The documents in this section support the Illumina MiSeq i100 Series integrations with on-premise Clarity LIMS setup.

The integration includes the following:

• Preconfigured MiSeq i100 Series On-Prem Sequencing v1.0 workflow that maps to lab protocols and instrument runs.

• Preconfigured protocols:

  • Assign Analysis Configuration Template (MiSeq i100 Series On-Prem Sequencing v1.0)

  • Make Bulk Pool (MiSeq i100 Series On-Prem Sequencing v1.0)

  • Dilute and Denature (MiSeq i100 Series On-Prem Sequencing v1.0)

  • Load to Dry Cartridge (MiSeq i100 Series On-Prem Sequencing v1.0)

  • AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0)

  • AUTOMATED - Analysis Run (MiSeq i100 Series On-Prem Sequencing v1.0)

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

• Automated planned run creation on MiSeq i100 Series instrument via Illumina Run Manager. The planned run created will be retrieved by MiSeq i100 Series Control Software and used to start a sequencing run.

• Automated tracking of the MiSeq i100 Series sequencing run status and parsing of run metrics.

• Automated tracking of the MiSeq i100 Series analysis run status, result summary and demultiplexing result files.

• Support for configuration of secondary analysis with library prep kit (LPK) and index adapter kit (IAK) via Analysis Configuration Template for the planned run. The analysis is performed by DRAGEN on-premise.

• (Optional) Preconfigured Library Prep Validation v2.3.5 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 MiSeq i100 Series On-Prem Sequencing v1.0 workflow.

Last Updated: December 2024

Release Date: December 2024

These Release Notes describe the key changes to software components for the Clarity LIMS MiSeq i100 Series On Premise Integration Package version 1.0.0.

Compatibility

Refer to Compatibility under Instruments & Integrations.

New Features

Features provided with this release of the integration package are as follows.

• Provides integration between the MiSeq i100 Series instrument and on-premise Clarity LIMS.

• Supports creation of planned run with secondary analysis configuration and generation of v2 sample sheet via Illumina Run Manager on instrument.

• Supports automated tracking of the sequencing run and parsing of run metrics for on-premise Clarity LIMS.

• Supports automated tracking of local analysis for analysis status and demultiplexing results.

• New MiSeq i100 Series On-Prem Sequencing v1.0 workflow available from Illumina Preset Protocols v2.10.

Known Limitations

• Tracks only the analysis configured with the planned run. Does not track analysis requeue or analysis triggered externally with the same planned run.

• The Assign Analysis Configuration Template step does not support pooled libraries.

• The integration does not support --bcl-sampleproject-subdirectories option of BCL Convert.

• The integration does not support pools with samples assigned with Analysis Configuration Templates from different instruments.

Known Issues

• On the Make Bulk Pool step, the log displays a warning when the Calculate Volume automation is triggered and at least one pool consists of multiple inputs. This issue is caused by the output custom field being reset multiple times at the end of the automation. This issue does not affect the Calculate Volume automation functionality.

The Illumina MiSeq i100 Series On-Prem Integration Package v1.0.0 supports the integration of Clarity LIMS to MiSeq i100 Series instruments that are on-premise via Illumina Run Manager.

Prerequisites

Installation Steps

Steps:

1. Install / Upgrade Clarity LIMS

2. Install and Configure Illumina Run Manager Integration Service

3. Install MiSeq i100 Series On-Prem Integration RPM

4. Install Illumina Preset Protocols (IPP) v2.10 RPM and Import Required IPP Workflows.

Step 1: Install / Upgrade Clarity LIMS

Install or upgrade to Clarity LIMS v6.2 or later. If you are using an older version of Clarity LIMS, please contact Illumina Support (techsupport@illumina.com) to schedule for upgrade.

Step 2: Install and Configure Illumina Run Manager Integration Service

Install and configure Illumina Run Manager Integration Service v1.0, please refer to Illumina Run Manager v1.0.0 Installation and User Interaction for details.

Step 3: Install MiSeq i100 Series On-Prem Integration RPM

Install ClarityLIMS-MiSeqi100Series-OnPremise RPM using yum or your preferred package manager.

Example with YUM:

yum install ClarityLIMS-MiSeqi100Series-OnPremise

Example with RPM:

rpm -i ClarityLIMS-MiSeqi100Series-OnPremise-{version}.86_64.rpm

Step 4: Install Illumina Preset Protocols (IPP) v2.10 and Import Required IPP Workflows

Install BaseSpaceLIMS-Illumina-Preset-Protocols RPM using yum or your preferred package manager.

Example with YUM:

yum install BaseSpaceLIMS-Illumina-Preset-Protocols

Example with RPM:

rpm -i BaseSpaceLIMS-Illumina-Preset-Protocols-{version}.86_64.rpm

Upon successful installation of IPP v2.10.0, install the following workflow configurations:

• MiSeq i100 Series On-Prem Sequencing v1.0 workflow

• (Optional) Library Prep Validation v2.3.5 workflow

For detailed options and steps on how to install IPP workflows and protocols, Installer Parameters and Operations section of Illumina Preset Protocols v2.10 Installation and User Configuration.

The Illumina MiSeq i100 Series On-Prem Integration Package v1.0.0 supports the integration of Clarity LIMS to Illumina MiSeq i100 Series sequencer connected to the Illumina Run Manager platform on premise.

For instructions on validating and troubleshooting the Illumina MiSeq i100 Series On-Premise Integration, refer to MiSeq i100 Series On-Premise Integration v1.0.0 User Interaction, Validation and Troubleshooting.

Prerequisites and Assumptions

Analysis Configuration Template

The MiSeq i100 Series On-Prem Sequencing v1.0 workflow uses analysis configuration templates (ACT) to configure secondary analysis for the planned runs. It is assumed that the required ACTs for your run has been pre-created using Illumina Run Manager UI. Please make sure that the index adapter kit (label group on Clarity) selected in the ACT has already been created on Clarity LIMS. The same label group shall be used in the library preparation step. Names of ACT must be unique. Please refer to the MiSeq i100 Series On-Premise Integration v1.0.0 User Interaction, Validation and Troubleshooting for details on creating ACT on Illumina Run Manager User Interface (UI).

Samples

It is assumed that samples that enter the MiSeq i100 Series On-Prem Sequencing v1.0 workflow have gone through library preparation and quantification processes before they are assigned to the workflow:

• Samples have been accessioned into Clarity LIMS.

• Sample and library names must contain only alphanumeric, dash, or underscore characters.

• Samples have been run through QC and library prep.

• Samples have the Molarity (nM) global field set to a valid value (required for the 'Calculate Volumes' automation in the Make Bulk Pool step).

Workflows, Protocols, and Steps

The MiSeq i100 Series On-Premise Integration Package v1.0.0 includes the following workflows:

• MiSeq i100 Series On-Prem Sequencing v1.0

• Library Prep Validation v2.3.5 (optional, but recommended for validation purposes)

The following describes the protocols and steps included in these workflows.

Library Prep Validation v2.3.5 Workflow

MiSeq i100 Series On-Prem Sequencing v1.0 Workflow

Validation Workflow

The Library Prep Validation v2.3.5 workflow allows for validation of the system after installation is complete. This workflow can be replaced by other custom library preparation workflows. For details, refer to MiSeq i100 Series On-Premise Integration v1.0.0 User Interaction, Validation and Troubleshooting.

[Optional] Configure a custom library preparation workflow. Routing of libraries from the custom workflow to the MiSeq i100 Series On-Premise Sequencing protocol can be enabled in the master step with the routing script available in the NGS package.

Step 1: Assign Analysis Configuration Template (MiSeq i100 Series On-Prem Sequencing v1.0)

In this step, secondary analyses are configured for the samples using ACTs. Each ACT contains details related to a particular secondary analysis (e.g., index adapter kit to use, reference genome setting). At this step, the user selects an ACT from a list of pre-constructed ACTs and assigns samples to it. A planned run can involve multiple ACTs and users have to repeat Step 1 for each ACT that is required for the planned run. For more information on creating ACTs and assigning samples to them, refer to MiSeq i100 Series On-Premise Integration v1.0.0 User Interaction, Validation and Troubleshooting.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -exp 'submittedSamples.each { sample -> if ( !sample.name.matches( ::[A-Za-z0-9_-]+:: ) ) { fail ( ::Submitted sample :: + sample.name + :: should only have alphanumeric, dash, and underscore characters. Please rename and try again.:: ) } }' -log {compoundOutputFileLuid1}

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/miseqi100series-onprem/automation/miseqi100series-onprem-automation.jar script:retrieve_act_list -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -n 'INSTRUMENT_USED' -t true

Master Step Fields

The following table lists the configuration details for custom fields that are defined on the Assign Analysis Configuration Template (MiSeq i100 Series On-Prem Sequencing v1.0) step.

Assign Analysis Configuration Template (MiSeq i100 Series On-Prem Sequencing v1.0) Master Step Field Configuration

Global Fields

The following lists the global custom fields that are configured to display on the Assign Analysis Configuration Template (MiSeq i100 Series On-Prem Sequencing v1.0) step. See Global Fields section for detail configuration.

• Molarity (nM)

• ACT Name

Step 2: Make Bulk Pool (MiSeq i100 Series On-Prem Sequencing v1.0)

Samples are pooled in the Make Bulk Pool (MiSeq i100 Series On-Prem Sequencing v1.0) step. You can manually create working pools based on the final loading concentration required.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp 'output.::Instrument ID:: = input.::Instrument ID::' -log {compoundOutputFileLuid1} && \
/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/miseqi100series-onprem/automation/miseqi100series-onprem-automation.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -m PerPool -n 'INPUT.Instrument ID' script:validate_analysis_config  script:validate_physical_logical_configurations

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} -log {compoundOutputFileLuid1} \
script:evaluateDynamicExpression -exp 'if (!input.hasValue(::Molarity (nM)::)){ return; }; output.::Run Mode:: = input.::Run Mode::; nextStep = ::ADVANCE::;'"

Master Step Fields

The following table lists configuration details for the custom fields that are defined on the Make Bulk Pool (MiSeq i100 Series On-Prem Sequencing v1.0) step.

Make Bulk Pool (MiSeq i100 Series On-Prem Sequencing v1.0) Master Step Field Configuration

Global Fields

The following lists the global custom fields that are configured to display on the Make Bulk Pool (MiSeq i100 Series On-Prem Sequencing v1.0) step. See Global Fields section for detail configuration.

• Final Loading Concentration (pM)

• RSB Volume (ul)

• MiSeq i100 Series Flowcell Type

Step 4: Load to Dry Cartridge (MiSeq i100 Series On-Prem Sequencing v1.0)

In this step, the user scans the dry cartridge barcode into the LIMS, and then manually places the working pools into the dry cartridge to be used in the MiSeq i100 Series run. In addition, this step validates the run setup and analysis information and generates samplesheet file and/or creates a planned run on instrument's Illumina Run Manager UI.

script:validateSampleCount -min 1 -max 1

 script:validate_output_containers -r 'MiSeq i100 Series Dry Cartridge:SC[0-9]{7}-SC3' -min 1 -max 1

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -t false -h false -exp 'nextStep = ::ADVANCE::' -log {compoundOutputFileLuid1}

Master Step Fields

The following table shows the master step fields that are configured on the Load to Dry Cartridge (MiSeq i100 Series On-Prem Sequencing v1.0) step. These fields are required for sample sheet generation and planned run creation in Illumina Run Manager.

Load to Dry Cartridge (MiSeq i100 Series On-Prem Sequencing v1.0) Master Step Field Configuration

Step 5: AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0)

In this step, the pooled samples in the dry cartridge are sequenced on the MiSeq i100 Series instrument.

There are no automation associated to this step.

Master Step Fields

The following tables show the master step fields that are configured on the AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0) step.

Master Step Field Information

All the fields above are Read-Only Text field, except Sequencing Log is a Read-Only Multiline Text.

Global Fields

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

• % Aligned R1

• % Aligned R2

• % Bases >=Q30 R1

• % Bases >=Q30 R2

• % Error Rate R1

• % Error Rate R2

• % Occupied

• % Phasing R1

• % Phasing R2

• % Prephasing R1

• % Prephasing R2

• % PF

• Intensity Cycle 1 R1

• Intensity Cycle 1 R2

• Reads PF

• Yield (Gb) R1

• Yield (Gb) R2

At the end of the step, the pools of samples are automatically route to next step.

Step 6: AUTOMATED - Analysis Run (MiSeq i100 Series On-Prem Sequencing v1.0)

Data from the analysis is parsed back to Clarity LIMS. In this step, the secondary analysis configured using the Analysis Configuration Template (ACT) is performed on DRAGEN on-premise.

There are no automation associated to this step.

Master Step Fields

The following tables show the master step fields that are configured on the AUTOMATED - Analysis Run (MiSeq i100 Series On-Prem Sequencing v1.0) step.

AUTOMATED - Analysis Run (MiSeq i100 Series On-Prem Sequencing v1.0) Master Step Field Information

All the fields above are Read-Only Text field, except Log is a Read-Only Multiline Text.

How the Integration Works

The following information summarizes how the MiSeq i100 Series On-Premise Integration works.

• After the 'Validate Run Setup and Create Planned Run' automation is triggered on the Load to Dry Cartridge (MiSeq i100 Series On-Prem Sequencing v1.0) step, the run parameters entered in the Run Details screen and the sample informations are sent to Illumina Run Manager. Illumina Run Manager validates the run and analysis configuration before the planned run is created, the automation also generates the sample sheet and attaches it to the step. In the event of incomplete information or misconfiguration on the planned run, an error will be displayed on the automation banner and details of the error will be logged in the automation log file.

• When the sequencing run starts on the instrument, the MiSeq i100 Series Control Software notifies the Illumina Run Manager integration. The events are processed and the integration service retrieves the run information from MiSeq i100 Illumina Run Manager. This information is used to populate the custom fields in the AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0) step.

• Other run events follow the same information flow. When sequencing is complete, the control software uploads the sequencing run data (primary metrics). Then, Illumina Run Manager integration retrieves the primary metrics and uses them to populate the fields in the Sample Details table (e.g., % Error Rate R1). The custom fields (e.g., Run Status, Current Read, and so on) on the AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0) step are updated using the run related information. If the sequencing run is successfully completed, the step automatically completes.

• The integration tracks the analysis events and results in the AUTOMATED - Analysis Run (MiSeq i100 Series On-Prem Sequencing v1.0) step. The secondary analysis results are in the external storage configured in Illumina Run Manager. The external storage information is found in the External Storage for Analysis Results configuration settings in Illumina Run Manager.

Start a Sequencing Run on MiSeq i100 Series Instrument

To start a planned run, refer to information on starting a planned run in the Illumina support site..

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.

Components Installed

The following sections describe the components (files, properties, reagent categories / label groups, reagent kits, and containers) that are installed by default as part of this integration.

Global Fields

Container Global Fields

Derived Sample Global Fields

Reagent Kits

• MiSeq i100 Series Dry Cartridge

• MiSeq i100 Series Library Denature (KLD)

• MiSeq i100 Series Wet Cartridge

• Resuspension Buffer (RSB)

Container Types

• MiSeq i100 Series Dry Cartridge

Instrument Types

• MiSeq i100 Series

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 the following information:

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

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

• All submitted samples must have an associated secondary analysis that is configured using the analysis configuration template (ACT). The ACT must be configured on MiSeq i100 Illumina Run Manager before starting the Assign Analysis Configuration Template step. The ACT names must be unique.

• Assign Analysis Configuration Template step does not support pool library as input.

• The dry cartridge barcode must be unique. There must not be multiple dry cartridge containers with the same name in the Clarity LIMS system.

• Reagent labels, or indexes, must be unique.

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

• The AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0) and AUTOMATED - Analysis Run (MiSeq i100 Series On-Prem Sequencing v1.0) steps must not be manually started or completed. These steps are fully automated and the integration service does not update samples correctly if they have been manually started.

• For the automated run to start successfully, you must click 'Validate Run Setup and Create Planned Run' in the Load to Dry Cartridge step and the automation must be ran successfully.

This section explains how to validate the installation of the Illumina MiSeq i100 Series On-Prem Integration Package v1.0.0.

The validation process involves the following actions:

• Running samples through the Library Prep Validation v2.3.5 workflow.

  • The workflow contains a single-step protocol that models the library prep required to produce libraries tagged with index sequences. At the end of the step, these libraries are routed to MiSeq i100 Series On-Prem Sequencing v1.0 workflow.

• Running libraries through the MiSeq i100 Series On-Prem Sequencing v1.0 workflow validates the following items:

  • Successful sequential step advancement of samples through the steps of the workflow.

    • Assign Analysis Configuration Template (MiSeq i100 Series On-Prem Sequencing v1.0)

    • Make Bulk Pool (MiSeq i100 Series On-Prem Sequencing v1.0)

    • Load to Dry Cartridge (MiSeq i100 Series On-Prem Sequencing v1.0)

    • AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0)

    • AUTOMATED - Analysis Run (MiSeq i100 Series On-Prem Sequencing v1.0)

  • Automated creation of a planned run on Illumina Run Manager (IRM).

    • Automatic validation of run setup information before planned run creation.

    • Automated generation of sample sheet as part of the planned run creation.

  • Automated tracking of the MiSeq i100 Series sequencing run and parsing of run statistics from Illumina Run Manager into the LIMS.

  • Automated tracking of the MiSeq i100 Series analysis run and demultiplexing result files from Illumina Run Manager into the LIMS.

The validation steps assume the following conditions have been met:

• Analysis configuration templates (ACTs) are created in Illumina Run Manager for your run.

• Index Adapter Kit used in selected ACT is created as label group in Clarity LIMS.

• This same label group must be used in the Library Prep Validation v2.3.5 step or your custom library prep step.

• Required DRAGEN analysis application is installed locally on instrument.

• The MiSeq i100 Series instrument to be integration is connected successfully via Illumina Run Manager UI.

Analysis Configuration Template Creation in Illumina Run Manager

MiSeq i100 Series On-Prem Sequencing v1.0 workflow requires Analysis Configuration Templates (ACTs) for configuring secondary analysis.

Create these ACTs in Illumina Run Manager (IRM) UI:

1. Select Analysis Configuration Templates from the dropdown list of the IRM UI menu icon

2. Select Add analysis template to create and add a new ACT.

3. Fill up details of the ACT and select Save.

For details, refer to the Illumina Run Manager help documentation on Illumina support site.

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 v2.3.5 and MiSeq i100 Series On-Prem Sequencing v1.0 workflows.

1. On the Configuration tab, under Workflows, activate both the Library Prep Validation v2.3.5 and MiSeq i100 Series On-Prem Sequencing v1.0 workflows.

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

   ⚠ Sample and library names must use only alphanumeric, dash, or underscore characters. Invalid characters can cause a sample sheet validation failure in the Load to Dry Cartridge step.

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

Library Prep Protocol

This single-step protocol models the library prep required to produce libraries tagged with index sequences that are ready for the MiSeq i100 Series On-Prem Sequencing v1.0 workflow.

• Label Group = same as the Index Adapter Kit selected in the ACT that is being used

• Sequencing Instrument = MiSeq i100 Series On-Prem

On exit from the step, the Routing Script automation is triggered. This automation assigns samples to the first step of the MiSeq i100 Series On-Prem Sequencing v1.0 workflow, Assign Analysis Configuration Template (MiSeq i100 Series On-Prem Sequencing v1.0).

Protocol: MiSeq i100 Series On-Prem Sequencing v1.0

The MiSeq i100 Series On-Prem Sequencing v1.0 protocol consists of the following steps:

• Assign Analysis Configuration Template (MiSeq i100 Series On-Prem Sequencing v1.0)

• Make Bulk Pool (MiSeq i100 Series On-Prem Sequencing v1.0)

• Load To Dry Cartridge (MiSeq i100 Series On-Prem Sequencing v1.0)

• AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0)

• AUTOMATED - Analysis Run (MiSeq i100 Series On-Prem Sequencing v1.0)

Step 1: Assign Analysis Configuration Template (MiSeq i100 Series On-Prem Sequencing v1.0)

1. In Lab View, locate the MiSeq i100 Series On-Prem Sequencing v1.0 protocol. The samples are queued for the Assign Analysis Configuration Template (MiSeq i100 Series On-Prem Sequencing v1.0) step.

2. Add the samples to the Ice Bucket and start the step. The Validate Sample Names automation is triggered upon step started, and validates the sample names.

3. Select the connected MiSeq i100 instrument to integrate with.

4. On Record Details screen, select 1. Retrieve ACT List to retrieve the list of ACTs created on Illumina Run Manager.

6. [Optional] Select 2. Retrieve ACT Information to trigger the automation.

   The Retrieve ACT Information automation retrieves the details of the selected ACT. High level information e.g., Library Prep Kit, Index Adapter Kit, Reference Genome etc. are populated on the step fields. For more details, refer to the ACTMetadata.csv file in the file placeholder section.

7. Select Next Steps to assign the ACT to the samples.

   This action triggers the Validate Reagent Labels and Apply Selected ACT to Samples and Set Next Step automation. This automation

   • ensures every sample has Molarity (nM) UDF value greater than zero

   • validates that the indexes applied to the libraries are valid for the selected ACT

   • assigns all samples in this step to the selected ACT

8. Select Finish Step.

Step 2: Make Bulk Pool (MiSeq i100 Series On-Prem Sequencing v1.0)

1. In Lab View, locate the MiSeq i100 Series On-Prem Sequencing v1.0 protocol. The samples are queued for the Make Bulk Pool (MiSeq i100 Series On-Prem Sequencing v1.0) step.

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

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

4. Create a pool of samples as follows.

   1. On the Pooling screen, create a pool by dragging samples into the Pool Creator.

   1. Enter a name for your pool or accept the default name (Pool #1).

   1. [Optional] If multiple pools are required, select the plus sign (+) next to Pool Creator to create a pool.

   ℹ Only a single pool is allowed in the Load to Dry Cartridge step for each run. Create multiple pools if required for subsequent runs.

   1. [Optional] To remove a pool, select the X in the top right corner of the pool.

5. Select Record Details to trigger the Validate Analysis Configurations automation.

   This automation performs the following checks on the analysis configuration for each pool:

   • Pooled samples are within the maximum configuration limit.

   • Pooled samples assigned with the same secondary analysis application must have the same analysis version (e.g. v3.8.4) and settings (e.g. Map/Align Output Format = CRAM).

6. [Optional] In the Sample Details table, select the pool next to the sample name to view details on the pool composition.

1. On the Record Details screen, navigate to the Reagent Lot Tracking section to track the lot information used in the step.
2. In the Step Details area, complete the following fields:

   • [Optional] % PhiX (100pM) Spike-In — The percentage of PhiX spike-in.

   • Final Loading Concentration (pM) — The final loading concentration of the pool in the flow cell.

   • Final Loading Volume (ul) — The final loading volume of the pool in the flow cell.

   • Bulk Pool Volume (ul) — The bulk pool volume is used in the Calculate Volumes script to determine RSB volumes required for pooling.

   • Minimum Per Sample Volume (ul) — The minimum volume for each sample. This field is prepopulated with the configured default value (2 µl) and can be edited. If the per sample volume is below the value set in this field, the Calculate Volumes script applies a rounding factor to affected samples so that the volume reaches the minimum volume.

   • Flowcell Type — The flow cell type that the run uses. Select from the following options:

     • 5M

     • 25M

3. Select Calculate Volumes to trigger the Calculate Volumes automation.

   This automation calculates the volumes required for each library to form a pool that has the concentration and volume specified in the Step Details field. It also generates the calculation file in a CSV format and attaches it to the step. Select the file name to download it.

4. Select Next Steps to trigger the Set Next Step automation.

   This automation sets the next step for samples to ADVANCE, which moves them to the Load to Dry Cartridge (MiSeq i100 Series On-Prem Sequencing v1.0) step.

5. Select Finish Step.

Step 4: Load to Dry Cartridge (MiSeq i100 Series On-Prem Sequencing v1.0)

1. In Lab View, locate the MiSeq i100 Series On-Prem Sequencing v1.0 protocol. The pool of samples are queued for the Load to Dry Cartridge (MiSeq i100 Series On-Prem Sequencing v1.0) step.

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

   ⚠ Only single pool is allowed at this step.

3. On the Ice Bucket screen, select the destination container type:

   • MiSeq i100 Series Dry Cartridge

4. Select Begin Work to trigger the Validate Single Pool automation.

   • The Validate Single Pool automation ensures that only one pool is selected for loading to the dry cartridge in this step.

5. On the Placement screen, do as follows.

   1. Drag the pool into the Placed Samples area on the right.

   2. Scan or type the barcode of the dry cartridge into the container name field.

   3. Select Record Details.

   After exiting the Placement screen, the Validate Dry Cartridge Barcode automation makes sure that the dry cartridge barcode conforms to the barcode mask. The barcode mask is SC[0-9]{7}-SC3.

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

   The analysis related information for the planned run comes from the ACT associated with the samples. Refer to the following table for details.

   Fields Displayed on Record Details Screen of Load to Dry Cartridge (MiSeq i100 Series On-Prem Sequencing v1.0) Step

   ¹ The Local run mode sends the Run/Analysis configuration to Illumina Run Manager. This configuration is downloaded to the MiSeq i100 Series instrument through Illumina Run Manager to start the run. The downstream secondary analysis is done on premise.

   ² The custom value must correspond to the longest index sequence of the samples in the pools in the library tube strip. Otherwise, the planned run creation fails and an error message displays.

7. Select Validate Run Setup and Create Planned Run to trigger the automation script. The script performs the following actions:

   • Validates the parameters entered on the Record Details screen.

   • Creates the planned run on Illumina Run Manager.

   • Generates the sample sheet and attaches it to the placeholder in the Files area on the Record Details screen.

8. Select Next Steps.

   On the Assign Next Steps screen, the next step for the pooled samples is set to the AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0) step.

Step 5: AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0)

User interaction is not required, but you can review the stages of the step in Clarity LIMS.

Review Run Data

Read summary metrics are recorded for the library pool. After the run is complete, open the step and review these metrics in the Step Details section and the Sample Details table.

Step 6: AUTOMATED - Analysis Run (MiSeq i100 Series On-Prem Sequencing v1.0)

User interaction is not required, but you can review the stages of the step in Clarity LIMS. If the run analysis tracking is successful, the integration completes the step automatically.

Review Analysis Data

After the analysis run is complete, open the step and review the following values in the Step Details section:

• Analysis Status - Status of the analysis run

• Analysis Result Location - Location of the secondary analysis data. This field is empty if transfer of secondary analysis data to an external location is disabled.

• Log - Log message when integration handling the event from IRM

File placeholder:

• Demultiplexing results file - demultiplexing statistics for each sample

At this point, the entire MiSeq i100 Series On-Prem Integration workflow is fully validated.

Troubleshooting

Validate Analysis Configuration Automation Check Fails

The Validate Analysis Configuration automation check is in the Make Bulk Pool (upon pooling) step. If a failure happens, an error message displays and the step can be aborted, or you might be prevented from continuing the step.

This check can fail for the following reasons:

• Pooled samples assigned with the same secondary analysis application must have the same analysis version (e.g. v3.8.4) and settings (e.g. Map/Align Output Format = CRAM).

  Resolve this error as follows.

  1. Pool the samples again. Make sure that samples with conflicting secondary analysis application, analysis versions or analysis settings, are in different pools.

Error When Creating Planned Runs

If you receive an error when creating a planned run, check the log message in the Load to Dry Cartridge step to identify the issue. If you cannot correct the issue, contact Illumina Support.

Incompatible Analyses in a Planned Run

Only compatible analysis versions should be combined in a single planned run. When incompatible analysis versions are combined, an error log message displays. An example of the error log message is shown below.

To resolve this error, check the ACTs that were used for the run. Create a new planned run with ACTs that have the same analysis and analysis versions.

Automated Step Does Not Start

If the AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0) step does not start, Clarity LIMS is likely not receiving sequencing run events from Illumina Run Manager correctly. Check for the following issues:

• The instrument must be reflected as Connected and Active in the Illumina Run Manager Integration UI. Reconnect the instrument if the status reflects otherwise.

• The container must be queued at the AUTOMATED - Sequencing Run step. It should not be in the ice bucket or in the previous step.

If the issues remain, contact Illumina Support.

Automated Step Starts, But Does Not Complete

If the AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0) step starts, but does not complete, do as follows.

1. Log in to the default user account and search for the in progress step in Clarity LIMS using the container name as the search term.

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

   If unable to reach the Record Details screen, or if the Sequencing Log field does not contain enough information to resolve the issue, contact Illumina Support. Provide the relevant information from the troubleshooting steps already performed.