Configuration

The Illumina MiSeqDx Integration Package v1.10.0 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 v1.10.0:

• 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

• Illumina SBS MiSeqDx (Universal Kit) 1.2

• Universal Kit Library Prep 1.2

There are three validation protocols. Each protocol is included in a workflow with the same name. The protocols are as follows.

• MiSeqDx Validation (CF 139-Variant Assay) 1.2

• MiSeqDx Validation (CF Clinical Sequencing Assay) 1.2

• MiSeqDx Validation (Universal Kit) 1.2

ℹ️ 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)

• Denature, Dilute and Load Sample (Illumina SBS MiSeqDx step)

• MiSeqDx Run (MiSeqDx) (Illumina SBS MiSeqDx step)

• Variant Calling (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 MiSeqDx Integration v1.10.0 User Interaction, Validation and Troubleshooting.

The following table lists the automations included in this integration, and the steps on which they are configured.

MiSeqDx Integration Default Automations

Protocol	Step	Automations on Step
CF 139-Variant Assay Library Prep 1.2 Universal Kit Library Prep 1.2	PCR Amplification (CF 139-Variant Assay/Universal Kit) 1.2	Auto Place Indexes Validate Index Placement
Illumina SBS MiSeqDx (CF 139-Variant Assay) 1.2 Illumina SBS MiSeqDx (CF Clinical Sequencing Assay) 1.2 Illumina SBS MiSeqDx (Universal Kit) 1.2	Library Normalization (MiSeqDx) 1.2	Create Normalization CSV
Illumina SBS MiSeqDx (CF 139-Variant Assay) 1.2 Illumina SBS MiSeqDx (CF Clinical Sequencing Assay) 1.2 Illumina SBS MiSeqDx (Universal Kit) 1.2 MiSeqDx Validation (CF 139-Variant Assay) 1.2 MiSeqDx Validation (CF Clinical Sequencing Assay) 1.2 MiSeqDx Validation (Universal Kit) 1.2	Denature, Dilute and Load Sample (CF 139-Variant Assay / CF Clinical Sequencing Assay / Universal Kit) 1.2	Generate MiSeqDx Sample Sheet Validate Container Name Validate Single Input
Illumina SBS MiSeqDx (CF 139-Variant Assay) 1.2 Illumina SBS MiSeqDx (CF Clinical Sequencing Assay) 1.2 Illumina SBS MiSeqDx (Universal Kit) 1.2 MiSeqDx Validation (CF 139-Variant Assay) 1.2 MiSeqDx Validation (CF Clinical Sequencing Assay) 1.2 MiSeqDx Validation (Universal Kit) 1.2	MiSeqDx Run (MiSeqDx) 1.2	AUTOMATED - Run Report Generation Verify Report Status

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 and the Universal Kit Library Prep 1.2 protocols, 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.

  ⚠️ For the Auto Place Indexes automation, if an 8-sample reagent category/label group is selected, index placement must be performed manually.

  ℹ️ 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 Installed Components.

⚠️ 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.

PCR Amplification (CF 139-Variant Assay) 1.2 Step

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.

  bash -l -c "/opt/gls/clarity/bin/java -cp /opt/gls/clarity/extensions/ngs-common/v5/EPP/PlacementHelper.jar place_indexes -i {stepURI:v2} -u {username} -p {password} -f /opt/gls/clarity/extensions/conf/miseqdx/placementpatterns/placement_pattern_index_cf139_01.tsv -ic 'CF 139-Variant Assay 8-Sample Indexes' -locked true"

• 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.

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:

  • 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.

  bash -l -c "/opt/gls/clarity/bin/java -cp /opt/gls/clarity/extensions/ngs-common/v5/EPP/PlacementHelper.jar place_indexes -i {stepURI:v2} -u {username} -p {password} -f /opt/gls/clarity/extensions/conf/miseqdx/placementpatterns/placement_pattern_index_cf139_01.tsv -ic 'CF 139-Variant Assay 8-Sample Indexes' -locked true -fc true"

PCR Amplification (Universal Kit) 1.2 Step

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.

  bash -l -c "/opt/gls/clarity/bin/java -cp /opt/gls/clarity/extensions/ngs-common/v5/EPP/PlacementHelper.jar place_indexes -i {stepURI:v2} -u {username} -p {password} -f /opt/gls/clarity/extensions/conf/miseqdx/placementpatterns/placement_pattern_index_universal_01.tsv -ic 'Universal Kit 8-Sample Indexes'"

• 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.

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:

  • Index placement is user-modifiable for Universal Kit. Any changes made by the user are preserved.

  bash -l -c "/opt/gls/clarity/bin/java -cp /opt/gls/clarity/extensions/ngs-common/v5/EPP/PlacementHelper.jar place_indexes -i {stepURI:v2} -u {username} -p {password} -f /opt/gls/clarity/extensions/conf/miseqdx/placementpatterns/placement_pattern_index_universal_01.tsv -ic 'Universal Kit 8-Sample Indexes' -fc true"

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.

• Secondary analysis results parsing in Variant Calling (MiSeqDx) 1.2 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 Normalization Buffer Volumes.

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.

bash -l -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:validateSampleCount -min 1 -max 1"

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.

  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.::) }

• The Container Name contains only letters and numbers, does not have special characters, and is in the format AB#######-XYZ (e.g., RG1234567-ABC).

  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.::) };

• Sets the Reagent Cartridge Barcode field attached to the output that is from the sample input.

  output.::Reagent Cartridge Barcode:: = output.container.name;

• 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.

  def match = output.container.name =~ /([a-zA-Z0-9]+-)([a-zA-Z0-9]{0,5})$/;
  if (match.matches()) {
      int barcodeSuffixLength = 5;
      int zerosToAdd = barcodeSuffixLength - match.group(2).size();
      output.container.::name:: = match.group(1).toString() + ::0::*zerosToAdd + match.group(2).toString()
  }

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 automation sets the value of the Progress field on the sample to 'Library ready for sequencing'.

  /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -u {username} -p {password} -i {processURI:v2} script:setUDF -f 'Progress' -t '//input/@uri->//sample/@uri' -v 'Library ready for sequencing'

• The automation starts a script that generates the sample sheet.

  && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/miseqdx/miseqdx-extensions.jar -u {username} -p {password} -i {processURI:v2} script:generate_miseqdx_sample_sheet -c {compoundOutputFileLuid1} -e {compoundOutputFileLuid2} --useSampleLimsID true --isMiSeqDx true

  ℹ️ 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.

  && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -u {username} -p {password} -i {processURI:v2} script: labelNonLabeledOutputs -l '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:

• Illumina MiSeqDx 1.10.0 Sample Sheet Generation — For details on the Diagnostic mode sample sheet designed for use with MOS software.

• Sample Sheet Generation — For details on the Research mode sample sheet designed for use with MCS software.

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

Item	Description
Lab Tracking Form	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.

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

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.

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

• 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

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

ℹ️ 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.

• 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.

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

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
Intensity Cycle 1	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

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.

  ⚠️ 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.

• 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.

  bash -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/miseqdx/miseqdx-sequencing-report.jar -i {processLuid} -r {compoundOutputFileLuid0}_RunReport.pdf -l {compoundOutputFileLuid1}_RunReportLogFile.html && /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 'step.::Report Status:: = ::Completed::' -t 'false' -log tempLog.html"

• 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.

  ⚠️ 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.

  

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).

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"

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:

netFolder = D:\Illumina\MiSeqOutput\161017_M99999_0047_FC1234567-ABCDErunNetworkLocation = /mnt/ussd-dev-mdx01/MiSeqOutput/161017_M99999_0047_FC1234567-ABCDEeventType = BeginSecondaryAnalysissoftwareType = MRScreationTime = Thu Oct 20 22:56:32 GMT 2016

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

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.

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

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.
log4j2.xml	/opt/gls/clarity/extensions/miseqdx/SequencingService/conf	File containing the settings for the sequencing jar logging.
miseqdx-sequencing-report.jar	/opt/gls/clarity/extensions/miseqdx/	Jar file containing API-based LIMS extensions used for report generation.
miseqdx-sequencing.jar	/opt/gls/clarity/extensions/miseqdx/SequencingService	Sequencing service jar file that captures the run results.
miseqdx_seqservice	/opt/gls/clarity/extensions/miseqdx/SequencingService/bin	Provides commands for interacting with the sequencing service. Accessible using systemctl. For example, use the following command to start the sequencing service: systemctl start miseqdx_seqservice The start keyword can be replaced with stop or status to operate on the sequencing service systemctl <start|stop|status> miseqdx_seqservice
run_miseqdx_seqservice	/opt/gls/clarity/extensions/miseqdx/SequencingService/bin	Called by miseqdx_seqservice-v1, this script performs some basic system validation, then starts the sequencing service.
MiSeqDxIntegrator.log	/opt/gls/clarity/extensions/miseqdx/SequencingService	Log file for Sequencing service.

Properties

The following table lists the properties installed with the Illumina MiSeqDx Integration Package. The following constraints are present when using the properties:

• Sequencing runs are matched using the flow cell ID and the base name of the sequencing step – MiSeqDx Run (MiSeqDx).

• Do not change this name – it is expected by the sequencing service that captures instrument run results. The base name is stored in the sequenceProcessBaseName property shown in Table 2. If this name is changed without the property being updated, the 'flow cell ID <-> sequencing step base name' matching system will fail.

• If necessary, you may modify the step name by editing or adding to the text after the base name portion. This part of the text is not used in the matching system. For example, you could change MiSeqDx Run (MiSeqDx) 1.2 to MiSeqDx Run (MiSeqDx) v1.2.

Several additional properties, each with the ‘99’ suffix appended to their name, are also installed. These properties are intended for use by the Clarity LIMS support team in automated validation tests and are not listed in the table.

ℹ️ Changes on miseqdx.seqservice.sequenceProcessBaseName and miseqdx.seqservice.variantCallingProcessBaseName properties take effect upon updates and do not require restart of the integration service. For all remaining properties, integration service has to be restarted for property changes to take effect.

Properties Installed with the Illumina MiSeqDx Integration Package

Property	Description	Default Value
miseqdx.seqservice.ignoreUnmatchedContainerIds	A flag indicating if event files that cannot be matched to flow cells in Clarity LIMS should be archived after a period of time (true), or continually reprocessed (false).	false ℹ️ To prevent the gls_events file directory from becoming cluttered, it is recommended that the value of this property is set to ‘true’.
miseqdx.seqservice.ignoreUnmatchedContainerIdsWaitDays	The number of days between when the event is created and the event file is archived.	14
miseqdx.seqservice.synchronizationPeriod	Invocation period in seconds.	60
miseqdx.seqservice.sequenceProcessBaseName	Sequencing process type base display name. Partial matching is used to look up the process type.	MiSeqDx Run (MiSeqDx)
miseqdx.seqservice.eventFileDirectory.1	A network location monitored for event files.	/mnt/gls_events
miseqdx.seqservice.netPathPrefixSearch.1	The network directory prefix contained in the event file.	\\nas\network\run_data
miseqdx.seqservice.netPathPrefixReplace.1	The mapped network directory mount name on the server used to access the run data directory.	/mnt/run_data
miseqdx.seqservice.eventFileDirectorySuffixes	A list of eventFileDirectory path entries to monitor for event files. The value is one or more comma-separated integers.	99
miseqdx.seqservice.runReportViewsVersion	The current version of the Run Report views in the database. The value 0 represents the state before the views are created. This property is automatically updated by the run report.	0
miseqdx.seqservice.netPathPrefixSearchReplaceSuffixes	A list of netPathPrefix search and replace entries for transforming Windows to Linux network paths. The value is one or more comma-separated integers.	99
miseqdx.seqservice.variantCallingProcessBaseName	Sequencing process type base display name. Partial matching is used to look up the process type.	Variant Calling (MiSeqDx)

It is possible to configure support for multiple, identical seqservice.netPathPrefixSearch property values. For details, refer to Configure Multiple Identical netPathPrefixSearch Values.

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

• Universal Kit Indexes

• Universal Kit 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

• EtOH

• Extension-Ligation Mix

• Hybridization Buffer

• Index Primers

• Library Beads

• Library Dilution Buffer

• Library Normalization Diluent

• Library Normalization Wash

• Library Storage Buffer

• MiSeqDx Flow Cell - CF 139-Variant Assay

• MiSeqDx Flow Cell - CF Clinical Sequencing Assay

• MiSeqDx Flow Cell - Universal kit

• MiSeqDx SBS Solution (PR2) - CF 139-Variant Assay

• MiSeqDx SBS Solution (PR2) - CF Clinical Sequencing Assay

• MiSeqDx SBS Solution (PR2) - Universal Kit

• NaOH

• PCR Clean-Up Beads

• PCR Master Mix

• PCR Polymerase

• Stringent Wash Buffer

• Universal Wash 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:

• MiSeqDx Reagent Cartridge - CF 139-Variant Assay

• MiSeqDx Reagent Cartridge - CF Clinical Sequencing Assay

• MiSeqDx Reagent Cartridge - Universal Kit

Instrument Integration

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.

1. 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.

2. Create a directory on the NAS to hold the event files.

3. Modify the software configuration files to call the batch files that create the event files.

4. Update sequencing service default properties to match the specifics of the installation.

ℹ️ 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 Metadata 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 Clarity LIMS (Clarity & LabLink Reference Guide) documentation.

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.

• For MOS sample sheet generation constraints, refer to Illumina MiSeqDx 1.10.0 Sample Sheet Generation.