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.
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.
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.
MiSeq i100 On-Prem v1.0.0
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:
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.
The integration does not support pools with samples assigned with Analysis Configuration Templates from different instruments.
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.
Installation
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
Prerequisite 1: Clarity LIMS v6.2 or later with SecretUtil v1.5.0.12 or later
The MiSeq i100 Series On-Prem integration v1.0 relies on secret management util version 1.5.0.12 or later to store the access token to Illumina Run Manager and its related secrets.
ℹ If using Vault, ensure that the appropriate ACL policies are added. Refer to Authentication ACL Policies under On-Premise Deployments > Configure Your HashiCorp Vault in .
Prerequisite 2: Illumina Run Manager Integration Service v1.0.0 or later
Illumina Run Manager Integration Service v1.0 or later is required for this integration.
Prerequisite 3: Illumina Preset Protocols (IPP) v2.10 with NGS Commons Package v5.25.0 or later
Illumina Preset Protocols (IPP) v2.10 package contains the workflow configuration and template files required to enable the MiSeq i100 Series On-Prem workflow to run properly.
Install the workflow configurations below on the same server on which you intend to install the MiSeq i100 Series On-Prem integration.
MiSeq i100 Series On-Prem Sequencing v1.0 workflow
Installation Steps
Steps:
Install / Upgrade Clarity LIMS
Install and Configure Illumina Run Manager Integration Service
Install MiSeq i100 Series On-Prem Integration RPM
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 ([email protected]) 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 for details.
Step 3: Install MiSeq i100 Series On-Prem Integration RPM
Install ClarityLIMS-MiSeqi100Series-OnPremise RPM using yum or your preferred package manager.
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)
The validation steps assume the following conditions have been met:
Integration package has been successfully installed. Refer to .
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.
ℹ For more information on creating a reagent label group, refer to Add and Configure Labels and Label Groups in the
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:
Select Analysis Configuration Templates from the dropdown list of the IRM UI menu icon
Select Add analysis template to create and add a new ACT.
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.
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.
On the Projects and Samples screen, create a project and add samples to it.
âš 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.
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.
Follow the steps in to run the Library Prep Validation workflow with the following:
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)
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.
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.
Select the connected MiSeq i100 instrument to integrate with.
Step 2: Make Bulk Pool (MiSeq i100 Series On-Prem Sequencing v1.0)
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.
Add the samples to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
On the Record Details screen, navigate to the Reagent Lot Tracking section to track the lot information used in the step.
[Optional] Create a new lot. For more information, refer to Add and Configure Reagent Kits and Lots in the .
In the Step Details area, complete the following fields:
Step 4: Load to Dry Cartridge (MiSeq i100 Series On-Prem Sequencing v1.0)
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.
Add the pool to the Ice Bucket and select View Ice Bucket.
âš Only single pool is allowed at this step.
Step 5: AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0)
Do not add samples to the Ice Bucket or start or complete the AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0) step. The integration does this automatically.
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 Details Section
The following values populate the master step fields:
Run Name
Run Status
Sample Details Table
Summary metrics populate the following global custom fields:
% Bases >=Q30 R1
% Bases >=Q30 R2
Step 6: AUTOMATED - Analysis Run (MiSeq i100 Series On-Prem Sequencing v1.0)
Do not add samples to the Ice Bucket or start or complete the AUTOMATED - Analysis Run (MiSeq i100 Series On-Prem Sequencing v1.0) step. The integration does this automatically.
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
Retrieve detailed analysis results from Illumina Run Manager.
At this point, the entire MiSeq i100 Series On-Prem Integration workflow is fully validated.
Troubleshooting
If an automation trigger does not appear to run its corresponding scripts, refer to the Troubleshooting Automation section in the .
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.
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.
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.
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.
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.
. For the adapter sequences for your library prep kits, refer to
.
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.
Assign the samples to the Library Prep Validation workflow.
AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0)
AUTOMATED - Analysis Run (MiSeq i100 Series On-Prem Sequencing v1.0)
On Record Details screen, select 1. Retrieve ACT List to retrieve the list of ACTs created on Illumina Run Manager.
[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.
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
Select Finish Step.
Create a pool of samples as follows.
On the Pooling screen, create a pool by dragging samples into the Pool Creator.
Enter a name for your pool or accept the default name (Pool #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.
[Optional] To remove a pool, select the X in the top right corner of the pool.
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).
[Optional] In the Sample Details table, select the pool next to the sample name to view details on the pool composition.
[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
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.
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.
Select Finish Step.
On the Ice Bucket screen, select the destination container type:
MiSeq i100 Series Dry Cartridge
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.
On the Placement screen, do as follows.
Drag the pool into the Placed Samples area on the right.
Scan or type the barcode of the dry cartridge into the container name field.
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.
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
Field
Description
Run Name
Experiment name.
Only alphanumeric characters, dashes, periods, spaces and underscores are permitted.
Maximum of 255 characters
Run Mode
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.
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.
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.
Select Finish Step to advance the pooled samples to the AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0) step. For more information on how to start the sequencing run for different run modes, refer to MiSeq i100 Series On-Prem Integration v1.0.0 Configuration.
Pool the samples again. Make sure that samples with conflicting secondary analysis application, analysis versions or analysis settings, are in different pools.
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.
The configuration provided in this integration has been established to support MiSeq i100 Series lab processes. Any configuration changes to protocols or workflows (including renaming protocols, steps, and fields) could break the integration.
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 for details on creating ACT on Illumina Run Manager User Interface (UI).
Clarity LIMS trims all leading and trailing spaces from the ACT names and ACTs created in Illumina Run Manager UI with names containing leading and/or trailing spaces may not be recognized properly on Clarity. Please ensure that the ACT do not have leading and/or trailing spaces.
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.
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
Protocol 1: Library Prep Validation v2.3.5
Purpose:
Included for validation purposes only, this protocol models the library preparation steps required to advance samples to the MiSeq i100 Series On-Prem Sequencing v1.0 workflow.
âš The label group (index adapter kit) used for library preparation must be the same as the index adapter kit specified in the ACT that is being used. For more information, refer to
MiSeq i100 Series On-Prem Sequencing v1.0 Workflow
Protocol 1: MiSeq i100 Series On-Prem Sequencing v1.0
Purpose:
This protocol models the lab processes of run setup for starting a MiSeq i100 Series sequencing and secondary analysis run on premise.
Steps:
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 .
[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 available in the NGS package.
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 .
Validate Sample Names Automation
Automatically triggered on entry of the step, this automation validates that the sample names contain only alphanumeric, dash and underscore characters.
1. Retrieve ACT List Automation
Triggered when you select '1. Retrieve ACT List' button on the Record Details screen, this automation retrieves the list of ACTS that has been created using Illumina Run Manager UI.
2. Retrieve ACT Information Automation
Triggered when you select '2. Retrieve ACT Information' button on the Record Details screen, this automation:
Retrieves the details of the selected ACT and populates the step UDFs (e.g., Analysis Version, Library Prep Kit, Index Adapter Kit, Reference Genome)
Validate Reagent Labels and Apply Selected ACT to Samples and Set Next Step Automation
Automatically triggered on exit of the Record Details screen, this automation:
Checks that the molarity of all samples are specified
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.
Validate Analysis Configurations Automation
Automatically triggered on exit of the Pooling screen, this automation performs analysis configuration validity and checks each pool for the following characteristics:
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).
Calculate Volumes Automation
Triggered when you select 'Calculate Volumes' button on the Record Details screen, this automation completes the following actions:
Checks to ensure all samples have molarity values assigned.
Uses Number of Samples in Pool to calculate the volumes needed for the 2 nM intermediate library pools.
Set Next Step Automation
Automatically triggered on exit of the Record Details screen, this automation completes the following actions:
Proceeds only if the sample has a molarity value.
Copies the Run Mode from input to output.
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.
Validate Single Pool Automation
Automatically triggered on entry to the step, this automation checks only single pool is use to start the step.
Validate Dry Cartridge Barcode Automation
Automatically triggered on exit of the Placement screen, this automation validates the dry cartridge barcode to ensure it conforms to the barcode mask SC[0-9]{7}-SC3.
Validate Run Setup and Create Planned Run Automation
Triggered when you select 'Validate Run Setup and Create Planned Run' on the Record Details screen, this automation completes the following actions:
Validates the parameters entered on the Record Details screen. These parameters are used to set up the run, generate the sample sheet file, and create the planned run on Illumina Run Manager.
Set Next Step Automation
Automatically triggered on exit of the Record Details screen, this automation completes the following actions:
Sets the next step for samples to ADVANCE, advancing them to the AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0) step.
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)
Do not add samples to the Ice Bucket, start or complete the AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0) step. The integration will do this automatically.
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
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)
Do not add samples to the Ice Bucket or start and complete the AUTOMATED - Analysis Run (MiSeq i100 Series On-Prem Analysis v1.0) step. The integration does this automatically.
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.
If a planned run with the same sample name and project name (case-insensitive) has been created previously in Illumina Run Manager, the sample sheet generated from the 'Validate Run Setup and Create Planned Run' automation can reflect the original case of the previous sample name. This can cause validation errors for analysis configurations with sample-level settings. To resolve this issue, change the sample name or the project name on Clarity LIMS and run the automation again.
Start a Sequencing Run on MiSeq i100 Series Instrument
To start a planned run, refer to information on .
Enabling Planned Run Generation for Samples Having Duplicate Name with Different Indexes
The library preparation workflow of the samples must be 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
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.
Samples have the Molarity (nM) global field set to a valid value (required for the 'Calculate Volumes' automation in the Make Bulk Pool step).
.
The protocol contains a single step - Library Prep Validation v2.3.5. After this step, a routing script sends the samples to the first step of the MiSeq i100 Series On-Prem Sequencing v1.0 workflow.
Steps:
Library Prep Validation v2.3.5
Assign Analysis Configuration Template (MiSeq i100 Series On-Prem Sequencing v1.0)
The secondary analysis of samples is configured using the ACT.
The labels applied to the samples are validated against the selected ACT to ensure that samples use valid labels.
Make Bulk Pool (MiSeq i100 Series On-Prem Sequencing v1.0)
Samples are pooled and resuspension buffers and reagents are added with the help of the 'Calculate Volumes' automation.
ACT of pooled samples are validated with Validate Analysis Configurations automation to ensure that the secondary analysis configurations remain valid after pooling.
Load To Dry Cartridge (MiSeq i100 Series On-Prem Sequencing v1.0)
The library pools from step 2 are now ready to be loaded to the MiSeq i100 Series dry cartridge.
Run and analysis information is validated.
Samplesheet is generated and planned run is created on MiSeq i100 Illumina Run Manager.
AUTOMATED - Sequencing Run (MiSeq i100 Series On-Prem Sequencing v1.0)
This is a fully-automated step that is started and completed automatically once the sequencing run is started and completed on the instrument side.
All the sequencing run metadata e.g. run configuration, primary run metrics etc. are recorded automatically.
AUTOMATED - Analysis Run (MiSeq i100 Series On-Prem Sequencing v1.0)
This is a fully-automated step that is started and completed automatically once the analysis run is started and completed on DRAGEN on premise.
All the analysis run metadata e.g. status and demultiplexing result files are recorded automatically.
Saves the details into a file stored in the Analysis Configuration Metadata file placeholder, which is available for user to download.
Checks that all samples are indexed, indexes are the same as the selected ACT.
Assign the ACT ID, ACT Name, Run Mode and Instrument ID to all the samples in the step.
Sets the next step for samples to ADVANCE, advancing them to the next step in the protocol - Make Bulk Pool (MiSeq i100 Series On-Prem Sequencing v1.0).
Text
Read Only
Reference Genome
Text
Read Only
Secondary Analysis Mode
Text
Read Only
Calculate the Final Loading Concentration (pM)
Copies Flowcell Type from the step inputs to the step outputs.
Calculates the per sample volume required for each library to make a 2 nM intermediate library pool and sets the total sample volume to zero.
Calculates the PhiX Spike-in volume if specified.
Calculates the adjusted per sample volume for the pools if any of the calculated volume is lesser than the value specified in Minimum Per Sample Volume (ul) step UDF.
Uses the Total Sample Volume (ul) to calculate the RSB volume (ul) required to top up the pools that are needed to create the 2 nM intermediate library pools. The RSB volume is saved to the file in the Calculation File placeholder in the Files section.
Uses the MiSeqi100Series_Bulk_Pool1.csv and MiSeqi100Series_Bulk_Pool2.csv template files to generate a single CSV file. This file contains information about the pools and the samples that they contain. The generated file is stored in the Calculation File placeholder in the Files section.
Resets the Number of Samples in Pool to null before exiting the step.
Sets the next step for samples to ADVANCE, advancing them to the Load to Dry Cartridge (MiSeq i100 Series On-Prem Sequencing v1.0) step.
Flowcell Loading Volume (ul)
Numeric
Required Field
Decimal places displayed: 2
Default: 250
Flowcell Type
Text Dropdown
Required Field
Dropdown Items
5M
25M
Minimum Per Sample Volume (ul)
Numeric
Required Field
Decimal places displayed: 2
Default: 2
Run Name may only contain alphanumeric, dash, underscore or period characters. Spaces are not permitted.
Run Name shall not exceed 255 characters.
Checks the Index 1 Cycles and Index 2 Cycles field values. If Index 2 Cycles is greater than 0, the Index 1 Cycles value must be greater than 0 or an error can occur.
Generates the sample sheet and creates the planned run on Illumina Run Manager. The sample sheet is attached to the step.
Planned Run ID
Text
Read Only
Hidden, use to store created planned run id
Read 1 Cycles
Numeric Dropdown
Required Field
Allow Custom Entries
Range: 1 – 301
Presets
101
Read 2 Cycles
Numeric Dropdown
Required Field
Allow Custom Entries
Range: 0 – 301
Presets
101
Run Mode
Text
Read Only
Default: Local
Run Name
Text
Required Field
Version of the instrument control software.
Instrument ID
Unique identification for the instrument.
Instrument Run ID
Unique identification for the instrument sequencing run.
Instrument Type
Type of the instrument, either MiSeqi100 or MiSeqi100Plus.
Output Folder
Output folder path for the sequencing run data.
Run End Time
Time completed for the sequencing run.
Run Name
Run name user configured for the sequencing run.
Run Start Time
Time started for the sequencing run.
Run Status
Status of the sequencing run.
Sequencing Log
Log messages by the Illumina Run Manager integration while handling the sequencing event from instrument.
Wet Cartridge Expiration Date
Wet cartridge expiration data.
Wet Cartridge Serial Number
Wet cartridge Serial Number.
% 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
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.
Numeric
Read Only
Decimal Places Displayed: 2
% Error Rate R2
Numeric
Read Only
Decimal Places Displayed: 2
% Occupied
Numeric
Read Only
Decimal Places Displayed: 2
% PF
Numeric
Read Only
Decimal Places Displayed: 2
% Phasing R1
Numeric
Read Only
Decimal Places Displayed: 3
% Phasing R2
Numeric
Read Only
Decimal Places Displayed: 3
% Prephasing R1
Numeric
Read Only
Decimal Places Displayed: 3
% Prephasing R2
Numeric
Read Only
Decimal Places Displayed: 3
Intensity Cycle 1 R1
Numeric
Read Only
Intensity Cycle 1 R2
Numeric
Read Only
Reads PF
Numeric
Read Only
Decimal Places Displayed: 2
Yield (Gb) R1
Numeric
Read Only
Decimal Places Displayed: 2
Yield (Gb) R2
Numeric
Read Only
Decimal Places Displayed: 2
Numeric Dropdown
Required
Preset:
225
Instrument ID
Text
Read Only
KLD Volume (ul)
Numeric
Read Only
Decimal Places Displayed: 1
MiSeq i100 Series Flowcell Type
Text Dropdown
Required
Preset:
5M
Molarity (nM)
Numeric
Decimal Places Displayed: 2
Number of Samples in Pool
Numeric
Per Sample Volume (ul)
Numeric
Read Only
Decimal Places Displayed: 2
PhiX Concentration (pM)
Numeric
PhiX Volume (ul)
Numeric
Decimal Places Displayed: 2
Required Bulk Pool Volume (ul)
Numeric
Read Only
Decimal Places Displayed: 1
RSB Volume (ul)
Numeric
Read Only
Decimal Places Displayed: 2
RSB Volume for Intermediate Pool (ul)
Numeric
Read Only
Decimal Places Displayed: 1
Run Mode
Text
Read Only
Total Sample Volume (ul)
Numeric
Read Only
Decimal Places Displayed: 1
Resuspension Buffer (RSB)
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.
Field Name
Field Type
Options
Analysis Configuration Template
Text Dropdown
Allow Custom Entries
Application
Text
Read Only
Application Version
Text
Read Only
Index Adapter Kit
Text
Read Only
Field Name
Field Type
Options
Additional Options and Dropdown Items
% PhiX (100 pM) Spike-In
Numeric
Range: 0 - 100
Bulk Pool Volume (ul)
Numeric
Required Field
Decimal places displayed: 2
Default: 1
Final Loading Concentration (pM)
Numeric Dropdown
Required Field
Allow Custom Entries
Dropdown Items
60
70
80
90
100
110
120
130
140
150
160
170
180
Field Name
Field Type
Options
Additional Options and Dropdown Items
Index 1 Cycles
Numeric Dropdown
Required Field
Allow Custom Entries
Range: 0 – 18
Presets
0
6
8
Index 2 Cycles
Numeric Dropdown
Required Field
Allow Custom Entries
Range: 0 – 18
Presets
0
6
8
Instrument
Text
Read Only
Field Name
Description
Current Cycle
Current completed cycle count.
Current Read
Current completed reads count.
Dry Cartridge Barcode Serial Number
Dry Cartridge Barcode Serial Number.
Dry Cartridge Expiration Date
Flow Cell expiration data.
Flow Cell ID
Serial number of the Flow Cell.
Flow Cell Type
Flow Cell Type. Value can be 5M or 25M.
Field Name
Description
Additional Information
Analysis Result Location
Analysis files final copy out location.
Analysis Run ID
Run ID associate with the Analysis run.
Hidden on Record Details screen by default.
Analysis Status
Status for the the analysis run.
Log
Log messages by the Illumina Run Manager integration while handling the analysis event from instrument.
-exp 'output.::Number of Samples in Pool:: = ::::;' -t true
'if (!step.::Run Name::.matches(::[a-zA-Z0-9-_]+[a-zA-Z0-9-_. ]*::)) { fail(::Run Name contains prohibited characters. Please check to make sure Run Name only contains alphanumeric characters, spaces, dashes, periods and underscores. Run Name must start with alphanumeric, a dash or an underscore::); };
if (step.::Run Name::.length() > 255) { fail(::Run Name shall not exceed 255 characters.::); };
if (step.::Index 2 Cycles:: > 0 && step.::Index 1 Cycles:: == 0) { fail(::Index 1 Cycles cannot be zero if Index 2 Cycles is non-zero::); };
if (output.hasValue(::Number of Samples in Pool::)) { output.::Number of Samples in Pool:: = output.::Number of Samples in Pool:: + 1; } else { output.::Number of Samples in Pool:: = 1; }' -t true
