Configuration
The Illumina MiSeq Integration Package v8.2.0 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).
For instructions on user interaction for each step, validating and troubleshooting the Illumina MiSeq Integration Package, refer to MiSeq Integration v8.2.0 User Interaction, Validation and Troubleshooting.
Compatibility
The MiSeq Sequencing v3.2 workflow is only compatible with MiSeq Integration Package v8.2.0.
MiSeq Integration v8.2.0 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. 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 Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
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 Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
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.
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
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-down Items
Comment
Multiline Text
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)
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
Sequencing Coverage
Text
Sequencing Method
Text Dropdown
Custom Entries
Presets
Single Read
Paired End Read
Indexed Single Read
Indexed Paired End Read
Global Field Configuration (Derived Sample)
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-Down Items
Normalized Molarity (nM)
Numeric
Decimal places displayed = 2
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.
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
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
Aligner-Key
ℹ️ Used for sample sheet generation
Text
Read Only
Hidden
Aligner-Value
ℹ️ Used for sample sheet generation
Text
Read Only
Annotation
Text Dropdown
Presets
None (default)
RefSeq
Ensembl
Applicable analysis fields for the selected Workflow
Multiline Text
Read Only
ℹ️ 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
Index
Read 2
Read 1, Index
Read 1, Index, Read 2
Read 1, Read 2
Index, Read 2
Description
Text
Experiment Name
Text
Required Field
Export to gVCF
Text Dropdown
Presets
None (default)
Yes
No
Flag PCR Duplicates
Text Dropdown
Presets
None (default)
Yes
No
Genome Folder
Text
Indel Realignment
Text Dropdown
Presets
None (default)
Yes
No
Indel Repeat Filter Cutoff
Text Dropdown
Presets
None (default)
Yes
No
Indel-Realignment-Key
ℹ️ Used for sample sheet generation
Text
Read Only
Hidden
Indel-Realignment-Value
ℹ️ Used for sample sheet generation
Text
Read Only
Hidden
Manifest
Text
Manifest Padding
Text Dropdown
Presets
None (default)
0
50
100
150
200
250
Manifest-Section
ℹ️ Used for sample sheet generation
Text
Read Only
Hidden
Picard HS Metric Reporting
Text Dropdown
Presets
None (default)
Yes
No
Read 1 Cycles
Numeric Dropdown
Required Field
Custom Entries
Presets
251 (Default)
151
101
76
51
Range = 1–1000
Decimal places displayed = 0
Read 2 Cycles
Numeric Dropdown
Required Field
Custom Entries
Presets
251 (Default)
151
101
76
51
Range = 0–1000
Decimal places displayed = 0
Read Stitching
Text Dropdown
Presets
None (default)
Yes
No
Reverse Complement
Text Dropdown
Presets
None (default)
Yes
No
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
ℹ️ Used for automation
Multiline Text
Required Field
Read Only
Hidden
Validation Script 2
ℹ️ Used for automation
Multiline Text
Required Field
Read Only
Hidden
Variant Caller
Text Dropdown
Presets
None (Default)
GATK
Germline
Somatic
Starling
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
ℹ️ Used for sample sheet generation
Text
Read Only
Hidden
Workflow
Text Dropdown
Required Field
Presets
GenerateFASTQ
LibraryQC
Resequencing
DNA Enrichment
DNA Amplicon
Groups of Defaults
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:
ℹ️ The Submitted Sample field, Progress, (added from previous MiSeq workflow) is obsolete in MiSeq v3.2.
Global Field Configuration (Submitted Sample)
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-Down Items
Read Length
ℹ️ Displays on Queue & Ice Bucket screens
Text
Sequencing Method
ℹ️ Displays on Queue & Ice Bucket screens
Text Dropdown
Custom Entries
Presets
Single Read
Paired End Read
Indexed Single Read
Indexed Paired End Read
Global Field Configuration (Derived Sample)
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-Down Items
Final Loading Concentration
ℹ️ Displays on Record Details screen
Numeric Dropdown
Required Field
Custom Entries
Presets
225
400
Decimal places displayed = 0
Step File Placeholders
Placeholders for the following files are configured on the Record Details screen of the Denature and Dilute (MiSeq v3.2) step.
Step 3: MiSeq Run (MiSeq v3.2)
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
Field Name
Field Type
Options
Additional Options and Drop-Down Items
Chemistry
Text
Read Only
Comment
Multiline Text
Experiment Name
Text
Read Only
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
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 Sequencing Results Parsing.
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)
Run Info (automatically attached)
Lab Tracking Form (manually uploaded)
Log File (automatically attached)
For details, refer to Generated and Captured Files.
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 Illumina Instrument Sample Sheets (NGS v5.17 & later).
For instructions on customizing the template, refer to Creating Template Files.
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
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.
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)
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
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
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)
% Aligned R1
% Aligned R1
% Aligned R2
% Aligned R2
% Bases >=Q30 R1
% Bases >=Q30 R1
% Bases >=Q30 R2
% Bases >=Q30 R2
% Error Rate R1
% Error Rate R1
% Error Rate R2
% Error Rate R2
% 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
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.
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.
Scripts and Files Installed
This integration requires components installed with the Illumina Preset Protocols (IPP).
The Illumina MiSeq Integration Package v8.2.0 RPM installs the scripts and files listed in the following table.
Component
Location
Description
configure_extensions_miseq_sequencingservice-v8.sh
/opt/gls/clarity/config/
Script that installs the service properties in the database.
log4j.xml
/opt/gls/clarity/extensions/Illumina_MiSeq/v8/SequencingService/conf
File containing the settings for logging the sequencing jar.
miseq-sequencing-v8.jar
/opt/gls/clarity/extensions/Illumina_MiSeq/v8/SequencingService
Jar file containing API-based Clarity LIMS extensions used for capturing run results and report generation.
InterOp libraries
/opt/gls/clarity/extensions/Illumina_MiSeq/v8/lib
Illumina shared library for parsing InterOp data files.
MiSeqSamplesheetv3.csv
ℹ️ Installed by IPP
/opt/gls/clarity/extensions/conf/driverfiletemplates
Template file used for file generation.
Properties Installed
The properties installed in the Illumina MiSeq Integration v8.2.0 are described in the following section.
The integration also installs several additional properties, each with the 99 suffix appended to their name, that are not listed in the table. These properties are intended for use by the Clarity LIMS support team in automated validation tests.
Sequencing runs are matched using the reagent cartridge ID and the base name of the sequencing step, MiSeq Run (MiSeq v3.2).
⚠️ Do not change the base name. The sequencing service that captures sequencing run results expects this name. The base name is stored in the sequenceProcessBaseName property. If the name is changed without the property being updated, the reagent cartridge ID <-> sequencing step base name matching system fails. If you need to rename the step, for example to add a version number, modify the text before or after the base name portion. This part of the name is not used in the matching system. For example, change MiSeq Run (MiSeq v3.2) to Demo-MiSeq Run (MiSeq v3.2)-Updated.
ℹ️ Changes on miseq.v8.seqservice.sequenceProcessBaseName property 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 MiSeq Integration Package
Property
Description
Default Value
miseq.v8.seqservice.sequenceProcessBaseName
Sequencing process type/master step base display name. Partial matching is used to look up the process type/master step. This property is added/updated by Illumina Preset Protocols (IPP) during the workflow installation.
MiSeq Run (MiSeq v3.2)
miseq.v8.seqservice.eventFileDirectory.1
A network location monitored for event files, e.g., /mnt/illumina/gls_events/
/mnt/gls_events
miseq.v8.seqservice.netPathPrefixSearch.1
The network directory prefix contained in the event file.
\\nas\network\run_data
miseq.v8.seqservice.netPathPrefixReplace.1
The mapped network directory mount name on the server used to access the run data directory, e.g., /mnt/network/data
/mnt/run_data
miseq.v8.seqservice.eventFileDirectorySuffixes
A list of eventFileDirectory path entries to monitor for event files. The value is one or more comma-separated integers.
99
ℹ️ Configured on install to point to 1
miseq.v8.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
ℹ️ Configured on install to point to 1
miseq.v8.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
miseq.v8.seqservice.ignoreUnmatchedContainerIds
A flag indicating if event files that cannot be matched to reagent cartridges in Clarity LIMS should be archived after a certain period (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
miseq.v8.seqservice.ignoreUnmatchedContainerIdsWaitDays
The number of days between when the event is created and the event file is archived.
14
miseq.v8.seqservice.synchronizationPeriod
Invocation period in seconds.
60
You can configure support for multiple identical seqservice.netPathPrefixSearch property values. For more information, refer to Configure Multiple Identical netPathPrefixSearch Values.
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.
Update sequencing service default properties to match the specifics of the installation.
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.
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