The following sections explain how to validate the installation of the Illumina MiSeq Integration Package v8.3.0.
The validation involves the following processes:
Running samples through the Library Prep Validation v2.3.4 workflow. The workflow contains a single-step protocol that models the library prep required to produce normalized libraries. At the end of the step, the normalized libraries automatically advance to the MiSeq Sequencing v3.2 workflow.
Running normalized libraries through the MiSeq Sequencing v3.2 workflow. This process validates the following information:
Automated generation of a sample sheet for use with the MiSeq Control Software (MCS) and Local Run Manager (LRM).
Automated tracking of the MiSeq sequencing run and parsing of run statistics into Clarity LIMS, including the following information:
Run status and metrics of sequencing run
Activate Workflow, Create Project, Add and Assign Samples
The following steps prepare Clarity LIMS to run samples through the Library Prep Validation and MiSeq Sequencing v3.2 workflows.
In the Clarity LIMS Configuration area, select each workflow and change the Workflow Status toggle to Active.
On the Projects & Samples screen, create a project. Add samples to the project.
Assign the samples to the Library Prep Validation workflow.
Library Prep Validation Protocol
This single-step protocol models the library prep required to produce normalized libraries that are ready for the MiSeq Sequencing v3.2 workflow.
Follow the steps in to run the Library Prep Validation workflow with the following:
Label Group = TruSeq HT Adapters v2 (D7-D5)
Sequencing Instrument = MiSeq
On exit from the step, the Routing Script automation is triggered. This automation assigns samples to the first step of the MiSeq Sequencing v3.2 workflow, which is Library Pooling (MiSeq v3.2) step.
MiSeq Sequencing v3.2 Protocol
Run Library Pooling (MiSeq v3.2)
In Lab View, locate the MiSeq Sequencing v3.2 protocol. Samples are queued for the Library Pooling (MiSeq v3.2) step. Select the step to proceed to the Queue screen.
On the Queue screen, do as follows.
Add the samples to the Ice Bucket.
Run Denature and Dilute (MiSeq v3.2)
In Lab View, locate the MiSeq Sequencing v3.2 protocol. The pooled samples are queued for the Denature and Dilute (MiSeq v3.2) step. Select the step to proceed to the Queue screen.
On the Queue screen, add the pool to the Ice Bucket, and then select View Ice Bucket.
âš Only one pool is allowed as an input.
Run MiSeq Run (MiSeq v3.2)
In Lab View, locate the MiSeq Sequencing v3.2 protocol. The pool of samples is queued for the MiSeq Run (MiSeq v3.2) step. Select the step to proceed to the Queue screen.
On the Queue screen, add the pool to the Ice Bucket, and then select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
Using Step Details Presets
Most of the custom fields in Step Details are analysis settings used by Local Run Manager (LRM) and are for advanced users only. The following fields are exceptions:
Workflow
Experiment Name
Description
Read 1 Cycles
To use the default analysis settings, select the analysis module from the Preset drop-down list.
The default values populate the respective fields and indicate the following actions are needed:
Required — The field is required for the specific analysis module selected. Replace the word Required with a proper value. For example, the Genome Folder field should contain the directory to the genome folder.
None — The field is not applicable for the specific analysis module selected. Leave the field as is.
For a list of fields that are applicable for the analysis module selected, refer to the Applicable analysis fields for the selected Workflow field in Step Details.
Validating Creation of Event Files
This validation checks the following information:
The destination path is correctly configured.
The instrument computer can access and write to the destination path.
There are no syntax errors in the Clarity LIMS batch file.
Use the following steps to confirm that event files are created by the batch file in the destination path. The steps assume that the default configuration has been successfully imported.
In C:\Illumina\gls, double-click the batch file gls_event_mcs_rta_lrm.bat.
Confirm that an empty event file appears in the configured DESTINATION_PATH.
Manual execution of the batch file produces an output resembling the following example:
Troubleshooting
If an automation trigger does not appear to run the corresponding scripts, see the following topics:
Troubleshooting Automation Worker in the .
Troubleshooting Automation in the .
If an error occurs that does not provide direction on how to proceed, confirm the version of the installed MiSeq Integration Package. To confirm the version, run the
command from the server console.
If the error is related to data parsing, retrieving run results data, or report values not appearing as expected, review the MiseqIntegrator.log file. The file is located at
Additional troubleshooting information for this integration is provided in the Illumina Instrument Integrations FAQ.
If you are unable to resolve the issue, contact the Clarity LIMS support team. Supply the relevant information from the troubleshooting already performed.
Sequencing run parameters
Real-Time Analysis (RTA) v1 run directory location and other run-specific information
In the Add Control Samples panel, add the PhiX v3 control sample to the Ice Bucket.
Select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
The Validate Maximum Number of Samples automation script runs. The maximum number of samples allowed is 1536.
On the Pool Samples screen, do as follows.
Create a pool of samples by dragging samples into the Pool Creator.
Name the pool or accept the default name (Pool #1).
Select Place Samples.
On the Placement screen, do as follows.
Select the pool from the Samples to be Placed area and drag it to the container.
Select Record Details.
On the Record Details screen, select Next Steps.
On the Assign Next Steps screen, next step is automatically set to Denature and Dilute (MiSeq v3.2).
Select Finish Step.
On the Ice Bucket screen, select Begin Work.
The Validate Single Input automation script runs.
On the Placement screen, do as follows.
Scan the MiSeq reagent cartridge barcode into the MiSeq Reagent Cartridge field.
Place the pool of samples into the reagent cartridge.
Select Record Details.
On the Record Details screen, do as follows.
In Reagent Lot Tracking, select the reagent lot used in the step.
If the reagent lot is not listed, add/activate the lot on the Reagents and Controls screen.
Use the Preset drop-down list to help populate the fields in Step Details, as needed.
For more information on presets, refer to . Workflow, Experiment Name, and Read 1 Cycles are required fields.
In the Sample Details table, enter the Final Loading Concentration. Select from the following preset options or enter a different value.
225 (for PCR-free workflows)
400 (for Nano workflows)
Select Validate Run Setup and Generate MiSeq SampleSheet.
Clarity LIMS generates the sample sheet and attaches it and a log file to placeholders in the Files area of the Record Details screen.
Download the files and validate the format and content.
Select Next Steps.
On the Assign Next Steps screen, samples are automatically assigned to the MiSeq Run (MiSeq v3.2) step.
Select Finish Step.
On the Record Details screen, the fields are read-only.
âš If the MiSeq Run master step is configured to track instruments, select the applicable instrument from the Instrument Used dropdown on Record Details screen and select Save.
For more information on adding instruments, refer to Add and Configure Instruments in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
When the run completes, the integration automatically performs the following actions:
Populates the fields.
Attaches files to the Illumina Run Report, Link to Run Folder, Run Parameters, and Run Info placeholders.
Populates the fields in the Sample Details table. For details, refer to .
The Log File is attached after the next step for samples is assigned by the Next Step - Advance automation.
âš Make sure the Illumina Run Report has been attached before continuing.
Select Next Steps.
On the Assign Next Steps screen, the next step is automatically set to Mark protocol as complete.
Refer to Compatibility under Instruments & Integrations.
New Features
Integration-related properties can now be accessed and updated via System Setting in Clarity v6.3. Refer to MiSeq Integration v8.3.0 Configuration for configurable properties.
Defects and Security Vulnerability Fixed
Fixed security vulnerabilities.
Revision History
Version
Changes
3
Renamed Defects Fixed section to Defects and Security Vulnerability Fixed section, and updated section contents.
2
Updated Compatibility section to reference Compatibility matrix table.
The MiSeq Integration Package v8.3.0 only supports Clarity LIMS v6.2 and above.
The Illumina MiSeq Integration v8.3.0 includes the following features:
Preconfigured MiSeq Sequencing v3.2 workflow that maps to lab protocol steps and instrument runs.
Automated sample sheet generation for use with MiSeq Control Software (MCS) and Local Run Manager.
The following supported Local Run Manager analysis modules:
Generate FASTQ
Library QC
Resequencing
DNA Enrichment
DNA Amplicon
Automated tracking of the sequencing run and the parsing of the following run statistics to Clarity LIMS:
Sequencing run metrics
Sequencing run parameters
Real-Time Analysis RTA v1 run directory location (and other run specific information)
Preconfigured Library Prep Validation workflow that is used for validation purposes only. This workflow contains a single step protocol that models the library prep required to produce normalized libraries that are ready for the MiSeq Sequencing v3.2 workflow. MiSeq Integration Package v8.3.0 works with Library Prep Validation v2.3.4, which is installed with Illumina Preset Protocols (IPP) v2.9. For details, refer to MiSeq Integration v8.3.0 User Interaction, Validation and Troubleshooting
MiSeq Integration v8.3.0 runs as independent service and can be on the same server as other Clarity LIMS integrations. The integration is only required for MCS v4.0. If you are running MCS v3.0 or below, do not upgrade to this integration.
Installation
The Illumina MiSeq Integration Package v8.3.0 supports the integration of Clarity LIMS to MiSeq instruments.
MiSeq Integration v8.3.0 runs as independent service and can be on the same server as other Clarity LIMS integrations. The integration is only required for MCS v4.0. If you are running MCS v3.0 or below, do not upgrade to this integration.
Compatibility
MiSeq Integration v8.3.0 is compatible with the following software:
Clarity LIMS v6.2.0 and later
Secret Util v1.0 and later
IPP v2.9 and later
MCS v4.0/LRM v3.0 and later
Oracle Linux (for compatibility, refer to )
Prerequisites
MiSeq Integration v8.3.0 has the following prerequisites:
Mount run data network-attached storage (NAS) share
IPP is installed
Prerequisite 1: Mount Run Data NAS Share
Mounting the NAS share of run data are needed to capture and generate files associated with the sequencing run. To mount NAS shares that contain data from the Clarity LIMS server, use Read/Write privileges as the glsjboss user. The following data can be mounted to the NAS share:
Run data (e.g., \\network-storage\run_data)
Prerequisite 2: IPP Installation
MiSeq Integration Package v8.3.0 depends on the QC Protocols configuration provided in IPP (v2.6 and later) for Clarity LIMS v6.2 and later.
If the base configuration is not installed, then install it on the Clarity LIMS server that is being used for the MiSeq integration. For details on IPP installation and configuration, refer to the Illumina Preset Protocols documentation.
If you are upgrading the base configuration, make sure that the IPP package is compatible with the version of Clarity LIMS you are installing.
If you do not have QC Protocols, then install them as follows.
Installation
MiSeq Integration v8.3.0 is distributed as the ClarityLIMS-Illumina-MiSeq-Package-v8 RPM package that must be installed on the Clarity LIMS server. This package installs the following items:
Bash scripts used to run the service
The miseq-sequencing.jar file
The configure_extensions_miseq_sequencingservice.sh script
The following installation steps are required for the installation of MiSeq Integration v8.3.0.
The MiSeq RPM must be installed on the Clarity LIMS server. This is the same location where the AI node or Automation Worker is installed. The automations and sequencing service use the existing Automation Worker.
Install the RPM
On the Clarity LIMS server, log in as the root user.
Run the following yum command to install the RPM:
ℹ You must use the --enablerepo command line argument to enable the repo. For the repo file and the correct name to use, contact Illumina Support.
Enter y to confirm that you want to proceed with the RPM installation.
Configure the Integration Properties
For more information on the configurable integration properties that enable capture and generation of files associated with the sequencing run, refer to .
Start the Sequencing Service
Run the following command to start the sequencing service:
Workflows, Protocols, and Steps Installed
MiSeq Integration v8.3.0 works with the MiSeq Sequencing v3.2, which contains a single protocol with the same name. This protocol includes the following steps:
Library Pooling (MiSeq v3.2)
Denature and Dilute (MiSeq v3.2)
MiSeq Run (MiSeq v3.2)
For descriptions of the protocol and the steps, refer to . For instructions on using the Library Prep Validation v2.3.4 workflow to validate the automated sample sheet generation, refer to .
Instrument Software
The MiSeq Control Software (MCS) is divided into the following modules:
MCS — Controls the instrument operation, including various configuration settings. This software is installed and runs on the instrument.
Local Run Manager — Analyzes sequencing data and integrates with MCS. This software can be accessed on the instrument through a web browser.
Real-Time Analysis (RTA) — Performs image processing and base calling (primary analysis). The software makes sure that data files are created and copied to the final destination folder and is installed and runs on the instrument.
For more information on MCS, refer to the MiSeq documentation at .
Instrument Integration
The instrument integration must be performed and maintained by Illumina Support. Illumina Support requires remote access to the instrument while it is idle.
The support team has created batch files that use custom scripts during the key events of a sequencing run. When these batch files are used, they read the event information and write it in a TXT event file at the same network share location that the instrument uses to write the run data. Another process running on the Clarity LIMS server receives the event files and takes the appropriate actions.
The sequencing service monitors the end of the run event. This event is used to capture key step data and files and parse run statistics for output custom fields.
Configure Batch Files
When the instrument is running, the final destination for the run data are a network storage path. The software is configured with a network storage path root (e.g., \\network-storage\illumina). Each sequencing run generates a unique run ID, which is appended to create a unique data run directory (e.g., \\network-storage\illumina\110419_InstrumentName_0001_ARG1234567).
The Clarity LIMS batch files must be configured to write to a directory within the network storage path root. This directory is typically named gls_events, but the directory name can be different as long as no spaces are used. The batch file used for MiSeq Control Software v4.0 is gls_event_mcs_rta_lrm.bat.
Before configuring the batch files, do the following:
Connect Batch Files to Instrument Events
Update the MiSeq Control Software configuration files as follows
Reboot the instrument.
Make a backup copy of the RTA configuration XML file and name it
Validation
Sample Sheet Generation
For instructions on how to validate the automated sample sheet generation, refer to .
Manual Invocation of Event Files
The manual invocation of an event file produces an output that contains the following information:
File name (e.g., event-EndRun-07295667.txt)
cycleNumber (e.g., 318)
Instrument Sequencing Run
The instrument sequencing run test validates that the Clarity LIMS batch file is connected properly and invoked on the instrument events. Before validating the batch file, make sure that the following prerequisites are met:
You have access to the NAS share.
Clarity LIMS-created events triggered by the End Run event of the Illumina sequencing run (e.g., \\network-storage\illumina\gls_events)
With Read access, the Clarity LIMS server reads the following information in individual sequencing run data folders:
Run information metadata from these files
<runFolderRoot>/RunInfo.xml
<runFolderRoot>/runParameters.xml
Run statistics from
<runFolderRoot>/InterOp/*.bin
The Clarity LIMS server generates the following files and information locally and stores them in Clarity LIMS:
Sample sheet (CSV file)
Run report (PDF file)
Run folder root link
The Clarity LIMS server copies and stores the following files from individual sequencing run data folders in Clarity LIMS:
As a glsjboss user, run the following command to view the complete list of IPP workflows:
To install the dependent QC Protocols, run the following command:
Smoke test directories
After confirmation, you are prompted to install the MiSeq workflow from the IPP script.
Install the MiSeq workflow as follows.
ℹ The following steps assume that the QC protocols from the IPP package have been installed. For more information, refer to Prerequisite 2: IPP Installation in Prerequisites.
As the glsjboss user, run the following command to view the complete list of IPP workflows:
Run the following command to install the MiSeq Sequencing v3.2 workflow configuration:
The installer does the following tasks:
Validates the import of the workflow and provides the Warning/Error details in STDOUT, which allows you proceed with the import or cancel it.
Automatically detects the version of Clarity LIMS and imports the applicable workflow configuration.
Creates or updates the miseq.v8.seqservice.sequenceProcessBaseName database property, which can be verified with the omxProps-ConfigTool utility. This property shows the sequencing master step base display name. It can also use partial matching to look up the master step by the sequencing service. The default value is MiSeq Run (MiSeq v3.2).
[Optional] Run the following command to install the Library Prep Validation v2.3.4 workflow that is used to validate the MiSeq Sequencing v3.2 workflow:
The installer does the following tasks:
Validates the import of the workflow and provides the Warning/Error details in STDOUT, which allows you proceed with the import or cancel it.
Automatically detects the version of Clarity LIMS and imports the applicable workflow configuration.
Run the following script to configure the service properties:
The script creates the configuration properties in the database. For a list of the configured properties, refer to Integration Properties Details.
Back up the MiSeq.Configuration.xml configuration file to
Make sure that the instrument is idle.
Shut down MiSeq Control Software.
Set up the directory structure as follows.
Create a directory (C:\Illumina\gls is recommended) on the local PC to hold the batch file.
âš For Windows 10, the folder must be under C:\Illumina instead of C:\Illumina\gls because of Windows software restriction policies. If the folder is not in that directory, the batch script does not run. For versions before Windows 10, C:\Illumina\gls is acceptable.
Create a directory (e.g., gls_events) on the NAS to hold the event files.
Configure the batch files as follows
Determine the site or instrument specific network storage path root.
Make the following changes to the gls_event_mcs_rta_lrm.bat batch file:
ℹ Make sure to include a trailing \ in the DESTINATION_PATH and DEFAULT_LOCAL_OUTPUT_PATH lines. Refer to the following examples:
Change the DESTINATION_PATH line to the name of the event file directory.
Change the DEFAULT_LOCAL_OUTPUT_PATH line to the name of the default local temp run folder directory.
The default directory is
Copy the DESTINATION_PATH and paste it into the Windows Explorer address bar.
Make sure that the network location is accessible and that it opens from the instrument.
The batch file must be deployed to the MiSeq instrument. This file must be deployed to its own directory (C:\Illumina\gls\ is recommended) on the instrument computer so that it is not overwritten or removed during an instrument software update.
Deploy the batch file as follows
From the Attachment section, download the batch file.
Copy the file to the directory that you created on the instrument computer (e.g., C:\Illumina\gls).
From the Command Prompt, run the following command to list the contents of the directory:
Make sure that the name of the batch file does not contain any special or hidden characters.
MiSeq.Configuration.xml
.
Edit the file to connect to the RTA End Run event as follows.
Open the MiSeq.Configuration.xml file at
Update the following content within the <ProcessCompleteEventFile> tags:
Save the edited file.
Validate the file as follows.
From C:\Illumina\RTA, create a backup of the RTA.exe.config file and name it Backup_RTA.exe.config.
Open RTA.exe.config and change the DefaultConfigFile value to MiSeq.Configuration.xml.
Save the changes and close the file.
From the Command Prompt, run the following command:
If the validation is successful, a warning message displays that says the output path cannot be placed into the run directory. Select OK.
In the Command Prompt, make sure the following error message displays:
Press Ctrl + C to terminate the RTA.exe application that is still running.
If the configuration file has an error, the command returns specifics about the problem. Refer to the following example:
After the validation is complete, go back to C:\Illumina\RTA and delete the current RTA.exe.config file.
âš Make sure that you rename the backup file. If you do not rename the file, it can cause the RTA not to work properly.
Restore the backup file from a by renaming it RTA.exe.config.
Start MiSeq Control Software.
Open the MiSeq.Configuration.xml file and make sure that the changes were saved.
If an event file is triggered automatically during a MiSeq run, then the completion event does not appear until the run has completed and RTA completes primary analysis.
The sequencing service processes and archives event files, which can cause validation issues while the service is running. You can make the following changes to avoid losing the event files that you are attempting to validate:
Modify the FINAL_EXTENSION value in the Clarity LIMS batch file so that the file extension is .test instead of .txt. The service only processes and archives TXT files. Make sure that you change FINAL_EXTENSION back to .txt after manual validation.
Monitor the MiSeqIntegrator.log file, which logs the file name and contents of each event file that is processed. The log file is at
Validate the sequencing run as follows
During the run, monitor the contents of the gls_events directory.
After the run is completed and the RTA completes primary analysis, make sure that a final EndRun event displays (e.g., event-EndRun-11043279.txt).
RunInfo.xml does not exist: System.ApplicationException: Error in the application.,0,0
Configuration
The Illumina MiSeq Integration Package v8.3.0 supports the integration of Clarity LIMS to Illumina MiSeq Sequencing Systems.
The integration allows for automated tracking of an Illumina sequencing run in Clarity LIMS. This capability includes tracking sequencing run status, generating run report, and capturing and parsing run statistics. In addition, this integration provides automated generation of a sample sheet file for use with the MiSeq Control Software (MCS) and Local Run Manager (LRM).
The MiSeq Sequencing v3.2 workflow is compatible with MiSeq Integration Package v8.2.0 and v8.3.0.
MiSeq Integration v8.2.0 and above is only required for MiSeq Control Software (MCS) v4.0. For MCS v3.1 or earlier, do not upgrade to MiSeq Integration v8.2.0 or above. This upgrade breaks the integration.
Prerequisites and Assumptions
Before samples are assigned to the MiSeq Sequencing v3.2 workflow, make sure that the following prerequisites are completed:
Samples have been accessioned into Clarity LIMS.
Samples have been run through QC and library prep.
Samples have been normalized, and the value is captured in a field called Normalized Molarity (nM).
For more information on sample accessioning, refer to Sample Accessioning and Upload and Modify Samples in the .
Samples can be assigned to the MiSeq Sequencing v3.2 workflow 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 Illumina MiSeq Integration includes the MiSeq Sequencing v3.2 workflow, which contains a single protocol of the same name.
Step 1: Library Pooling (MiSeq v3.2)
In this step, the lab scientist manually places libraries into pools in the Clarity LIMS Placement screen.
Set Next Step - Advance Automation
This automation advances samples to the next step in the protocol. The automation is automatically triggered on exit of the Record Details screen.
The default command line is as follows.
Validate Maximum Number of Samples Automation
This automation checks the maximum number of samples that is allowed in a single pool. The default maximum for Illumina MiSeq Control Software (MCS) is 1536 samples. The automation is automatically triggered on entry of the Pooling screen.
The default command line is as follows.
Master Step Fields
The following field is configured on the Library Pooling (MiSeq v3.2) master step. The field displays on the Record Details screen at run time.
Library Pooling (MiSeq v3.2) Master Step Field Configuration
Global Fields
The following table lists the global fields that are displayed on the Queue and Ice Bucket screens of the Library Pooling (MiSeq v3.2) step. Most fields display in expanded view only.
Global Field Configuration (Submitted Sample)
Global Field Configuration (Derived Sample)
Step 2: Denature and Dilute (MiSeq v3.2)
In this step, pooled libraries are denatured and diluted, and then placed into the reagent cartridge loaded into the MiSeq instrument.
Validate Single Input Automation
This automation checks that there is only one pooled input in the step. The automation is automatically triggered when starting the step.
The default command line is as follows.
Validate Run Setup and Generate MiSeq SampleSheet Automation
This automation validates the information entered on the Record Details screen, generates the sample sheet (refer to ), and attaches the sample sheet to the Denature and Dilute step.
Select a button on the Record Details screen to trigger this automation.
Validation Rules
Set Next Step - Advance Automation
This automation advances samples to the next step in the protocol. The automation is automatically triggered by exiting the Record Details screen.
The default command line is as follows.
Master Step Fields
Most fields configured on the Denature and Dilute (MiSeq v3.2) step display on the Record Details screen in the Step Data table.
These fields are manually populated at run time. The values are then used to generate the sample sheet.
Denature & Dilute (MiSeq v3.2) Master Step Field Configuration
Groups of Defaults
Resequencing
Workflow = Resequencing
Read 1 Cycles = 251
Library QC
Workflow = Library QC
Read 1 Cycles = 251
GenerateFastQ
Workflow = GenerateFastQ
Read 1 Cycles = 251
DNA Enrichment
Workflow = DNA Enrichment
Read 1 Cycles = 251
DNA Amplicon
Workflow = DNA Amplicon
Read 1 Cycles = 251
Global Fields
The following table shows the global fields that are configured to display on the Queue, Ice Bucket, and Record Details screens of the Denature and Dilute (MiSeq v3.2) step:
The Submitted Sample field, Progress, (added from previous MiSeq workflow) is obsolete in MiSeq v3.2.
Global Field Configuration (Submitted Sample)
Global Field Configuration (Derived Sample)
Step File Placeholders
Placeholders for the following files are configured on the Record Details screen of the Denature and Dilute (MiSeq v3.2) step.
Lab Tracking Form
Manually uploaded
This item in Clarity LIMS allows the lab scientist to attach a lab-specific tracking form to the step manually.
MiSeq SampleSheet
Automatically attached
This CSV file is automatically generated by Clarity LIMS for use with the MiSeq system. The file can be opened as a text file or an Excel spreadsheet.
MiSeq SampleSheet Generation Log
Automatically attached
This log file is automatically generated by Clarity LIMS. The log file captures any errors that Clarity LIMS might encounter when generating the sample sheet.
Log File
Automatically attached
This log file is automatically generated by Clarity LIMS. The log file captures the status of the EvaluateDynamicExpression script that is invoked by the Set Next Step - Advance automation.
Step 3: MiSeq Run (MiSeq v3.2)
Set Next Step - Advance Automation
This automation advances samples to the next step in the protocol. The automation is automatically triggered by exiting the Record Details screen.
The default command line is as follows.
Master Step Fields
The following fields are configured on the MiSeq Run (MiSeq v3.2) step. These fields display on the Record Details screen at run time. The read-only fields are automatically populated at the end of the run.
MiSeq Run (MiSeq v3.2) Master Step Field Configuration
Global Fields
There are several sample and measurement global fields that are displayed on the Record Details screen of the MiSeq Run (MiSeq v3.2) step. These fields are autopopulated at the end of the sequencing run.
For more information, refer to .
Step File Placeholders
Placeholders for the following files are configured on the Record Details screen of the MiSeq Run (MiSeq v3.2) step:
Illumina Run Report (automatically attached)
Link to Run Folder (automatically attached)
Run Parameters (automatically attached)
For details, refer to .
Sample Sheet Generation
Sample sheet generation occurs in the Denature & Dilute (MiSeq v3.2) step. This step places samples on the container loaded in the system.
The default configuration provides only the Validate Run Setup and Generate MiSeq SampleSheet automation. This automation uses the Template File Generator (DriverFileGenerator.jar) and a template file to generate a CSV format file for use with the MiSeq Control Software (MCS).
The sample sheet content is determined by the fields on the Record Details screen of the step in the Step Data table. The values entered into these fields are used to populate the sample sheet.
To customize the template used to create the sample sheet, you can insert additional columns.
The following additional details are available:
For a sample template that you can download and customize for the lab, refer to .
For instructions on customizing the template, refer to .
Sequencing Results Parsing
The MiSeq Run (MiSeq v3.2) step records information for the flow cell lane and generates a report summarizing the results. In addition, run parameters, run info, and a link to the run folder are automatically captured.
Generated and Captured Files
The following table describes the run information files, reports, placeholders, and links that Clarity LIMS automatically generates or captures during a sequencing run:
Run Information Generated or Captured by MiSeq Run (MiSeq v3.2) Step
Metadata
The following list shows metadata that Clarity LIMS automatically captures from the Illumina sequencing software as part of a sequencing run. This information is gathered from various run result files and events.
Chemistry
Experiment Name (entered in software)
Finish Date (run completion date)
Primary Analysis Metrics
The following table lists the real-time analysis (RTA) primary analysis metrics Clarity LIMS automatically captures and records per read, for samples in each flow cell lane. These metrics are captured upon run completion and are stored as fields in the Sample Details table on the Record Details screen.
To see both per read and per lane metrics, expand the output.
RTA Primary Analysis Metrics Captured by MiSeq Run (MiSeq v3.2) Step
How It Works
The sequencing service runs on the Clarity LIMS server. The service detects event files that instrument RTA produces as the run progresses. The event files let the service know where to find the run data.
As the run data are written out and the End Run event is detected, the following events occur:
The data are matched to the step based on the reagent cartridge ID that was entered or scanned on the Denature and Dilute (MiSeq v3.2) step.
Scripts and Files Installed
This integration requires components installed with the Illumina Preset Protocols (IPP).
The Illumina MiSeq Integration Package v8.3.0 RPM installs the scripts and files listed in the following table.
Properties Installed
Refer to for the properties installed with Illumina MiSeq Integration v8.3.0.
Consumables Installed
Reagent Categories and Kits
Reagent categories or label groups are installed with the IPP workflow configuration slices.
The MiSeq Reagent Kit is included in the Illumina MiSeq Integration.
Control Types
The PhiX v3 control type is included in the Illumina MiSeq Integration.
Container Types
The following container types are included in the Illumina MiSeq Integration:
MiSeq Reagent Cartridge
96 well plate
Tube
All one-dimensional container types with both numeric rows and numeric columns are supported.
Instrument Integration
To make sure that the Illumina instrument warranty remains valid, the instrument integration must be performed and maintained by the Clarity LIMS Support team. To perform this integration, the Support team requires remote access to the system while it is idle.
The following steps are performed by the Clarity LIMS Support team when configuring the sequencing for use with the Illumina MiSeq Integration.
Create a directory on the local computer to hold the batch files. These batch files write event files to the network attached storage (NAS) shares.
Create a directory on the NAS to hold the event files.
Modify Illumina software configuration files to call the batch files that create the event files.
Rules and Constraints
The Illumina MiSeq Integration operates with the following constraints:
The reagent cartridge ID must be unique. There should not be multiple reagent cartridge containers in the system with the same name.
The reagent cartridge ID must be scanned as the reagent cartridge Container Name on the Denature & Dilute (MiSeq v3.2) step.
Sequencing Coverage
Text
Sequencing Method
Text Dropdown
Custom Entries
Presets
Single Read
Paired End Read
Indexed Single Read
The Experiment Name custom field cannot exceed 40 characters.
The Experiment Name field can only contain letters, numbers, periods, nonconsecutive spaces, and the following special characters: `~!@#%-_}{.
If Starling is selected as the variant caller, the Export to gVCF field must be set to Yes.
If the Read 2 Cycles field is empty or 0, the following rules apply:
The Custom Primers field cannot have a selection that contains Read 2.
The Adapter Read 2 field cannot have a value.
The UMI - Read 2 Length field and the UMI - Read 2 Start From Cycle field cannot have values.
The UMI - Read 1 Length field and the UMI - Read 1 Start From Cycle field cannot be used independently.
The UMI - Read 2 Length field and the UMI - Read 2 Start From Cycle field cannot be used independently.
If the UMI - Read 2 Length field is greater than 0, the UMI - Read 1 Length field must be greater than 0.
The Adapter Read 1 and Adapter Read 2 fields can only contain ACGT+ characters. The adapter sequence cannot start or end with + or contain more than one +.
For DNA Enrichment analysis, if the Variant Caller value is Somatic, the Variant Frequency Percentage must be between 1 and 100.
For DNA Amplicon analysis, if the Variant Caller value is Somatic, the Variant Frequency Percentage field must contain a value between 0.05 and 30.
If the Aligner value is BWA or TruSeq Amplicon Aligner, an error occurs when the Workflow value is not equal to DNA Amplicon.
If the Aligner value is BWA-MEM or BWA-Backtrack Legacy, an error occurs when the Workflow value is equal to DNA Amplicon.
If the Variant Caller value is Starling or GATK, an error occurs when the Workflow value is equal to DNA Amplicon.
If the Variant Caller value is Somatic, an error occurs when the Workflow value is not equal to DNA Enrichment or DNA Amplicon.
If the Variant Caller value is Germline, an error occurs when the Workflow value is not equal to DNA Amplicon.
The default command line is as follows.
There is an 8000 character limit in the Oracle database used for the automation storage. Because of this character limit, the automation splits and saves the validation expressions to the Validation Script 1 and Validation Script 2. These fields are configured as master step fields.
Validation Script 1 Command Line
Validation Script 2 Command Line
Aligner-Key
ℹ Used for sample sheet generation
Text
Read Only
Hidden
Aligner-Value
ℹ Used for sample sheet generation
Text
Read Only
Annotation
Text Dropdown
Presets
None (default)
RefSeq
Applicable analysis fields for the selected Workflow
Multiline Text
Read Only
ℹ Select a value from the drop-down list in the upper-right corner (below NEXT STEPS).
Comment
Multiline Text
Custom Primers
Text Dropdown
Presets
None (Default)
Read 1
Description
Text
Experiment Name
Text
Required Field
Export to gVCF
Text Dropdown
Presets
None (default)
Yes
Flag PCR Duplicates
Text Dropdown
Presets
None (default)
Yes
Genome Folder
Text
Indel Realignment
Text Dropdown
Presets
None (default)
Yes
Indel Repeat Filter Cutoff
Text Dropdown
Presets
None (default)
Yes
Indel-Realignment-Key
ℹ Used for sample sheet generation
Text
Read Only
Hidden
Indel-Realignment-Value
ℹ Used for sample sheet generation
Text
Read Only
Hidden
Manifest
Text
Manifest Padding
Text Dropdown
Presets
None (default)
0
Manifest-Section
ℹ Used for sample sheet generation
Text
Read Only
Hidden
Picard HS Metric Reporting
Text Dropdown
Presets
None (default)
Yes
Read 1 Cycles
Numeric Dropdown
Required Field
Custom Entries
Presets
251 (Default)
151
Read 2 Cycles
Numeric Dropdown
Required Field
Custom Entries
Presets
251 (Default)
151
Read Stitching
Text Dropdown
Presets
None (default)
Yes
Reverse Complement
Text Dropdown
Presets
None (default)
Yes
UMI - Read 1 Length
Numeric
Minimum value: 1
UMI - Read 1 Start From Cycle
Numeric
Minimum value: 1
UMI - Read 2 Length
Numeric
Minimum value: 1
UMI - Read 2 Start From Cycle
Numeric
Minimum value: 1
Validation Script 1
ℹ Used for automation
Multiline Text
Required Field
Read Only
Hidden
Validation Script 2
ℹ Used for automation
Multiline Text
Required Field
Read Only
Hidden
Variant Caller
Text Dropdown
Presets
None (Default)
GATK
Variant Caller Depth Filter
Numeric
Minimum value: 10
Maximum value: 10000
Variant Quality Filter
Numeric
Minimum value: 2
Maximum value: 1000
Variant Frequency Percentage
Numeric
Minimum value: 0.05
Maximum value: 100
Decimal places displayed = 2
Variant-Caller-Value
ℹ Used for sample sheet generation
Text
Read Only
Hidden
Workflow
Text Dropdown
Required Field
Presets
GenerateFASTQ
LibraryQC
Custom Primers = None
Aligner = BWA-MEM
Annotation = None
Export to gVCF = No
Flag PCR Duplicates = Yes
Genome Folder = Required
Indel Realignment = No
Indel Repeat Filter Cutoff = None
Manifest = None
Manifest Padding = None
Picard HS Metric Reporting = None
Reverse Complement = None
Read Stitching = None
Variant Caller = GATK
Applicable analysis fields for the selected Workflow =
Aligner (BWA-Backtrack Legacy, BWA-MEM)
Export to gVCF
Flag PCR Duplicates
Genome Folder (fill in the folder path)
Indel Realignment
Variant Caller (Starling, GATK)
Custom Primers = None
Aligner = None
Annotation = None
Export to gVCF = None
Flag PCR Duplicates = Yes
Genome Folder = Required
Indel Realignment = None
Indel Repeat Filter Cutoff = None
Manifest = None
Manifest Padding = None
Picard HS Metric Reporting = None
Reverse Complement = None
Read Stitching = None
Variant Caller = None
Applicable analysis fields for the selected Workflow =
Flag PCR Duplicates
Genome Folder (fill in the folder path)
Custom Primers = None
Aligner = None
Annotation = None
Export to gVCF = None
Flag PCR Duplicates = None
Genome Folder = None
Indel Realignment = None
Indel Repeat Filter Cutoff = None
Manifest = None
Manifest Padding = None
Picard HS Metric Reporting = None
Reverse Complement = None
Read Stitching = None
Variant Caller = None
Applicable analysis fields for the selected Workflow = None
Custom Primers = None
Aligner = BWA-MEM
Annotation = None
Export to gVCF = None
Flag PCR Duplicates = Yes
Genome Folder = Required
Indel Realignment = Yes
Indel Repeat Filter Cutoff = None
Manifest = Required
Manifest Padding = 150
Picard HS Metric Reporting = No
Reverse Complement = None
Read Stitching = None
Variant Caller = Starling
Applicable analysis fields for the selected Workflow =
Aligner (BWA-Backtrack Legacy, BWA-MEM)
Flag PCR Duplicates
Genome Folder (fill in the folder path)
Indel Realignment
Indel Repeat Filter Cutoff (if Variant Caller is Somatic)
Manifest (fill in the file name)
Manifest Padding
Picard HS Metric Reporting
Variant Caller (Starling, Somatic, GATK)
Variant Frequency Percentage (if Variant Caller is Somatic)
Custom Primers = None
Aligner = BWA
Annotation = RefSeq
Export to gVCF = None
Flag PCR Duplicates = None
Genome Folder = Required
Indel Realignment = Yes
Indel Repeat Filter Cutoff = None
Manifest = Required
Manifest Padding = None
Picard HS Metric Reporting = None
Reverse Complement = None
Read Stitching = None
Variant Caller = Germline
Variant Caller Depth Filter = 10
Variant Quality Filter = 30
Applicable analysis fields for the selected Workflow =
Aligner (BWA, TruSeq Amplicon Aligner)
Annotation (RefSeq, Ensembl)
Genome Folder (fill in the folder path)
Indel Realignment
Manifest (fill in the file name)
Read stitching (if Aligner is TruSeq Amplicon Aligner)
Variant Caller (Germline, Somatic)
Variant Caller Depth Filter (10–10000)
Variant Frequency Percentage (if Variant Caller is Somatic)
Variant Quality Filter (2–1000)
Finish Date
Date
Read Only
Flow Cell ID
Text
Read Only
Flow Cell Version
Text
Read Only
Index 1 Read Cycles
Numeric
Read Only
Decimal Places Displayed: 0
Index 2 Read Cycles
Numeric
Read Only
Decimal Places Displayed: 0
Output Folder
Text
Read Only
PR2 Bottle ID
Text
Read Only
Read 1 Cycles
Numeric
Read Only
Decimal Places Displayed: 0
Read 2 Cycles
Numeric
Read Only
Decimal Places Displayed: 0
Reagent Cartridge ID
Text
Read Only
Reagent Cartridge Part #
Text
Read Only
Run ID
Text
Read Only
Run Type
Text
Read Only
Status
Text
Read Only
Workflow
Text
Read Only
Run Info (automatically attached)
Lab Tracking Form (manually uploaded)
Log File (automatically attached)
If the End Run event contains a date in the format YYYY-MM-DD, Finish Date is set to the date in the event file.
If the End Run event does not contain a date or the date is in the wrong format, Finish Date is set to the date when the event file is processed.
Flow Cell ID
Flow Cell Version
Index 1 Read Cycles (intended Index cycles)
Index 2 Read Cycles (intended Index cycles)
Output Folder (run folder root)
PR2 Bottle ID
Reagent Cartridge ID
Reagent Cartridge Part #
Read 1 Cycles
Read 2 Cycles
Run ID (the unique run ID)
Run Type
Status (current status of the sequencing run on the instrument)
Workflow
% Phasing R1
% Phasing R2
% Prephasing R1
% Prephasing R2
%PF R1
%PF R2
Cluster Density (K/mm^2) R1
Cluster Density (K/mm^2) R2
Intensity Cycle 1 R1
Intensity Cycle 1 R1
Intensity Cycle 1 R2
Intensity Cycle 1 R2
Reads PF (M) R1
Reads PF (M) R2
Yield PF (Gb) R1
Yield PF (Gb) R1
Yield PF (Gb) R2
Yield PF (Gb) R2
Read-only field values on the Record Details screen are populated accordingly.
When the service has finished processing the end run event and updating the fields in Clarity LIMS, the sequencing service generates the report and attaches it to the step.
Update sequencing service default properties to match the specifics of the installation.
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-down Items
Comment
Multiline Text
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-Down Items
Application
Text Dropdown
Custom Entries
Presets
TruSeq mRNA sequencing
TruSeq DNA sequencing (large genome de novo)
TruSeq DNA sequencing (large genome re-seq)
TruSeq DNA sequencing (small genome de novo)
TruSeq DNA sequencing (small genome re-seq)
Nextera DNA sequencing
TruSeq Custom Amplicon sequencing
ChIP-sequencing
Exome sequencing
Mate pair sequencing
Small RNA sequencing
Pooling
Text Dropdown
Custom Entries
Presets
Yes
No
Read Length
Text
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-Down Items
Normalized Molarity (nM)
Numeric
Decimal places displayed = 2
Field Name
Field Type
Options
Additional Options and Drop-Down Items
Adapter Read 1
Text
Adapter Read 2
Text
Aligner
Text Dropdown
Presets
None (default)
BWA-MEM
BWA-Backtrack Legacy
BWA
TruSeq Amplicon Aligner
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-Down Items
Read Length
ℹ Displays on Queue & Ice Bucket screens
Text
Sequencing Method
ℹ Displays on Queue & Ice Bucket screens
Text Dropdown
Custom Entries
Presets
Single Read
Paired End Read
Indexed Single Read
Indexed Paired End Read
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-Down Items
Final Loading Concentration
ℹ Displays on Record Details screen
Numeric Dropdown
Required Field
Custom Entries
Presets
225
400
Decimal places displayed = 0
Field Name
Field Type
Options
Additional Options and Drop-Down Items
Chemistry
Text
Read Only
Comment
Multiline Text
Experiment Name
Text
Read Only
Item
Description
Run Info
Run Parameters
These XML files are automatically captured by Clarity LIMS from the run folder of the system. They include the key run parameters that are parsed out into step custom fields in Clarity LIMS.
Link to Run Folder
This link is the path to the network run folder where the data that was captured from the system during the run is stored. The link is automatically generated by Clarity LIMS.
Illumina Run Report
This report provides key information about the run and the samples on the flow cell. The report is automatically generated by Clarity LIMS.
Information includes the flow cell ID, run directory location, and primary analysis metrics for the sequencing run. Information is summarized per flow cell lane for the entire run and individual reads for paired-end runs.
These metrics are compared against the per lane averages of the sequencing run. The per lane averages are calculated using metrics from the last five sequencing runs. Any values outside of one standard deviation are highlighted.
Lab Tracking Form
This item in Clarity LIMS allows you to manually attach a lab-specific tracking form to the step.
Per Read LIMS Field Name(Stored on derived sample/analyte input to the step)
Per Lane LIMS Field Name(Stored in measurement placeholders in the Sample Details table on the Record Details screen)
if (step.::Experiment Name::.length() > 40) { fail(::Experiment Name cannot exceed 40 characters.::) }; if (!step.::Experiment Name::.matches(::^(?!.*[ ]{2})[a-zA-Z0-9-_`.~!#@%{ }]+::)) { fail(::Experiment Name contains prohibited characters. Allowed characters are letters, numbers, periods, non-consecutive spaces and the following special characters: `~!@#%-_}{::) }; if (step.::Workflow:: == ::Resequencing:: && step.::Variant Caller:: == ::Starling:: && (!step.hasValue(::Export to gVCF::) || step.::Export to gVCF:: != ::Yes::)) { fail(::Export to gVCF must be set to Yes for Starling variant caller.::) }; if (step.::Workflow:: == ::DNA Enrichment:: && step.::Variant Caller:: == ::Somatic:: && (!step.hasValue(::Variant Frequency Percentage::) || step.::Variant Frequency Percentage:: > 100 || step.::Variant Frequency Percentage:: < 1)) { fail(::In the Variant Frequency Percentage field, please enter values between 1 and 100.::) }; if (step.::Workflow:: == ::DNA Amplicon:: && step.::Variant Caller:: == ::Somatic:: && (!step.hasValue(::Variant Frequency Percentage::) || step.::Variant Frequency Percentage:: > 30 || step.::Variant Frequency Percentage:: < 0.05)) { fail(::In the Variant Frequency Percentage field, please enter values between 0.05 and 30.::) }; if (!step.hasValue(::Read 2 Cycles::) || step.::Read 2 Cycles:: == 0) { if (step.::Custom Primers::.contains(::Read 2::)) { fail(::Custom Primers setting selected is invalid and can only be used in a Paired-End run.::) }; if (step.hasValue(::Adapter Read 2::)) { fail(::Adapter Read 2 is only applicable for a Paired-End run.::) }; if (step.hasValue(::UMI - Read 2 Length::) || step.hasValue(::UMI - Read 2 Start From Cycle::)) { fail(::UMI - Read 2 Length and UMI - Read 2 Start From Cycle are only applicable for a Paired-End run.::) }; }; if (step.hasValue(::UMI - Read 1 Length::) && !step.hasValue(::UMI - Read 1 Start From Cycle::)) { fail(::UMI - Read 1 Start From Cycle must be greater than 0 if UMI - Read 1 Length is greater than 0.::) }; if (!step.hasValue(::UMI - Read 1 Length::) && step.hasValue(::UMI - Read 1 Start From Cycle::)) { fail(::UMI - Read 1 Length must be greater than 0 if UMI - Read 1 Start From Cycle is greater than 0.::) }; if (step.hasValue(::UMI - Read 2 Length::) && !step.hasValue(::UMI - Read 2 Start From Cycle::)) { fail(::UMI - Read 2 Start From Cycle must be greater than 0 if UMI - Read 2 Length is greater than 0.::) }; if (!step.hasValue(::UMI - Read 2 Length::) && step.hasValue(::UMI - Read 2 Start From Cycle::)) { fail(::UMI - Read 2 Length must be greater than 0 if UMI - Read 2 Start From Cycle is greater than 0.::) }; if (!step.hasValue(::UMI - Read 1 Length::) && step.hasValue(::UMI - Read 2 Length::)) { fail(::UMI - Read 1 Length must be greater than 0 if UMI - Read 2 Length is greater than 0.::) }; if (step.hasValue(::Adapter Read 1::) && !step.::Adapter Read 1::.matches(::^[ACGT]+(\\+[ACGT]+){0,1}$::)) { fail(::Adapter Read 1 contains prohibited characters. Allowed characters are: ACGT+ and the adapter sequence cannot start or end with + or contain more than one +.::) }; if (step.hasValue(::Adapter Read 2::) && !step.::Adapter Read 2::.matches(::^[ACGT]+(\\+[ACGT]+){0,1}$::)) { fail(::Adapter Read 2 contains prohibited characters. Allowed characters are: ACGT+ and the adapter sequence cannot start or end with + or contain more than one +.::) };
if ((step.::Aligner:: == ::BWA:: || step.::Aligner:: == ::TruSeq Amplicon Aligner::) && step.::Workflow:: != ::DNA Amplicon::) { fail(::Aligner field contains invalid value. Please refer to the \u005c\u0022Applicable analysis fields for the selected Workflow:\u005c\u0022 section for more information.::) }; if ((step.::Aligner:: == ::BWA-MEM:: || step.::Aligner:: == ::BWA-Backtrack Legacy::) && step.::Workflow:: == ::DNA Amplicon::) { fail(::Aligner field contains invalid value. Please refer to the \u005c\u0022Applicable analysis fields for the selected Workflow:\u005c\u0022 section for more information.::) }; if ((step.::Variant Caller:: == ::Starling:: || step.::Variant Caller:: == ::GATK::) && step.::Workflow:: == ::DNA Amplicon::) { fail(::Variant Caller field contains invalid value. Please refer to the \u005c\u0022Applicable analysis fields for the selected Workflow:\u005c\u0022 section for more information.::) }; if (step.::Variant Caller:: == ::Somatic:: && step.::Workflow:: != ::DNA Enrichment:: && step.::Workflow:: != ::DNA Amplicon::) { fail(::Variant Caller field contains invalid value. Please refer to the \u005c\u0022Applicable analysis fields for the selected Workflow:\u005c\u0022 section for more information.::) }; if (step.::Variant Caller:: == ::Germline:: && step.::Workflow:: != ::DNA Amplicon::) { fail(::Variant Caller field contains invalid value. Please refer to the \u005c\u0022Applicable analysis fields for the selected Workflow:\u005c\u0022 section for more information.::) }; step.::Indel-Realignment-Key:: = ::::; step.::Indel-Realignment-Value:: = ::::; step.::Aligner-Key:: = ::::; step.::Aligner-Value:: = ::::; step.::Variant-Caller-Value:: = ::::; if (step.::Workflow:: == ::DNA Amplicon::) { if (step.::Indel Realignment:: == ::Yes::) { step.::Indel-Realignment-Key:: = ::variantcallerrealignindels::; step.::Indel-Realignment-Value:: = ::1::; }; else if (step.::Indel Realignment:: == ::No::) { step.::Indel-Realignment-Key:: = ::variantcallerrealignindels::; step.::Indel-Realignment-Value:: = ::0::; }; if (step.::Aligner:: == ::TruSeq Amplicon Aligner::) { step.::Aligner-Key:: = ::aligner::; step.::Aligner-Value:: = ::Amplicon::; }; else if (step.::Aligner:: == ::BWA::) { step.::Aligner-Key:: = ::aligner::; step.::Aligner-Value:: = ::BWA::; }; }; if (step.::Workflow:: != ::DNA Amplicon::) { if (step.::Indel Realignment:: == ::Yes::) { step.::Indel-Realignment-Key:: = ::indelrealignment::; step.::Indel-Realignment-Value:: = ::GATK::; }; else if (step.::Indel Realignment:: == ::No::) { step.::Indel-Realignment-Key:: = ::indelrealignment::; step.::Indel-Realignment-Value:: = ::None::; }; if (step.::Aligner:: == ::BWA-Backtrack Legacy::) { step.::Aligner-Key:: = ::runbwaaln::; step.::Aligner-Value:: = ::1::; }; else if (step.::Aligner:: == ::BWA-MEM::) { step.::Aligner-Key:: = ::runbwaaln::; step.::Aligner-Value:: = ::0::; }; }; if (step.hasValue(::Variant Caller::)) { if (step.::Variant Caller:: == ::None::) { step.::Variant-Caller-Value:: = ::::; }; if (step.::Variant Caller:: == ::GATK::) { step.::Variant-Caller-Value:: = ::GATK::; }; else if (step.::Variant Caller:: == ::Starling::) { step.::Variant-Caller-Value:: = ::Starling::; }; else if (step.::Variant Caller:: == ::Germline::) { step.::Variant-Caller-Value:: = ::PiscesGermline::; }; else if (step.::Variant Caller:: == ::Somatic:: && step.::Workflow:: == ::DNA Amplicon::) { step.::Variant-Caller-Value:: = ::PiscesSomatic::; }; else if (step.::Variant Caller:: == ::Somatic:: && step.::Workflow:: != ::DNA Amplicon::) { step.::Variant-Caller-Value:: = ::Somatic::; }; }; if (step.hasValue(::Manifest::)) { step.::Manifest-Section:: = ::[Manifests]::; }; else { step.::Manifest-Section:: = ::::; }
