Next Step field for all samples is prepopulated with Mark protocol as complete.
5. Select Finish Step.

At this point, the whole NextSeq 1000/2000 Integration workflow is fully validated.

Troubleshooting

If an automation trigger does not appear to run its corresponding scripts, refer to Troubleshooting Automation in the Clarity LIMS (API & Database) documentation.

If the AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3) step starts but does not finish, complete the following steps:

1. Log in to Clarity LIMS using the default user account and use one of the following methods to open the step in Clarity LIMS:

   • Method 1: In Lab View, find the step in the Recent Activities pane.

   • Method 2: Search for the step in Clarity LIMS using reagent cartridge barcode as the search term.

2. On the Record Details screen, locate the Sequencing Log field. The multiline text field contains logging information.

If you cannot reach the Record Details screen, or if the Sequencing Log field does not contain enough information to resolve the issue, contact the Clarity LIMS support team. Supply the relevant information from the troubleshooting steps already performed.

The Illumina NextSeq 1000/2000 Integration Package v2.5.0 supports the integration of Clarity LIMS to Illumina NextSeq 1000/2000 sequencing systems.

For instructions on validating and troubleshooting the NextSeq 1000/2000 Integration, refer to NextSeq 1000/2000 Integration v2.5.0 User Interaction, Validation and Troubleshooting.

The configuration provided in this integration has been established to support NextSeq 1000/2000 lab processes. Any configuration changes to protocols or workflows — including renaming protocols, steps, and fields — could break the process.

Prerequisites and Assumptions

The Illumina NextSeq 1000/2000 Integration Package v2.5.0 is compatible with Illumina cloud hosted deployments only.

This integration requires a Personal, Professional, or Enterprise BaseSpace Sequence Hub subscription.

It is assumed that samples entering the NextSeq 1000/2000 Sequencing v2.4 workflow have gone through library preparation and quantification processes. Before they are assigned to the workflow, samples have completed the following actions:

• Samples have been accessioned into Clarity LIMS.

• Samples have been run through QC and library prep.

• Samples have the Molarity (nM) global field set to some value. The Calculate Volumes automation in the Library Pooling and Dilution step requires a value in the Molarity (nM) global field.

For more information on sample accessioning, refer to Sample Accessioning and Upload and Modify Samples in the Getting Started section of the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.

You can assign samples to workflows automatically, using a routing script, or manually—from the Projects & Samples dashboard. Refer to Assign and Process Samples in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.

Workflows, Protocols, and Steps

The NextSeq 1000/2000 Integration Package v2.5.0 includes the following workflows:

• NextSeq 1000/2000 Sequencing v2.4

• [Optional] Library Prep Validation v2.3.3 (recommended for validation purposes)

The following protocols and steps are included in these workflows.

Library Prep Validation v2.3.3 Workflow

NextSeq 1000/2000 Sequencing v2.4 Workflow

Validation Workflow

The Library Prep Validation v2.3.3 workflow allows for validation of the system after installation is complete. For details, refer to NextSeq 1000/2000 Integration v2.5.0 User Interaction, Validation and Troubleshooting.

Step 1: Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4)

In this step, the addition of RSB dilutes pooled samples. Manually create a working pool based on the final loading concentration required.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -s 'com/illumina/upa/scripts/common/step_started.groovy'

nextStep = ::ADVANCE::

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -s 'com/illumina/upa/scripts/common/pooling.groovy' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -s 'com/illumina/upa/scripts/common/step_completed.groovy'

¹ These automations are required for CLPA support only.

Master Step Fields

The following fields are defined on the Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4) step.

Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4) Master Step Field Configuration

Global Fields

The following table lists the global fields that are configured to display on the Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4) step.

Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4) Global Field Configuration

Step 2: Load to Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.4)

In this step, scan the reagent cartridge barcode into Clarity LIMS, then manually place the working pool into the reagent cartridge for the NextSeq 1000/2000 run. This step validates the run setup and analysis information and generates the sample sheet file and/or creates a planned run depending on the Run Mode selected.

if (!output.container.name.matches(::[A-Z]{2}[0-9]{7}-[A-Z0-9]{4}::)){
    fail (::Invalid Reagent Cartridge Barcode. Please verify and try again.::) 
}

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/SIS/SISServices/extensions/automation/nextseq1k2k-automation.jar script:retrieve_analysis_workflow_versions -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid2}

nextStep = ::ADVANCE::

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -s 'com/illumina/upa/scripts/common/step_completed.groovy'

¹ These automations are required for CLPA support only.

² These automations are required for the NextSeq 1000/2000 Sequencing v2.4 workflows and contain additional logic needed for CLPA support. If you would like to remove CLPA support, contact Illumina Support.

Master Step Fields

There are 24 fields defined on the Load to Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.4) step. These fields are required for sample sheet generation and planned run creation.

Load to Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.4) Master Step Fields Configuration

Step 3: AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.4)

This step is fully automated.

The integration starts and completes the step automatically. Data from the run is parsed back to Clarity LIMS. No user interaction is required. In this step, the pooled samples in the reagent cartridge are sequenced on the NextSeq 1000/2000 instrument.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid0} -s 'com/illumina/upa/scripts/common/step_started.groovy' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid0} -s 'com/illumina/upa/scripts/nextseq1k2k/register_nextseq1k2k_run.groovy' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid0} -s 'com/illumina/upa/scripts/nextseq1k2k/nextseq1k2k_associate_seqrun.groovy' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid0} -s 'com/illumina/upa/scripts/nextseq1k2k/nextseq1k2k_seqrun_started.groovy'

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid0} -s 'com/illumina/upa/scripts/nextseq1k2k/nextseq1k2k_seqrun_completed.groovy' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid0} -s 'com/illumina/upa/scripts/common/step_completed.groovy'

¹ These automations are required for CLPA support only.

Master Step Fields

There following fields are defined on the AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.4) step. These fields are used to display the run status and sequencing run and analysis configuration parsed from the RunParameters.xml file of the sequencing run.

Run Parameters and Corresponding Clarity LIMS Step Fields

The following table shows how some of the step fields map to the fields on the RunParameters.xml file, and whether the field is visible on the Record Details screen.

Additional Master Step Fields and Values

The following table shows how the other step fields derive their values, and whether the step field is visible on the Record Details screen.

¹ For information on how the integration works, refer to Run Status, Primary Metrics, and Analysis Results Parsing and Recording in How the NextSeq 1000/2000 Integration Works.

Global Fields

The following global fields are used to capture the run metrics in Clarity LIMS.

• % Bases >=Q30 R1

• % Bases >=Q30 R2

• % Error Rate R1

• % Error Rate R2

• Yield (Gb) R1

• Yield (Gb) R2

• Reads PF R1

• Reads PF R2

• %PF R1

• %PF R2

• % Aligned R1

• % Aligned R2

• % Phasing R1

• % Phasing R2

• % Prephasing R1

• % Prephasing R2

• Intensity Cycle 1 R1

• Intensity Cycle 1 R2

• Cluster Density R1

• Cluster Density R2

At the end of this step, the pool of samples is automatically advanced to (and queued for) the Demultiplexing (NextSeq 1000/2000 Sequencing v2.4) step.

Step 4: Demultiplexing (NextSeq 1000/2000 Sequencing v2.4)

This step is a semi-automated step.

The integration starts the step automatically and demultiplexing data from the GenerateFASTQ secondary analysis is parsed back to Clarity LIMS. The lab scientist reviews the demultiplexing result parsed into Clarity LIMS, assigns QC flags, and completes the step.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -s 'com/illumina/upa/scripts/common/step_started.groovy'

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -u {username} -p {password} script:assignQCForDemuxStep -i {processURI:v2} -log {compoundOutputFileLuid1} -qcResult {compoundOutputFileLuid2}

nextStep = ::ADVANCE::

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -s 'com/illumina/upa/scripts/common/step_completed.groovy' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -s 'com/illumina/upa/scripts/nextseq1k2k/nextseq1k2k_analysisrun_completed.groovy'

¹ These automations are required for CLPA support only.

Master Step Fields

The following table lists the master step fields that are configured on the Demultiplexing (NextSeq 1000/2000 Sequencing v2.4) master step.

Master Step Field Configuration for Demultiplexing (NextSeq 1000/2000 Sequencing v2.4) Step

Global Fields

The following table lists the global fields that are configured on the Demultiplexing (NextSeq 1000/2000 Sequencing v2.4) step. These fields are used to display the demultiplexing result metrics for individual library in the sample pool.

The demultiplexing statistics are aggregated for multi-lane flow cells. For each demultiplexing field (for example, # Reads) displayed in the Sample Details table for the multi-lane flow cell (for example, P3), the displayed value is the sum of the statistics across all lanes.

Global Field Configuration for Demultiplexing (NextSeq 1000/2000 Sequencing v2.4) Step

How the NextSeq 1000/2000 Integration Works

Sample Sheet Generation and Planned Run Creation

On the Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.4) step, when the Validate Run Setup and Create Planned Run automation is triggered, the run and analysis parameters entered in the Run Details screen are used to create a planned run. Then, the run and analysis configuration is validated. If the validation fails, an error message is sent to Clarity LIMS. If the validation passes, the sample sheet is generated (and sent back to Clarity LIMS) and/or a planned run is created depending on the Run Mode selected.

• Local run mode: Only the sample sheet is generated and stored on Clarity LIMS. The sample sheet contains all the run and analysis configuration required to start the run on the instrument.

• Hybrid/Cloud run mode: A planned run is created. This planned run contains all the run and analysis configuration required to start the run on the instrument. The sample sheet is also generated and stored on Clarity LIMS for reference purposes.

For details on how to start a run using different run modes, refer to Start A Sequencing Run On Instrument With Different Run Modes.

If a planned run with the same sample and project name has been created previously, the original case with the previous sample name can be reflected in the sample sheet generated from the Validate Run Setup and Create Planned Run automation. To resolve this issue, change the sample or project name in Clarity LIMS and run the automation again.

Run Status, Primary Metrics, and Analysis Results Parsing and Recording

After the sequencing run is started on the instrument, NextSeq 1000/2000 Control Software (NCS) notifies BaseSpace Sequence Hub that the sequencing run has started.

BaseSpace Sequence Hub then does the following actions:

• Local run mode: Creates a run on the BaseSpace Sequence Hub database with the run configuration information received from NCS.

• Hybrid/Cloud run mode: Creates a run on the BaseSpace Sequence Hub database by copying the run configuration information from the planned run using a special Cloud Run ID received from NCS.

After the run is created on BaseSpace Sequence Hub, a run update event message that carries the RunStarted equivalent message is created on BaseSpace Sequence Hub and delivered to Clarity LIMS SQS queue service. The message is captured and processed by the integration.

Based on the reagent cartridge barcode information in the event message (refer to the following example), the integration service looks in the queue of AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.4) step for a container with the same reagent cartridge barcode. When identified, the step is started automatically and the Run Status field on Record Details screen is updated to RunStarted. The integration service then updates all the other step fields (for example, Current Read, Output Folder) by parsing the details from the RunParameters.xml uploaded to BaseSpace Sequence Hub by NCS.

#Example of an run update event
{
"acl": [
"wid:61dc14e8-d823-3bd1-bea3-66f9c8388254"
],
"apiUrl": "https://api.basespace.illumina.com/v2/runs/r.ZvTfDXsNhUyh2kA5S4Zewg",
"dateModified": "2020-03-26T09:19:58.5546497Z",
"flowcellBarcode": "11M11COMC",
"gdsFolderPath": "/Runs/200326_MP2-15_87_11M11COMC_r.ZvTfDXsNhUyh2kA5S4Zewg",
"gdsVolumeName": "bssh.61dc14e8d8233bd1bea366f9c8388254",
"id": "r.ZvTfDXsNhUyh2kA5S4Zewg",
"instrumentRunId": "200326_MP2-15_87_11M11COMC",
"name": "200326_MP2-15_87_11M11COMC",
"reagentBarcode": "EC0001990-EC5",
"sampleSheetName": "SampleSheet.csv",
"status": "Running",
"v1pre3Id": "194424236"
}

How the Integration Service Handles Run Status

The NextSeq 1000/2000 Control Software (NCS) continues to notify BaseSpace Sequence Hub (BSSH) of any run status change throughout the course of the sequencing run. The run update event delivery process continues as described in the previous section.

The following table lists the run status details and how the integration service handles each status.

Start A Sequencing Run On Instrument With Different Run Modes

Refer to the NextSeq 1000/2000 Product Documentation for the following information:

• Configuring a run with the Proactive, Run Monitory and Storage option. Local runs are done in Local mode, and cloud or hybrid runs are done in Cloud or Hybrid mode.

• Starting a local, cloud, or hybrid run.

Enabling Planned Run Generation for Samples Having Duplicate Name with Different Indexes

The library preparation workflow of the samples must be configured to ensure unique derived sample names before routing the samples through the library preparation workflow.

Re-queue Samples for Cloud Run

Clarity LIMS requires unique library names when samples are re-queued to the workflow in the NextSeq 1000/2000 integration. Assign unique names to libraries after going through library preparation.

The following steps are used to configure the library preparation workflow correctly before routing the samples for re-queuing:

1. From Configuration, select the Lab Work tab.

2. Search for the library preparation workflow used for the re-queued samples.

3. In the Master Step of the library preparation workflow, modify the naming convention under Step Type to generate unique library names (for example, appending LIMS ID to the default naming convention, like OutputItemLIMSID).

4. Select Save.

Components Installed

The following sections describe the various components that are installed by default as part of this integration. These components include the following items:

• Reagent categories/label groups

• Reagent kits

• Control types

• Containers

Information on installed workflows, protocols, steps, and automation points is provided in the Workflows, Protocols, and Steps section of NextSeq 1000/2000 Integration v2.5.0 User Interaction, Validation and Troubleshooting.

Reagent Categories/Label Groups

• TruSeq HT adapters v2 (D7-D5)

Reagent Kits

• Resuspension Buffer (RSB)

• NextSeq 1000/2000 reagent cartridge

Container Types

• Tube

• 96-well plate

• NextSeq 1000/2000 reagent cartridge

Control Types

• PhiX v3

This integration supports the NextSeq 1000/2000 reagent cartridge with barcode provided in the format [A-Z]{2}[0-9]{7}-[A-Z0-9]{4} (for example, EC1234567-EC03).

Rules and Constraints

• The workflow configuration contains several validation checks. To make sure that the calculations work properly, it is important that you do not disable any of this validation logic. The validation checks determine:

  • Which samples, and how many, can enter each step together.

  • Which samples, and how many, can be pooled together.

• The NextSeq 1000/2000 reagent cartridge barcode must be unique. There should not be multiple NextSeq 1000/2000 reagent cartridge containers in the system with the same name.

• Reagent labels (indexes) must be unique.

• One library pool can only contain one library or control with no label (index).

• Do not manually start or complete the AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.4) step. This step is a fully automated step, and the integration service does not update samples correctly if they have been manually started.

• Do not manually start the Demultiplexing (NextSeq 1000/2000 Sequencing v2.4) step. This step is semi-automated, and the integration service does not update the demultiplexing result correctly if they have been manually started.

• For the automated run to start successfully, select Validate Run Setup and Create Planned Run in the Load to Reagent Cartridge step.

Last Updated: November 2024

Release Date: September 2023

Document Version: 2

These release notes describe the key changes to software components for the Clarity LIMS NextSeq 1000/2000 Integration Package v2.4.0.

Compatibility

Refer to under Instruments & Integrations.

New Features

• Updates third-party dependency libraries.

Defect Repairs

• The NextSeq 1000/2000 sample sheet now reflects the submitted sample name instead of the library name.

• The scope for the JWT token in the integration is restricted.

• Fixed SIS core service not being properly registered in systemctl.

Known Issues

• Sequencing run error triggered by disk space full on instrument causes RunParameters.xml to be empty and the run event cannot be processed on Clarity LIMS. This error is unlikely to occur as pre-run checks on the instrument check for sufficient disk space before run is started.

Limitations

For the demultiplexing information in the Sample Details table to display correctly, the following conditions must be met:

• The DRAGEN version must be earlier than v3.9.3.

• The samples in the run must not repeat.

• The run must be for the single lane flow cell (P1 or P2).

Revision History

BSSH subscription (personal/professional/enterprise) is required for NextSeq 1000/2000 Integration v2.4.0

The Illumina NextSeq 1000/2000 Integration Package v2.4.0 supports the integration of Clarity LIMS to Illumina NextSeq 1000/2000 sequencing systems.

For instructions on user interaction for each step, validating and troubleshooting the NextSeq 1000/2000 Integration, refer to .

The configuration provided in this integration has been established to support NextSeq 1000/2000 lab processes. Any configuration changes to protocols or workflows — including renaming protocols, steps, and fields (global or master step) — could break the process.

Prerequisites and Assumptions

The Illumina NextSeq 1000/2000 Integration Package v2.4.0 is compatible with Illumina cloud hosted deployments only.

It is assumed that samples entering the NextSeq 1000/2000 Sequencing v2.3 workflow have gone through library preparation and quantification processes. Before they are assigned to the workflow, samples have completed the following actions:

• Samples have been accessioned into Clarity LIMS.

• Samples have been run through QC and library prep.

• Samples have the Molarity (nM) global field set to some value. The Calculate Volumes automation in the Library Pooling and Dilution step requires a value in the Molarity (nM) global field.

For more information on sample accessioning, refer to Sample Accessioning and Upload and Modify Samples in the Getting Started section of the .

You can assign samples to workflows automatically, using a routing script, or manually—from the Projects & Samples dashboard. Refer to Assign and Process Samples in the .

Workflows, Protocols, and Steps

The NextSeq 1000/2000 Integration Package v2.4.0 includes the following workflows:

• [Optional] Library Prep Validation v2.3.1 (recommended for validation purposes).

• NextSeq 1000/2000 Sequencing v2.3

  Samples are routed to this protocol after they have gone through Library Prep Validation v2.3.1 (or any other library prep workflow). Samples are pooled, diluted to final loading concentration, and added to a reagent cartridge in preparation for the sequencing run.

  The workflow configuration includes a global field named Illumina Universal Sample Identifier. This text field is reserved for Clarity LIMS Product Analytics support. The field is optional and not required for this integration.

Library Prep Validation v2.3.1 Workflow

NextSeq 1000/2000 Sequencing v2.3 Workflow

Validation Workflow

Step 1: Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3)

In this step, the addition of RSB dilutes pooled samples. Manually create a working pool based on the final loading concentration required.

¹ These automations are required for Clarity LIMS Product Analytics support only.

Master Step Fields

The following fields are defined on the Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) step.

Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) Master Step Field Configuration

Global Fields

The following table lists the global fields that are configured to display on the Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) step.

Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) Global Field Configuration

Step 2: Load to Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3)

In this step, scan the reagent cartridge barcode into Clarity LIMS, then manually place the working pool into the reagent cartridge for the NextSeq 1000/2000 run. This step validates the run setup and analysis information and generates the sample sheet file and/or creates a planned run on Illumina Connected Analytics, depending on the Run Mode selected.

¹ These automations are required for Clarity LIMS Product Analytics support only.

² These automations are required for the NextSeq 1000/2000 Sequencing v2.3 workflows and contain additional logic needed for Clarity LIMS Product Analytics support. If you would like to remove Clarity LIMS Product Analytics support, contact Illumina Support.

Master Step Fields

There are 24 fields defined on the Load to Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3) step. These fields are required for sample sheet generation and planned run creation on Illumina Connected Analytics (ICA).

Load to Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3) Master Step Fields Configuration

Step 3: AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3)

This step is fully automated.

The integration starts and completes the step automatically. Data from the run is parsed back to Clarity LIMS. No user interaction is required. In this step, the pooled samples in the reagent cartridge are sequenced on the NextSeq 1000/2000 instrument.

¹ These automations are required for Clarity LIMS Product Analytics support only.

Master Step Fields

There following fields are defined on the AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3) step. These fields are used to display the run status and sequencing run and analysis configuration parsed from the RunParameters.xml file of the sequencing run.

Run Parameters and Corresponding Clarity LIMS Step Fields The following table shows how some of the step fields map to the fields on the RunParameters.xml file, and whether the field is visible on the Record Details screen.

Additional Master Step Fields and Values The following table shows how the other step fields derive their values, and whether the step field is visible on the Record Details screen.

Global Fields

The following global fields are used to capture the run metrics in Clarity LIMS.

• % Bases >=Q30 R1

• % Bases >=Q30 R2

• % Error Rate R1

• % Error Rate R2

• Yield (Gb) R1

• Yield (Gb) R2

• Reads PF R1

• Reads PF R2

• %PF R1

• %PF R2

• % Aligned R1

• % Aligned R2

• % Phasing R1

• % Phasing R2

• % Prephasing R1

• % Prephasing R2

• Intensity Cycle 1 R1

• Intensity Cycle 1 R2

• Cluster Density R1

• Cluster Density R2

At the end of this step, the pool of samples is automatically advanced to (and queued for) the Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) step.

Step 4: Demultiplexing (NextSeq 1000/2000 Sequencing v2.3)

This step is a semi-automated step.

• The DRAGEN version must be earlier than v3.9.3.

• The samples in the run must not repeat.

• The run must be for the single lane flow cell (P1 or P2).

The integration starts the step automatically and demultiplexing data from the GenerateFASTQ secondary analysis is parsed back to Clarity LIMS. The lab scientist reviews the demultiplexing result parsed into Clarity LIMS, assigns QC flags, and completes the step.

Master Step Fields

The following table lists the 12 master step fields that are configured on the Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) master step.

Master Step Field Configuration for Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) Step

Global Fields

The following table lists the global fields that are configured on the Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) step. These fields are used to display the demultiplexing result metrics for individual library in the sample pool.

Global Field Configuration for Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) Step

How the NextSeq 1000/2000 Integration Works

Sample Sheet Generation and Planned Run Creation

Run Status, Primary Metrics, and Analysis Results Parsing and Recording

After the sequencing run is started on the instrument, NextSeq 1000/2000 Control Software (NCS) notifies BaseSpace Sequence Hub (BSSH) that the sequencing run has started.

BSSH then does the following actions:

• Local run mode: Creates a run on the BSSH database with the run configuration information received from NCS.

• Hybrid/Cloud run mode: Creates a run on the BSSH database by copying the run configuration information from the planned run created in ICA, using a special Cloud Run ID received from NCS.

After the run is created on BSSH, a run update event message that carries the RunStarted equivalent message is created on BSSH and sent to the integration service which processes the message.

Based on the reagent cartridge barcode information in the event message (refer to the following example), the integration looks in the queue of AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3) step for a container with the same reagent cartridge barcode. When identified, the step is started automatically and the Run Status field on Record Details screen is updated to RunStarted. The integration then updates all the other step fields (eg, Current Read, Output Folder) by parsing the details from the RunParameters.xml uploaded to BSSH by NCS.

How the Integration Service Handles Run Status

The NextSeq 1000/2000 Control Software (NCS) continues to notify BaseSpace Sequence Hub (BSSH) of any run status change throughout the course of the sequencing run. The run update event delivery process continues as described in the previous section.

The following table lists the run status details and how the integration service handles each status.

Start A Sequencing Run On Instrument With Different Run Modes

• Configuring a run with the Proactive, Run Monitory and Storage option. Local runs are done in Local mode, and cloud or hybrid runs are done in Cloud or Hybrid mode.

• Starting a local, cloud, or hybrid run.

Components Installed

This section describes the various components that are installed by default as part of this integration.

Reagent Categories/Label Groups

• TruSeq HT adapters v2 (D7-D5)

Reagent Kits

• Resuspension Buffer (RSB)

• NextSeq 1000/2000 reagent cartridge

Container Types

• Tube

• 96-well plate

• NextSeq 1000/2000 reagent cartridge

Control Types

• PhiX v3

This integration supports the NextSeq 1000/2000 reagent cartridge with barcode provided in the format [A-Za-z]{2}[0-9]{7}-[A-Za-z0-9]{4} (eg, EC1234567-EC03).

Enabling Planned Run Generation for Samples Having Duplicate Name with Different Indexes

Re-queue Samples for Cloud Run

Clarity LIMS requires unique library names when samples are re-queued to the workflow in the NextSeq 1000/2000 integration. Assign unique names to libraries after going through library preparation.

The following steps are used to configure the library preparation workflow correctly before routing the samples for re-queuing:

1. From Configuration, select the Lab Work tab.

2. Search for the library preparation workflow used for the re-queued samples.

3. In the Master Step of the library preparation workflow, modify the naming convention under Step Type to generate unique library names (E.g., appending LIMS ID to the default naming convention, like OutputItemLIMSID).

4. Select Save.

Rules and Constraints

• The workflow configuration contains several validation checks. To make sure that the calculations work properly, it is important that you do not disable any of this validation logic. The validation checks determine:

  • Which samples, and how many, can enter each step together.

  • Which samples, and how many, can be pooled together.

• The NextSeq 1000/2000 reagent cartridge barcode must be unique. There should not be multiple NextSeq 1000/2000 reagent cartridge containers in the system with the same name.

• Reagent labels (indexes) must be unique.

• One library pool can only contain one library or control with no label (index).

• Do not manually start or complete the AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3) step. This step is a fully automated step, and the integration service does not update samples correctly if they have been manually started.

• Do not manually start the Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) step. This step is semi-automated, and the integration service does not update the demultiplexing result correctly if they have been manually started.

• For the automated run to start successfully, select Validate Run Setup and Create Planned Run in the Load to Reagent Cartridge step.

• The NextSeq 1000/2000 Reagent Cartridge barcode should not be modified after it has been successfully validated. Modifying the barcode would result in Clarity failing to update subsequent steps and sample details properly.