The Illumina MiSeqDx Integration v1.11.0 includes the following features:
MiSeqDx RPM that supports Diagnostic (Dx) mode.
MiSeq RPM that supports Research (RUO) mode.
Preconfigured Clarity LIMS protocols that map to library prep and sequencing lab protocols and instrument runs.
Preconfigured protocols that allow for validation of the sample sheet generation and index placement functionality.
Automatic generation of:
A sample sheet file for use with the MOS (Diagnostic mode) instrument software.
A run report, which includes the current run statistics.
Automated tracking of the following information in Clarity LIMS:
Progress and metrics of a MiSeqDx sequencing run
Per-instrument sequencing runs
The ability to monitor the run status (cycle number) across multiple instruments from within Clarity LIMS.
The ability to attach Variant Call Format (VCF) files to Clarity LIMS.
Sequencing run parameters
Real-Time Analysis (RTA) run directory location and other run specific information
Configuration
The Illumina MiSeqDx Integration Package v1.11.0 supports the integration between Clarity LIMS and MiSeqDx instruments.
This documentation describes the integration between Clarity LIMS and the MiSeqDx system. It includes information about protocols and automations, configuration options, installed components, and rules and constraints.
Workflows, Protocols, and Steps
The following protocols are included in MiSeqDx Integration Package v1.11.0:
CF 139-Variant Assay Library Prep 1.2
CF Clinical Sequencing Assay Library Prep 1.2
Illumina SBS MiSeqDx (CF 139-Variant Assay) 1.2
Illumina SBS MiSeqDx (CF Clinical Sequencing Assay) 1.2
There are three validation protocols. Each protocol is included in a workflow with the same name. The protocols are as follows.
In Assay workflows, include a QC validation protocol between the Library Prep and Illumina SBS protocols. The default workflow does not include a QC protocol.
Each validation protocol includes the following steps from the Library Prep and Illumina SBS MiSeqDx protocols:
Extension-Ligation of Bound Oligos (Library Prep step)
PCR Amplification (Library Prep step)
Library Pooling (MiSeqDx) (Illumina SBS MiSeqDx step)
For instructions on user interaction for each step and using the MiSeqDx Validation (CF 139-Variant Assay) 1.2 protocol to validate automated sample sheet generation, refer to .
The following table lists the automations included in this integration, and the steps on which they are configured.
MiSeqDx Integration Default Automations
Library Prep Protocol - PCR Amplification Step
This section discusses the index placement and validation automations configured on the PCR Amplification 1.2 Library Prep steps.
The example workflow uses the CF 139-Variant Assay Library Prep 1.2 protocol.
By default, in the CF 139-Variant Assay Library Prep 1.2 protocol, the PCR Amplification 1.2 step includes two automations. Both automations invoke the place_indexes script with different options.
Auto Place Indexes — Automatically invoked on entry to the Add Reagents screen.
âš For the Auto Place Indexes automation, if an 8-sample reagent category/label group is selected, index placement must be performed manually.
ℹ The CF Clinical Sequencing Assay Library Prep 1.2 protocol only uses 8-sample reagent categories/label groups. Hence, index placement must be performed manually.
Validate Index Placement — Automatically invoked on exit from the Add Reagents screen.
A list of reagents installed with this configuration is provided in .
Good laboratory practices mandate that a positive control DNA sample and a negative (no-template) control sample are included in every run. The positive control DNA sample should be a well-characterized sample with a known CFTR mutation.
Auto Place Indexes Automation
Automatically triggered on entry to Add Reagents screen, this automation completes the following actions:
Invokes place_indexes script to place reagent indexes into the container based on the pattern defined in a specified index placement pattern file.
Reagent indexes are assigned to each sample in the destination container according to the specified index placement pattern file.
Validate Index Placement Automation
Automatically triggered on exit from Add Reagents screen, this automation completes the following actions:
Invokes place_indexes script to validate the index placement:
Illumina SBS MiSeqDx v1.2 Protocols
This section describes the features of the key steps in the Illumina SBS MiSeqDx v1.2 protocols.
Library normalization CSV file generation in Library Normalization (MiSeqDx) step.
Reagent cartridge name validation and sample sheet generation in Denature, Dilute and Load Sample 1.2 step.
Primary analysis (sequencing) results parsing, which includes generation of the run report, in MiSeqDx Run (MiSeqDx) step.
Library Normalization (MiSeqDx) Step
In each of the Illumina SBS MiSeqDx protocols, the Library Normalization (MiSeqDx) v1.2 step includes automated calculation of normalization buffer volumes. The results are provided in an autogenerated comma-separated file that is attached to the step.
Create Normalization CSV Automation
Buffer volumes are calculated by the normalizationBufferVolumes script. The script is invoked by the Create Normalization CSV automation when you select a button on the Record Details screen.
For details on the normalizationBufferVolumes script, refer to .
Denature, Dilute and Load Sample 1.2 Step
In each of the Illumina SBS MiSeqDx and Validation protocols, the Denature, Dilute and Load Sample 1.2 step includes automations to:
Validate single input
Validate the MiSeqDx reagent cartridge name
Generate sample sheet
Validate Single Input
Automatically initiated on entry to the step, the Validate Single Input automation validates that there is only 1 pool added to this step.
Validate Container Name Automation
Automatically initiated on exit from the Placement screen of the step, the Validate Container Name automation validates the reagent cartridge name to ensure:
The reagent cartridge Container Name has been renamed and is not the same as its Clarity LIMS ID.
Generate MiSeqDx Sample Sheet Automation
The sample sheet generation automation is configured on the Record Details screen of the Denature, Dilute and Load Sample 1.2 step.
This automation is triggered by a button on the Record Details screen.
The integration allows for generation of a sample sheet designed to be used with the MOS (Diagnostic mode) instrument software.
By default, one MiSeqDx instrument sample sheet is created for the reagent cartridge loaded during the step. The placeholders and sample sheet files in the following table are generated by the Denature, Dilute and Load Sample 1.2 step.
Generated Placeholders and Sample Sheet Files
MiSeqDx Run (MiSeqDx) Step
The MiSeqDx Run (MiSeqDx) step records information for each lane of the flow cell 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 MiSeqDx Run (MiSeqDx) v1.2 Step
Metadata
The following metadata are stored as custom fields tagged to the MiSeqDx Run (MiSeqDx step). These fields are all read-only.
Finish Date* – run completion date
Run Type – Diagnostic or Research mode
Status – current status of the sequencing run on the instrument
There are two additional master step fields in this step:
Comments — multiline text field used for any comments that are attached to the run
Report Status — hidden read-only field that tracks if the run report has been successfully uploaded
Date Run is populated with the date that the Begin Run event file is first picked up and associated with a step in Clarity LIMS, not the date of the Begin Run event itself (the date the run was performed on the instrument). This property is provided by default in Clarity LIMS and is hidden. It is used in the AUTOMATED - Run Report Generation automation.
Finish Date is populated as follows.
If the End Run event contains a date in the format YYYY-MM-DD, Finish Date will be set to the date in the event file.
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.
RTA Primary Analysis Metrics Captured by MiSeqDx Run (MiSeqDx) Step
How it Works
The sequencing service may run on Clarity LIMS server in an on-premise environment or on a remote server in a SaaS environment. The service detects event files that the instrument software (RTA) is producing as the run progresses. These event files let the service know where to find the run data.
As the run data are written out and the events come in (Begin Run, Cycle Complete, End Run), the data are matched to the step based on the reagent cartridge ID. This value was entered as the Container Name on the Denature, Dilute and Load Sample 1.2 step. The read-only field values on the Record Details screen are populated accordingly.
AUTOMATED - Run Report Generation Automation
The automation is configured to be invoked by a button on the Record Details screen. However, it is automatically started by the sequencing service when it has finished processing the end run event.
âš It is not intended for the user to select the AUTOMATED - Run Report Generation button. The run report is generated and attached to the step automatically.
Verify Report Status Automation
The Verify Report Status automation prevents movement to the Assign Next Steps screen unless the Report Status field is set to Completed (this is done by the previous automation after the run report is attached to the step).
Variant Calling (MiSeqDx) 1.2 Step
The Variant Calling (MiSeqDx) 1.2 step attaches secondary analysis output files for each of the samples.
In addition, a Combined Variant Call File, a text file, and a variant call file for each individual sample are automatically captured following the completion of secondary analysis.
How it Works
Following primary analysis, secondary analysis runs on the MiSeqDx instrument. Clarity LIMS detects the completion of secondary analysis and lets the service know where to find the result files. While the EndRun event is processed successfully for primary analysis, the sequence service generates a BeginSecondaryAnalysis event file in the same directory with the miseqdx.seqservice.eventFileDirectory property.
An example of how Clarity LIMS shows the location of the secondary analysis event file is:
This event file signals that secondary analysis has started. The sequence service periodically checks if the analysis is complete by looking for the CompletedJobInfo.xml file. This file is in the folder specified by the runNetworkLocation parameter of the event file.
The frequency of checking is defined by the miseqdx.seqservice.synchronizationPeriod property.
If the completion file appears, the sequence service processes the event. The event is archived in a similar way to how the EndRun event file is handled. If the event completion does not appear within the number days specified by the miseqdx.seqservice.ignoreUnmatchedContainerIdsWaitDays property, the service stops monitoring the event file and the file is archived.
Captured Files
The following table describes the Variant Call Format (VCF) files that Clarity LIMS captures and attaches to the step after the secondary analysis.
Variant Call Format Files Captured by the LIMS After Secondary Analysis
Installed Components
The Illumina MiSeqDx Integration RPM package installs the following components.
This integration requires installation of the associated NGS Extensions Package (refer to Release Notes).
Scripts and Files
Properties
Refer to for the properties installed with the Illumina MiSeqDx Integration Package.
Reagent Categories/Label Groups
The following reagent categories/label groups are included in the default configuration for the MiSeqDx Integration Package:
CF 139-Variant Assay Indexes
CF 139-Variant Assay 8-Sample Indexes
CF Clinical Sequencing Assay 8-Sample Indexes
Reagent Kits
The following reagent kits are included in the default configuration for the Illumina MiSeqDx Integration Package:
CF 139-Variant Assay-Oligo Pool
CF Clinical Sequencing Assay-Oligo Pool
Custom Oligo Pool
Elution Buffer
Controls
The following controls are included in the default configuration for the MiSeqDx Integration Package:
Negative Control for MiSeqDx
PhiX Internal Control
Positive Control for MiSeqDx
Container Types
All one-dimensional container types with both numeric rows and numeric columns are supported.
The following container types are included in the default configuration for the MiSeqDx Integration Package:
The following information provides an overview of the steps performed by the Clarity LIMS support team when configuring the instrument for use with the MiSeqDx Integration to Clarity LIMS.
Configure the MiSeqDx as follows.
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 the software configuration files to call the batch files that create the event files.
To make sure that your Illumina instrument warranty remains valid, the instrument integration must be performed and maintained by the Clarity LIMS support team. To perform this integration, the Clarity LIMS support team will require direct access to the instrument via WebEx or Remote Desktop while the instrument is idle.
Configuring Instrument Names
We recommend configuring instrument names in Clarity LIMS to track the specific lab instrument on which a run was completed.
This information should be configured on the MiSeqDx Run (MiSeqDx) 1.2 step, and should be set to the same name as is configured in the Illumina sequencing software (ie, this will be value of the Instrument Run ID field described in the section).
When the integration reads the Instrument Run ID field in the run results, it will update the instrument on the step in Clarity LIMS. This update eliminates the need to enter this information manually.
For instructions on configuring instrument names, refer to the .
Configuring Sample Sheet Generation
Sample sheet generation occurs on the step prior to the sequencing run—Denature, Dilute and Load Sample 1.2—which is the step where samples are placed on the container that will be loaded in the instrument.
By default, an instrument sample sheet is created for the container loaded during the Denature, Dilute and Load Sample 1.2 step.
Rules and Constraints
This integration operates with the following constraints:
There must only be a single input on the Denature, Dilute and Load Sample 1.2 step.
The Reagent Cartridge ID must be unique. There should not be multiple containers in the system with the same name.
The reagent cartridge ID must be scanned as the Container Name on the Denature, Dilute and Load Sample 1.2 step.
Denature, Dilute and Load Sample (Illumina SBS MiSeqDx step)
MiSeqDx Run (MiSeqDx) (Illumina SBS MiSeqDx step)
Variant Calling (MiSeqDx) (Illumina SBS MiSeqDx step)
The automatic index placement pattern for CF 139-Variant Assay should not be modified.
Upon completion of the reagent index placement, a success message displays. Select OK to close the message. Indexes with the pattern specified in the placement file are then assigned to the samples.
Index placement cannot be modified for CF 139-Variant Assay. Any changes made by the user are disregarded and the automatic index placement is restored.
Secondary analysis results parsing in Variant Calling (MiSeqDx) 1.2 step.
The Container Name contains only letters and numbers, does not have special characters, and is in the format AB#######-XYZ (e.g., RG1234567-ABC).
Sets the Reagent Cartridge Barcode field attached to the output that is from the sample input.
Pads the suffix (the characters after the dash) of the Container Name so that it is at least five characters long. The Container Name is used by the MiSeqDx Sequencing Service to match the run data to the Clarity LIMS step in the MiSeqDx Run (MiSeqDx) step.
The automation sets the value of the Progress field on the sample to 'Library ready for sequencing'.
The automation starts a script that generates the sample sheet.
ℹ To maintain unique values in the Sample_Name column of the sample sheet, the sample sheet generation automation script has the --useSampleLimsID parameter set to true. The --appendLimsID parameter must not be set to true.
For the automation to display the correct instrument name when it is completed, the --isMiSeqDx parameter must be set to true.
The automation labels the derived samples that do not have reagent labels. The label value applied is NoIndex.
For more information on sample sheet generation, including details on file format and contents, master step and global fields, script parameters, and configuration options, refer to the following documentation:
Sample Sheet Generation — For details on the Research mode sample sheet designed for use with MCS software.
Flow Cell ID
Flow Cell Version
Experiment Name – entered in software
Read 1 Cycles
Index 1 Read Cycles – intended Index cycles
Index 2 Read Cycles – intended Index cycles
Read 2 Cycles
Output Folder – run folder root
Run ID – the unique run ID
Reagent Cartridge ID
Reagent Cartridge Part #
PR2 Bottle ID
Chemistry
Workflow
If the End Run event does not contain a date or the date is in the wrong format, Finish Date will be set to the date when the event file is processed.
Intensity Cycle 1 R1
Intensity Cycle 1 R2
% Intensity Cycle 20
% Intensity Cycle 20 R1
% Intensity Cycle 20 R2
% Phasing
% Phasing R1
% Phasing R2
% Prephasing
% Prephasing R1
% Prephasing R2
% Aligned
% Aligned R1
% Aligned R2
% Error Rate
% Error Rate R1
% Error Rate R2
The automatin generates the run report and sets the Report Status field to completed. Run report generation is asynchronous. The sequencing service does not wait for the report generation to complete before processing the next event file.
An additional shared result-file placeholder is configured on the step. This placeholder is for a new log file — Illumina Run Report Log File — that is generated and attached to the step when the report runs.
The run report can be successfully generated in a hosted deployment of Clarity LIMS where the sequencing service is running on a remote server on the customer network.
âš Following the sequencing run, the run report is automatically generated by the AUTOMATED - Run Report Generation automation. Do not change this name. This name is expected by the sequencing service that captures instrument run results. The base name is stored in the sequenceProcessBaseName property. If this name is changed without the property being updated, the 'flow cell ID <-> sequencing step base name' matching will fail.
/opt/gls/clarity/extensions/miseqdx/
Jar file containing API-based LIMS extensions used for report generation.
Provides commands for interacting with the sequencing service.
Accessible using systemctl. For example, use the following command to start the sequencing service:
The start keyword can be replaced with stop or status to operate on the sequencing service
This item allows you to manually attach a lab-specific tracking form to the step.
MiSeqDx Sample Sheet file
This CSV file is automatically generated by the LIMS for use with the MOS or MCS software.
May be opened as a text file, or as an MS Excel spreadsheet.
MiSeqDx Sample Sheet Generation Log
Automatically generated by Clarity LIMS, this log file captures any errors encountered when generating the MiSeqDx sample sheet.
Item
Description
Run Info
Run Parameters
These XML files are automatically captured by Clarity LIMS from the instrument run folder. They include the key run parameters, many of which are parsed out into key master step and global fields.
Link to Run Folder
Automatically generated by Clarity LIMS, this is a link to the network run folder where the data that was captured from the instrument during the run is stored.
Illumina Run Report
Automatically generated by Clarity LIMS, this report provides key information about the run and the samples on the flow cell.
Information includes the flow cell ID, run directory location, instrument name, as well as primary analysis metrics for the instrument – summarized per flow cell lane for the entire run, and individual reads in the case of paired-end runs.
These metrics are compared against the instrument's per lane averages, calculated using metrics from the last five sequencing runs. Any values outside of one standard deviation are highlighted.
The run report calculations assume that the flow cell has 8 lanes. If you are using a two-lane rapid-run flow cell, the calculated values will be incorrect.
Lab Tracking Form
This item allows you to manually attach a lab-specific tracking form to the step.
RTA Primary Analysis Metrics
Corresponding Fields
Raw Yield (Gb)
Yield PF (Gb) R1
Yield PF (Gb) R2
% Bases >Q30
% Bases >=Q30 R1
% Bases >=Q30 R2
Cluster Density (K/mm^2)
Cluster Density (K/mm^2) R1
Cluster Density (K/mm^2) R2
Clusters Raw
Clusters Raw R1
Clusters Raw R2
Clusters PF
Clusters PF R1
Clusters PF R2
%PF
%PF R1
%PF R2
File Type
Description
Combined Variant Call File
Variant call VCF files for all samples are compressed into a zip file and attached to the step for every run. Available for all 3 assays.
Combined Output Text File
For CF Clinical Sequencing Assay and CF 139-Variant Assay only, a text file containing the summary of variant call information for all samples is attached for every run.
Variant Call File per sample
The general variant call VCF file (not CFTR VCF file) for each sample is attached. Available for all 3 assays.
Files Installed
Location
Description
configure_extensions_miseqdx_workflow.sh
/opt/gls/clarity/config/
Script that imports the integration configuration.
miseqdx-extensions.jar
/opt/gls/clarity/extensions/miseqdx
Jar file containing API-based LIMS extensions used throughout the protocols.
configure_extensions_miseqdx_sequencingservice.sh
/opt/gls/clarity/config/extensions/miseqdx
Script that imports the properties for the integration.
These Release Notes describe the key changes to software components for the Clarity LIMS MiSeqDx Integration Package version 1.11.0. This is an optional software update.
if (output.container.name == output.container.node.@limsid) { fail(::The Container Name cannot be the same as its LIMS ID. Please scan the Reagent Cartridge RFID.::) }
bash -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -i {stepURI:v2} -u {username} -p {password} script:evaluateDynamicExpression -exp 'if (!step.hasValue( ::Report Status:: ) || step.::Report Status:: != ::Completed::) { fail(::Cannot advance because expected files are missing, please wait until the RunReport, RunInfo and RunParameters files are generated and attached before completing the step.::) } ' -t 'false' -log tempLog.html"
else if (!output.container.name.matches(::^[a-zA-Z0-9-]*$::)) { fail(::The Container Name can contain letters, numbers and dashes only. No other special characters are allowed. Please scan the Reagent Cartridge RFID.::) };
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 MiSeqDx Integration v1.11.0 Configuration for configurable properties.
Removal of the following obsoleted workflows and protocols:
Illumina SBS MiSeqDx (Universal Kit)
Universal Kit Library Prep
MiSeqDx Validation (Universal Kit)
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.
1
Initial release.
systemctl start miseqdx_seqservice
systemctl <start
Installation
The Illumina MiSeqDx Integration Package v1.11.0 supports MiSeqDx instruments running in Diagnostic mode. For MiSeqDx instruments running in Research mode, install the MiSeq Integration Package. For more information, refer to MiSeq Integration v8.3.0 Installation.
Compatibility
MiSeqDx Integration v1.11.0 is compatible with the following software:
Clarity LIMS v6.2 and later
Secret Util v1.0 and later
IPP v2.6 and later
Prerequisites
MiSeqDx Integration v1.11.0 has the following prerequisites.
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)
Installation
MiSeqDx Integration v1.11.0 supports both on-premise and cloud integrations. This integration is distributed as the following RPM packages:
BaseSpaceLIMS-miseqdx-extensions
BaseSpaceLIMS-miseqdx-sequencing-service
The BaseSpaceLIMS-miseqdx-extensions RPM installs the following items:
Protocols and workflows
Integration properties that configure the service
Placement pattern files that determine reagent index assignment
The BaseSpaceLIMS-miseqdx-sequencing-service RPM installs the following items:
If not found, user configuration (the glsjboss user and the glsjdk8 and claritylims user groups)
If not installed, Java 8
Bash scripts to run the miseqdx_seqservice
On-Premise Installation
Use the following instructions to install the BaseSpaceLIMS-miseqdx-extensions and BaseSpaceLIMS-miseqdx-sequencing-service RPMs on the Clarity LIMS server.
Install the RPMs
On the Clarity LIMS server, log in as the root user.
Run the following yum commands to install the RPMs:
Import Workflow Configurations for MiSeqDx
ℹ If you are upgrading from an earlier version of the MiSeqDx integration package and the system is configured with the configure_extensions_miseqdx_workflow.sh and configure_extensions_miseqdx_sequencingservice.sh scripts, refer to .
When prompted by the RPM instructions to import the workflow configuration, run the following command as the glsjboss user:
Configure the Integration Properties
For more information on the configurable integration properties, refer to .
Start the Sequencing Service
Run the following command to start the sequencing service:
Cloud Installation
The BaseSpaceLIMS-miseqdx-extensions RPM must be installed on the Clarity LIMS server. The BaseSpaceLIMS-miseqdx-sequencing-service RPM can be installed remotely on another server within the network.
Specifications
The following hardware, operating system, and network specifications must be met to install the BaseSpaceLIMS-miseqdx-sequencing-service RPM:
Hardware requirements:
Install the RPM
On the applicable server, log in as the root user.
Run the following yum command to install the RPM:
Import Workflow Configurations for MiSeqDx
ℹ If you are upgrading from an earlier version of the MiSeqDx integration package and the system is configured with the configure_extensions_miseqdx_workflow.sh and configure_extensions_miseqdx_sequencingservice.sh scripts, refer to .
When prompted by the RPM instructions to import the workflow configuration, run the following command as the glsjboss user:
Configure the Integration Properties
For more information on the properties that must be configured, refer to .
Install Sequencing Service RPM on Remote Server
On the applicable server, log in as the root user.
Run the following yum command to install the RPM:
Copy API Connection Properties File from the Clarity LIMS Server to the Remote Server
Make sure that the extensions RPM is installed on the Clarity LIMS server.
Run the following command to generate the integration.properties API connection properties file:
Start the Sequencing Service
Run the following command to start the sequencing service:
Workflows, Protocols, and Steps Installed
MiSeqDx Integration v1.11.0 installs the following protocols:
CF 139-Variant Assay Library Prep 1.2
CF Clinical Sequencing Assay Library Prep 1.2
Illumina SBS MiSeqDx (CF 139-Variant Assay) 1.2
The integrations also install the following validation protocols that are included in a workflow with the same name:
For descriptions of the protocol and the steps, refer to . For instructions on user interaction for each step and using the MiSeqDx validation workflows to validate the automated sample sheet generation, refer to .
Instrument Software
The instrument software is divided into the following modules:
MiSeqDx Operating Software (MOS) — Controls the instrument operation, including various configuration settings. This software is installed and runs on the instrument.
MiSeqDx Reporting Software (MRS) — Performs the following secondary analysis functions:
Demultiplexing
For more information on the MiSeqDx software, refer to the MiSeqDx documentation at .
Instrument Integration
Illumina provides a supported mechanism for using custom scripts on key events during a sequencing run. The Clarity LIMS support team has created batch files that plugs into these events. When the batch file is used, it reads the event information and writes 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 server where the sequencing service RPM is installed receives the event files and takes the appropriate actions.
The sequencing service monitors the following events (the actual event names may be different):
End Run — This event is used to update the sequencing steps in Clarity LIMS, captures key process data and files, and parses run statistics for output custom fields.
Begin Secondary Analysis — Indicates that secondary analysis in the MRS has started so that the sequencing service can start to monitor for results. After secondary analysis is complete, the VCF files are uploaded to Clarity LIMS.
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.
To avoid inadvertently removing or overwriting the batch file when updating the instrument software, the batch file can be placed in its own directory on the instrument computer.
Before configuring the batch files, do the following:
Configure RTA
Update the MOS configuration files as follows.
Turn off the instrument and restart the instrument computer.
Using Task Manager, make sure that MOS is not auto-launched by Windows.
Validation
Sample Sheet Generation
For instructions on how to validate the automated sample sheet generation, refer to .
Manual Invocation of Event Files
For instructions on validating the creation of event files, refer to .
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 you have 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:
The gls_events_mos_rta.bat batch file that configures RTA
Smoke test directories
Enter y to confirm that you want to proceed with the RPM installations. After confirmation, the following scripts and files are installed:
configure_extensions_miseqdx_workflow.sh
configure_extensions_miseqdx_sequencingservice.sh
miseqdx-extensions.jar (contains the sample sheet generation and other scripts)
miseqdx-sequencing-report.jar (generates the sequencing run report)
These files are installed at the following locations:
When prompted by the RPM instructions to configure the sequencing service (which includes properties with default values set for the integration), run the following command as the glsjboss user:
64-bit processor (dual core 2.0 GHz)
OS requirements, plus at least an additional 512 MB RAM
SSL access to the Clarity LIMS server from the network
A mounted network folder where the sequencing runs are written
Enter y to confirm that you want to proceed with the RPM installations. After confirmation, the following scripts and files are installed:
configure_extensions_miseqdx_workflow.sh
configure_extensions_miseqdx_sequencingservice.sh
miseqdx-extensions.jar (contains the sample sheet generation and other scripts)
miseqdx-sequencing-report.jar (generates the sequencing run report)
These files are installed at the following locations:
When prompted by the RPM instructions to configure the sequencing service (which includes properties with default values set for the integration), run the following command as the glsjboss user:
Enter y to confirm that you want to proceed with the RPM installation.
Copy the integrations.properties file from the Clarity LIMS server to the following location on the remote server:
Illumina SBS MiSeqDx (CF Clinical Sequencing Assay) 1.2
Alignment
Variant calling
Report generation
The specific functions that are supported vary by the kit. This software is installed on or off the instrument.
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.
Illumina User Management (IUM) — Contains a user database file that is used with the MiSeqDx instrument. This file controls user passwords and privileges for MOS.
Back up the MiSeq.Configuration.xml configuration file to
Make sure that the instrument is idle.
Shut down MOS.
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 file as follows
Determine the site or instrument specific network storage path root.
Change the DESTINATION_PATH line to use the storage path root and the name of the event file directory.
ℹ Make sure to include the trailing \ in the DESTINATION_PATH line. Refer to the following example:
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.
Deploy the batch file as follows
On the server where the sequencing service RPM is installed, copy the batch file from /opt/gls/clarity/extensions/miseqdx/InstrumentIntegrations to C:\Illumina\gls on the local computer.
If necessary, create the gls_events_mos_rta.bat batch file in C:\Illumina\gls. For more information, contact Illumina Support.
From the command prompt, list the contents on the C:\Illumina\gls directory using the following command:
Make sure that the name of the batch file you created does not contain any special (hidden) characters.
[Optional] If MOS is auto-launched, remove it from the auto-launch list and restart the computer.
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 <RTAConfiguration> tags:
Save and close the edited file.
Validate the file as follows.
Open MiSeq.Configuration.xml configuration file in Explorer to perform some of the XML validation.
To further validate the file, run the following command from the Command Prompt:
If the validation is successful, the following message displays:
If the configuration files contain an error, the command returns specifics about the problem. The following example shows an error that occurs when a <GLS> key is added to the file at line 83:
Start MOS.
Open the MiSeq.Configuration.xml file and make sure that the changes were saved.
The default configuration has been successfully imported.
Manual invocation of the event files has been validated. This validation checks for the following information:
The DESTINATION_PATH is configured correctly.
The instrument computer can access and write to the DESTINATION_PATH.
There are no syntax errors in the Clarity LIMS batch file.
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 *MiSeqDxIntegrator.log_ file, which logs the file name and contents of each event file that is processed.
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).
This section explains how to use the MiSeqDx Validation workflow that is included in the Illumina MiSeqDx Integration Package. The workflow allows for simple validation of the following functionality:
Auto-placement of reagent indexes on the PCR Amplification step
Generation of a sample sheet file for use with the MiSeqDx Operating Software (MOS) on the Denature, Dilute and Load Sample step
In addition, this section provides tips to help you troubleshoot the system.
All validation steps assume that you have installed the MiSeqDx Integration Package and have imported the default Clarity LIMS configuration. The validation steps are based on MiSeqDx Validation (CF 139-Variant Assay) 1.2, where the autoplacement of indexes validation occurs in Run PCR Amplification step and sample sheet generation validation occurs in Run Denature, Dilute and Load Samples step.
For compatibility information, refer to . Only Clarity LIMS v6.2 or later is supported in MiSeqDx Integration Package v1.11.0 or in subsequent releases.
Instructions for configuring sample sheet generation are provided in .
Protocols
The following protocols are included in MiSeqDx Integration Package v1.11.0.
Library Prep Protocols:
CF 139-Variant Assay Library Prep 1.2
CF Clinical Sequencing Assay Library Prep 1.2
Each validation protocol includes the following steps from the Library Prep and Illumina SBS MiSeqDx protocols:
Extension-Ligation of Bound Oligos (Library Prep step)
PCR Amplification (Library Prep step)
Library Pooling (MiSeqDx) (Illumina SBS MiSeqDx step)
Library Prep Protocols
This section discusses the Library Prep protocols included in the MiSeqDx v1.11.0 Integration Package.
Step 1: Hybridization of Oligo Pool (CF 139-Variant Assay) 1.2
The following process is used to hybridize the oligo pool.
Add samples to Ice Bucket.
Add Negative Control and Positive Control samples to Ice Bucket.
ℹ Good laboratory practices mandate that a positive control DNA sample and a negative (no-template) control sample are included in every run. The positive control DNA sample should be a well-characterized sample with a known CFTR mutation.
Step 2: Removal of Unbound Oligos (CF 139-Variant Assay) 1.2
The process below is used to remove unbound oligos.
Add samples to Ice Bucket.
Select Begin Work.
On the Placement screen, select all samples and place in container.
Step 3: Extension-Ligation of Bound Oligos (CF 139-Variant Assay) 1.2
The process below is used to add reagents to the Extension-Ligation Mix field.
Add samples to Ice Bucket. Select Begin Work.
On the Placement screen, select all samples and place in container.
In Lab View, locate the MiSeqDx Validation protocol. You will see your samples queued for the Extension-Ligation of Bound Oligos step. Prepare the samples as follows.
Add the samples to the Ice Bucket.
On the Placement screen, place the samples into the output container.
PCR Amplification Step
In Lab View, locate the MiSeqDx Validation protocol. You will see your samples queued for the PCR Amplification step.
Add the samples to the Ice Bucket.
In the Reagent Label Options panel, in the Group of labels list, select CF 139-Variant Assay Indexes (or the equivalent for the workflow you activated).
Library Pooling (MiSeqDx) Step
Return to Lab View and locate the MiSeqDx Validation protocol. You will see your samples queued for the Library Pooling (MiSeqDx) 1.2 step.
Prepare the samples as follows.
Add samples to the Ice Bucket.
In the Add Control Samples panel, select PhiX Internal Control. Select Begin Work.
Denature, Dilute and Load Sample Step
Return to Lab View and locate the MiSeqDx Validation protocol.
You will see your pooled samples queued for the Denature, Dilute and Load Sample step.
Place the pooled samples as follows.
Add the pool to the Ice Bucket.
Validation of auto-placement of indexes and sample sheet generation is completed at the end of this step.
MiSeqDx Run (MiSeqDx) Step
On the Record Details screen, do not select the AUTOMATED - Run Report Generation button. The run report is generated and attached to the step automatically. This process may take a while.
On the Record Details screen of the MiSeqDx Run (MiSeqDx) 1.2 step:
Run the instrument. The read-only field values are automatically populated as the instrument runs.
After the run has completed, observe the Record Details screen of the MiSeqDx Run (MiSeqDx) 1.2 step for the following.
The autopopulated read-only fields.
Variant Calling (MiSeqDx) Step
On the Record Details screen of the Variant Calling (MiSeqDx) 1.2 step:
Make sure that the VCF has the correct name based on the sample sheet.
The file-naming format for the VCF file is SampleName_S#.vcf. In this format, the SampleName is the value in the Sample_Name column of the sample sheet, and # is the sample number determined by the order in which samples are listed in the sample sheet.
After secondary analysis has completed, the following files are attached to the step:
Illumina SBS MiSeqDx Protocol
Sort MiSeqDx Samples (MiSeqDx) Step
This step routes your samples to the appropriate next step. Assign each sample to one of the following steps:
Library Normalization (MiSeqDx)
Library Pooling (MiSeqDx)
Denature, Dilute and Load Sample
Library Normalization (MiSeqDx) Step
On the Record Details screen, enter values for the following items:
Library volume (µl) transferred to destination plate
Normalized conc (nM)
Library Pooling (MiSeqDx) Step
Refer to under MiSeqDx Validation Protocol for details.
Denature, Dilute and Load Sample Step
On the Placement screen, set up the reagent cartridge as follows.
Using an RFID scanner, scan the RFID of the MiSeqDx reagent cartridge into the Container Name field.
Place the pool of samples into the reagent cartridge.
Refer to under MiSeqDx Validation Protocol for more details.
On exit from the Placement screen, the automation is automatically triggered and validates the Container Name.
MiSeqDx Run (MiSeqDx) Step
Refer to under MiSeqDx Validation Protocol for details.
Variant Calling (MiSeqDx) Step
Refer to under MiSeqDx Validation Protocol for details.
Validate Creation of Event Files
Validating the creation of the event files confirms the following:
Your 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.
Follow the steps below 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 gls_event_mos_rta.bat.
Confirm that an empty event file appears in the configured DESTINATION_PATH.
Manually invoke the gls_event_mos.bat file.
The file produces an output similar to the following example:
Troubleshooting
If an automation trigger does not appear to run its corresponding scripts, see:
Troubleshooting Automation Worker in the .
Troubleshooting Automation in the .
If an error occurs that does not provide direction on how to proceed, troubleshoot the error as follows.
Confirm the version of the installed Illumina MiSeqDx Integration Package by running the following command from the server console.
If the error is related to retrieving run results data, review the MiseqDxIntegrator.log.
Contact the Clarity LIMS Support team, supplying the relevant information from the troubleshooting already performed.
Error: While loading Configuration "c:\illumina\rta\configs\MiSeq.Configuration.xml"
There is an error in XML document <83, 5>
Error loading xml file: Unknown node 'GLS' at line 83 pos 5
Illumina SBS MiSeqDx Protocols:
Illumina SBS MiSeqDx (CF 139-Variant Assay) 1.2
Illumina SBS MiSeqDx (CF Clinical Sequencing Assay) 1.2
Denature, Dilute and Load Sample (Illumina SBS MiSeqDx step)
MiSeqDx Run (MiSeqDx) (Illumina SBS MiSeqDx step)
Variant Calling (MiSeqDx) (Illumina SBS MiSeqDx step)
Select Begin Work.
On the Placement screen, select all samples and place in container. Select Record Details.
On the Record Details screen, under Reagent Lot Tracking, set the following values:
gDNA Concentration (ng/uL): 50 ng/ul
gDNA Volume (uL): 5 uL
Select the reagent lots for CF 139-Variant Assay-Oligo Pool and Hybridization Buffer.
Select Next Steps.
Select Removal of Unbound Oligos (CF 139-Variant Assay) 1.2 as the next step.
Select Apply.
Select Finish Step.
Select Record Details.
On the Record Details screen, under Reagent Lot Tracking, select the reagent lots for Stringent Wash Buffer and Universal Wash Buffer.
Select Next Steps.
Select Extension-Ligation of Bound Oligos (CF 139-Variant Assay) 1.2 as the next step.
Select Apply.
Select Finish Step.
On the Record Details screen, under Reagent Lot Tracking, select the reagent lots for Extension-Ligation Mix.
Select Next Steps.
Select PCR Amplification (CF 139-Variant Assay) 1.2 as the next step.
Select Apply.
Select Finish Step.
If you are working with more than 8 samples, select CF 139-Variant Assay Indexes.
Select Begin Work.
On the Placement screen, select all samples and place in container. Select Add Reagents.
On entry to the Add Reagents screen, the Auto Place Indexes automation is invoked and the reagents are placed on the samples.
Once placement is complete, a message displays indicating that the index pattern has been applied successfully. Select OK.
On Add Reagents screen, reagents are already placed. Select Record Details.
On exit from the Add Reagents screen, the index placement is validated. Once validation is complete, a message displays indicating that the index pattern has been validated on all samples successfully. Select OK.
On the Record Details screen, under Reagent Lot Tracking, enter the lots for the following fields:
Index Primers
NaOH
PCR Master Mix
PCR Polymerase
Select Next Steps.
Select PCR Clean-Up (CF 139-Variant Assay) 1.2 as the next step.
Select Apply.
Select Finish Step.
On the Record Details screen, under Reagent Lot Tracking, select the reagent lots for the following:
Elution Buffer
EtOH
PCR Clean-up Beads
Select Next Steps.
Select Mark protocol as complete as the next step. Select Apply.
Select Finish Step.
ℹ The validation steps are based on MiSeqDx Validation (CF 139-Variant Assay) 1.2
On the Projects and Samples screen, create a project and assign samples as follows.
Create a test project and add samples to it.
âš The project must contain between 9 and 96 samples.
Assign your samples to the MiSeqDx Validation workflow.
Select Record Details.
On the Record Details screen, under Reagent Lot Tracking, select the Extension-Ligation Mix reagent lot used in the step.
If required, activate the lot on the Reagents / Reagents and Controls screen.
Select Next Steps.
On the Assign Next Steps screen, assign the samples to the PCR Amplification step.
Select Finish Step.
Select Begin Work.
On the Placement screen, place the samples into the output container.
Select Add Reagents.
On entry to the Add Reagents screen, the Auto Place Indexes automation is automatically invoked and the reagents are placed on the samples.
After placement is complete, a message displays indicating that the index pattern has been applied successfully. Select OK.
On the Add Reagents screen, reagents are already placed. Select Record Details.
On exit from the Add Reagents screen, the Validate Index Placement automation is automatically invoked and index placement is validated.
When validation is complete, a message displays indicating that the index pattern has been validated on all samples successfully. Select OK.
On the Record Details screen, under Reagent Lot Tracking, select the lots for the following items:
Index Primers
NaOH
PCR Master Mix
PCR Polymerase
Select Next Steps.
On the Assign Next Steps screen, assign samples to the Library Pooling (MiSeqDx) 1.2 step.
Select Finish Step.
On the Pool Samples screen, create a pool of samples. Select Place Samples.
On the Placement screen, place the pool into the output container. Select Record Details.
Under Reagent Lot Tracking, select the reagent lot used in the step.
Under Step Details, enter the concentration value in the Normalized conc. (nM) field.
Select Next Steps.
On the Assign Next Steps screen, assign the pool to the Denature, Dilute and Load Samples step.
On the Place Samples screen, the Validate Single Input script runs and validates only one pool that is entered into this step as the input sample. If validation fails, Clarity LIMS prompts you that it is only able to proceed with the step with one pool.
On the Placement screen, place the pool into a MiSeqDx reagent cartridge.
Select Record Details.
On entry to the Record Details screen, a script validates the Container Name and prompts you to scan the reagent cartridge barcode.
On the Record Details screen of the Denature, Dilute and Load Sample step, generate the sample sheet as follows.
ℹ All read-only files are autopopulated.
Enter the folder path for the Reference Genomes that will be used for secondary analysis in the GenomeFolder field.
Under Reagent Lot Tracking, select the reagent lots used in the step.
Enter the lots for MiSeqDx Flow Cell - CF 139-Variant Assay and MiSeqDx SBS Solution (PR2) - CF 139-Variant Assay.
In the Step Details section, review the step parameters.
Select Generate MiSeqDx Sample Sheet to generate the MiSeqDx sample sheet.
Clarity LIMS attaches the generated sample sheet and log file to placeholders in the Files area of the Record Details screen. Download the files and validate their format and content.
[Optional] In the Files section, upload the Lab Tracking Form to the appropriate placeholder.
Select Next Steps.
On the Assign Next Steps screen, assign the samples to the MiSeqDx Run (MiSeqDx) 1.2 step.
The generated and attached Illumina Run Report and Log File.
The attached Run Parameters and Run Info files.
The generated and attached Link to Run Folder.
QC flags set on each lane.
[Optional] In the Files section, upload the Lab Tracking Form.
Select Next Steps.
The Verify Report Status automation should run and allow you to go to the Assign Next Steps screen.
On the Assign Next Steps screen, assign the samples to the Variant Calling (MiSeqDx) 1.2 step.
Select Finish Step.
Combined Variant Call File — a compressed VCF file (zip file) containing all the variant call files, including the CFTR specific VCF files.
Combined Output Text File — a combined text file containing a summary of all the sample metrics.
Variant Call File per individual sample
QC flags are set on each lane.
Maximum destination container volume (µl)
Any other required fields
Select Create Normalization CSV to start the automation.
When the normalizationBufferVolumes script has completed, the Normalization buffer volumes CSV file is generated and attached to the step. This file is in the Files area of the Record Details screen.
