Configuration
Last updated
Last updated
The Illumina NextSeq 500/550 Integration Package v2.3.0 supports the integration of Clarity LIMS to Illumina NextSeq 500 and 550 sequencing systems.
The integration allows for automated tracking of an Illumina sequencing run in Clarity LIMS, which includes tracking instrument 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 bcl2fastq2 v2.20.0 analysis software.
This document describes the integration between Clarity LIMS and the NextSeq system. It includes information about protocols and automations, configuration options, installed components, and rules and constraints.
For instructions on user interaction for each step, validating and troubleshooting the NextSeq 500/550 Integration Package, refer to .
It is assumed that samples enter the NextSeq 500/550 Sequencing v1.1 workflow as normalized libraries and have reagent labels attached.
That is, before they are assigned to the workflow:
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 Getting Started section of the .
You can assign samples to workflows automatically, using a routing script, or manually — from the Projects & Samples dashboard. Refer to Assign and Process Samples in the .
NextSeq Integration Package v2.3.0 includes the NextSeq 500/550 Sequencing v1.1 workflow, which contains a single protocol of the same name.
The NextSeq 500/550 Sequencing v1.1 protocol includes the following steps:
Library Pooling (NextSeq 500/550 v1.1)
Denature & Dilute (NextSeq 500/550 v1.1)
NextSeq 500/550 Run (NextSeq 500/550 v1.1)
The Library Pooling (NextSeq 500/550 v1.1) step is derived from the Library Pooling v1.0 master step. Libraries are placed into pools manually.
This field is configured on the Library Pooling (NextSeq 500/550 v1.1) step and displays on the Record Details screen at run time.
The following table shows field configuration details.
Library Pooling (NextSeq 500/550 v1.1) Master Step Field Configuration
The following table lists the global custom fields configured to display on the Queue and Ice Bucket screens of the Library Pooling (NextSeq 500/550 v1.1) step. Most of these fields show in the expanded view only.
Global Custom Field Configuration (Submitted Sample)
Global Custom Field Configuration (Derived Sample)
In this step, pooled libraries are denatured and diluted and placed into the reagent cartridge that is loaded into the NextSeq instrument.
There are 10 fields configured on the Denature and Dilute (NextSeq 500/550 v1.0) step. At run time, these fields display on the Record Details screen, in the Step Data table. The fields are manually populated. Their values are used to generate the sample sheet.
The following table lists field configuration details.
Denature and Dilute (NextSeq 500/550 v1.1) Master Step Field Configuration
Groups of Defaults
The following table lists the global fields that are configured to display on the Queue, Ice Bucket, and Record Details screens of the Denature and Dilute (NextSeq 500/550 v1.1) step.
Global Custom Field Configuration (Submitted Sample)
Global Custom Field Configuration (Derived Sample)
Placeholders for the following files are configured on the Record Details screen of the Denature and Dilute (NextSeq 500/550 v1.1) step
In this step, pooled samples are sequenced on the NextSeq 500/550 instrument and the run metrics are recorded in Clarity LIMS.
There are 16 fields configured on the NextSeq 500/550 Run (NextSeq 500/550 v1.1) step. These fields display on the Record Details screen at run time. Some of the field values must be completed manually, and the remaining fields are automatically populated at the end of the run.
The following table lists field configuration details.
NextSeq 500/550 Run (NextSeq 500/550 v1.1) Master Step Field Configuration
There are several sample and measurement global fields configured to display on the Record Details screen of the NextSeq 500/550 Run (NextSeq 500/550 v1.1) step. These fields are populated at the end of the sequencing run.
Placeholders for the following files are configured on the Record Details screen of the NextSeq 500/550 Run (NextSeq 500/550 v1.1) 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)
Sample sheet generation occurs on the step before the sequencing run Denature and Dilute (NextSeq 500/550 v1.1) step. Samples are placed on the container to be loaded in the instrument. The default configuration provides the Generate bcl2fastq2 NextSeq Samplesheet automation.
The Generate bcl2fastq2 NextSeq Samplesheet automation uses the Template File Generator (DriverFileGenerator.jar) and a template file to generate a CSV format file for use with bcl2fastq2 v2.20.0 analysis software. The sample sheet content is determined by the fields that display on the Record Details screen of the step (in the Step Data table) and the values entered into the fields. Templates can be customized to create the sample sheet. If additional columns are required by the lab, then they can be inserted.
The NextSeq 500/550 Run (NextSeq 500/550 v1.1) step records information for the flow cell lanes and generates a report summarizing the results. In addition, run parameters, run info, and a link to the run folder are automatically captured.
The following table lists the run information files, reports, placeholders, and links that Clarity LIMS automatically generates or capture during a sequencing run.
Run Information Generated or Captured by NextSeq 500/550 Run (NextSeq 500/550 v1.1) Step
The following list includes the 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
Flow Cell ID
Index 1 Read Cycles — Configured Index 1 length
Index 2 Read Cycles — Configured Index 2 length
Output Folder — Run folder root
PR2 Bottle ID
Reagent Cartridge ID
Read 1 Cycles - Configured Read 1 cycle
Read 2 Cycles - Configured Read 2 cycle
Run ID — The unique run ID
Run Type
Status — Displays the completed vs configured aggregated (i.e., Read 1 and Read 2) read cycles. Example: Cycle 10 of 100.
Workflow
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.
The following table lists the Real-Time Analysis v2 (RTA2) primary analysis metrics that Clarity LIMS automatically captures and records, per read, for samples in each flow cell lane. These metrics are captured after run completion and are stored as global custom fields in the Record Details screen Sample Details table. Per read and per lane metrics are viewable by expanding the output.
RTA Primary Analysis Metrics Captured by NextSeq 500/550 Run (NextSeq 500/550 v1.0) Step
The sequencing service runs on the Clarity LIMS server. The service detects event files that the instrument software (RTA2) produces as the run progresses, which tells the service where to find the run data. As the run data is written out and the End Run event is detected, the data is matched to the step. This matching is based on the reagent cartridge ID that was entered/scanned in the Denature and Dilute (NextSeq 500/550 v1.1) step. Read-only field values on the Record Details screen are populated accordingly. When 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.
The following table lists the scripts and files installed in the Illumina NextSeq 500/550 Integration Package v2.3.0 RPM.
Illumina NextSeq 500/550 Integration Package v2.3.0 Scripts and Files Installed
The following table lists the properties installed with Illumina NextSeq 500/550 Integration Package v2.3.0.
Additional properties, each with the 99 suffix appended to their name, are also installed and intended for use by the Clarity LIMS Support team in automated validation tests. Those properties are not listed in the table.
Properties Installed with the Illumina NextSeq 500/550 Integration Package
¹ It is possible to configure support for multiple, identical seqservice.netPathPrefixSearch property values.
Reagent categories/label groups are installed with the IPP workflow configuration slices.
The NextSeq Reagent Kit is included in the NextSeq Integration.
The PhiX v3 control type is included in the NextSeq Integration.
The NextSeq Reagent Cartridge container type is included in the NextSeq Integration.
All one-dimensional container types with both numeric rows and numeric columns are supported.
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 instrument while it is idle.
To configure the Illumina instrument for use with the Illumina NextSeq Integration, the Support team:
Creates a directory on the local computer to hold the batch files. These batch files write event files to the network attached storage (NAS) shares.
Creates a directory on the NAS to hold the event files.
Modifies Illumina software configuration files to call the batch files that create the event files.
Updates sequencing service default properties to match the specifics of the installation.
This integration operates with the following constraints:
The reagent cartridge ID must be unique. Avoid multiple reagent cartridge containers in the system with identical names.
The reagent cartridge ID must be scanned as the reagent cartridge Container Name on the Denature and Dilute (NextSeq 500/550 v1.1) step.
For more information, see the section below.
This integration requires installation of the Illumina Preset Protocols (IPP). For details, refer to .
Sequencing runs are matched using the reagent cartridge ID and the sequencing steps base name. The base name is NextSeq 500/550 Run (NextSeq 500/550 v1.1). Do not change the base name. The 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 'reagent cartridge ID <-> sequencing step base name' matching system fails. If necessary, modify the step name by editing or adding text after the base name portion, as it is not used in the matching system. For example, change NextSeq 500/550 Run (NextSeq 500/550 v1.1) to NextSeq 500/550 Run (NextSeq 500/550 v1.1) v2.
Changes on nextseq.v2.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.
Field Name | Field Type | Field Constraints/Options | Preset Values/Additional Options and Drop-down Items |
Comment | Multiline Text |
Field Name | Field Type | Field Constraints/Options | Preset Values/Additional Options and Dropdown Items |
Application | Text Dropdown | Custom Entries | Presets
|
Pooling | Text Dropdown | Custom Entries | Presets
|
Read Length | Text |
Sequencing Coverage | Text |
Sequencing Method | Text Dropdown | Custom Entries | Presets
|
Field Name | Field Type | Field Constraints/Options | Preset Values/Additional Options and Dropdown Items |
Normalized Molarity (nM) | Numeric | Decimal places displayed = 2 |
Field Name | Field Type | Field Constraints/Options | Preset Values/Additional Options and Drop-Down Items |
Adapter | Text |
Adapter Read 2 | Text |
Experiment Name | Text |
Mask Adapter | Text |
Mask Adapter Read 2 | Text |
Read 1 Cycles | Numeric Dropdown |
|
|
Read 2 Cycles | Numeric Dropdown |
|
|
SampleSheet Template | Text Dropdown |
|
Workflow | Text Dropdown |
|
|
Field Name | Field Type | Field Constraints/Options | Preset Value/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 |
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 |
Run ID | Text | Read Only |
Run Type | Text | Read Only |
Status | Text | Read Only |
Workflow | Text | Read Only |
Item | Description |
Run Info Run Parameters |
|
Link to Run Folder |
|
Illumina Run Report |
|
Lab Tracking Form |
|
Per Read Clarity LIMS Field Name (stored on derived sample/analyte input to the step) | Per Lane Clarity LIMS Field Name (stored in measurement placeholders in Record Details screens Sample Details table) |
% 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 |
Files Installed | Location | Description |
configure_extensions_nextseq_sequencingservice.sh | /opt/gls/clarity/config/ | Script that installs the service properties in the database. |
log4j2.xml | /opt/gls/clarity/extensions/Illumina_NextSeq/v2/SequencingService/conf | File containing the settings for the sequencing jar logging. |
nextseq-sequencing.jar | /opt/gls/clarity/extensions/Illumina_NextSeq/v2/SequencingService | Jar file containing API-based Clarity LIMS extensions used for capturing run results and report generation. |
InterOp libraries | opt/gls/clarity/extensions/Illumina_NextSeq/v2/lib | Shared library for parsing InterOp data files. |
Installed from IPP
| /opt/gls/clarity/extensions/conf/driverfiletemplates | Template file used for file generation. In this integration, the reverse complement template is used by default. |
Field Name | Field Type | Field Constraints/Options | Preset Values/Additional Options and Drop-Down Items |
Read Length | Text |
Sequencing Method | Text Dropdown |
|
|
Field Name | Field Type | Field Constraints/Options | Preset Values/Additional Options and Drop-Down Items |
Final Loading Concentration | Numeric Dropdown |
|
|
Property | Description | Default Value |
nextseq.v2.seqservice.sequenceProcessBaseName | Sequencing process type / master step base display name. Partial matching is used to look up the process type / master step. | NextSeq 500/550 Run (NextSeq 500/550 v1.1) |
nextseq.v2.seqservice.eventFileDirectory.1 | A network location monitored for event files (e.g., /mnt/illumina/gls_events/) | /mnt/gls_events |
nextseq.v2.seqservice.netPathPrefixSearch.1¹ | The network directory prefix contained in the event file - most likely in Windows format. | \\nas\network\run_data |
nextseq.v2.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 |
nextseq.v2.seqservice.eventFileDirectorySuffixes | A list of eventFileDirectory path entries to monitor for event files. The value is one or more comma-separated integers. | 99 |
nextseq.v2.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 |
nextseq.v2.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 |
nextseq.v2.seqservice.ignoreUnmatchedContainerIds | A flag indicating if event files that cannot be matched to reagent cartridges in Clarity LIMS should be archived after a certain time (true), or continually reprocessed (false). | false |
nextseq.v2.seqservice.ignoreUnmatchedContainerIdsWaitDays | The number of days between when the event is created and the event file is archived. | 14 |
nextseq.v2.seqservice.synchronizationPeriod | Invocation period in seconds. | 60 |
Displays on Queue & Ice Bucket screens
Displays on Queue & Ice Bucket screens
Displays on Record Details screen
Installed from the IPP
Configured on install to point to 1.
Configured on install to point to 1.
To prevent the gls_events file directory from becoming cluttered, it is recommended that the value of this property is set to true.