Configuration

The Illumina NextSeq 500/550 Integration Package v2.4.0 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 NextSeq 500/550 Integration v2.4.0 User Interaction, Validation and Troubleshooting.

Prerequisites and Assumptions

It is assumed that samples enter the NextSeq 500/550 Sequencing v1.2 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 Clarity LIMS (Clarity & LabLink Reference Guide) documentation.

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 Clarity LIMS (Clarity & LabLink Reference Guide) documentation.

Workflows, Protocols, and Steps

NextSeq Integration Package v2.4.0 includes the NextSeq 500/550 Sequencing v1.2 workflow, which contains a single protocol of the same name.

The NextSeq 500/550 Sequencing v1.2 protocol includes the following steps:

• Library Pooling (NextSeq 500/550 v1.2)

• Denature & Dilute (NextSeq 500/550 v1.2)

• NextSeq 500/550 Run (NextSeq 500/550 v1.2)

Step 1: Library Pooling (NextSeq 500/550 v1.2)

The Library Pooling (NextSeq 500/550 v1.2) step is derived from the Library Pooling v1.0 master step. Libraries are placed into pools manually.

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:evaluateDynamicExpression \
-t false \
-h false \
-exp 'nextStep = ::ADVANCE::' \
-log {compoundOutputFileLuid0}"

Master Step Fields

This field is configured on the Library Pooling (NextSeq 500/550 v1.2) step and displays on the Record Details screen at run time.

The following table shows field configuration details.

Library Pooling (NextSeq 500/550 v1.2) Master Step Field Configuration

Global Fields

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 v1.2) step. Most of these fields show in the expanded view only.

Global Custom Field Configuration (Submitted Sample)

Global Custom Field Configuration (Derived Sample)

Step 2: Denature and Dilute (NextSeq 500/550 v1.2)

In this step, pooled libraries are denatured and diluted and placed into the reagent cartridge that is loaded into the NextSeq instrument.

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {processURI:v2} -u {username} -p {password} \
script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/{udf:Samplesheet Template} \
-o {compoundOutputFileLuid1}.csv \
-q true \
-destLIMSID {compoundOutputFileLuid1} \
-l {compoundOutputFileLuid2}"

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:evaluateDynamicExpression \
-t false \
-h false \
-exp 'nextStep = ::ADVANCE::' \
-log {compoundOutputFileLuid3}"

Master Step Fields

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 v1.2) Master Step Field Configuration

Groups of Defaults

Global Fields

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 v1.2) step.

Global Custom Field Configuration (Submitted Sample)

Global Custom Field Configuration (Derived Sample)

Step File Placeholders

Placeholders for the following files are configured on the Record Details screen of the Denature and Dilute (NextSeq 500/550 v1.2) step

Step 3: NextSeq 500/550 Run (NextSeq 500/550 v1.2)

In this step, pooled samples are sequenced on the NextSeq 500/550 instrument and the run metrics are recorded in Clarity LIMS.

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:evaluateDynamicExpression \
-t false \
-h false \
-exp 'nextStep = ::ADVANCE::' \
-log {compoundOutputFileLuid5}"

Master Step Fields

There are 16 fields configured on the NextSeq 500/550 Run (NextSeq 500/550 v1.2) 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 v1.2) Master Step Field Configuration

Global Fields

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 v1.2) step. These fields are populated at the end of the sequencing run.

For more information, see the Sequencing Results Parsing section below.

Step File Placeholders

Placeholders for the following files are configured on the Record Details screen of the NextSeq 500/550 Run (NextSeq 500/550 v1.2) 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)

Sample Sheet Generation

Sample sheet generation occurs on the step before the sequencing run Denature and Dilute (NextSeq 500/550 v1.2) 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.

Sequencing Results Parsing

The NextSeq 500/550 Run (NextSeq 500/550 v1.2) 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.

Generated and Captured Files

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 v1.2) Step

Metadata

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.

Primary Analysis Metrics

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 v1.2) 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.

Components Installed

This integration requires installation of the Illumina Preset Protocols (IPP). For details, refer to NextSeq 500/550 Integration v2.4.0 Release Notes.

Scripts and Files Installed

The following table lists the scripts and files installed in the Illumina NextSeq 500/550 Integration Package v2.4.0 RPM.

Illumina NextSeq 500/550 Integration Package v2.4.0 Scripts and Files Installed

Properties Installed

Refer to Integration Properties Details for the properties installed with Illumina NextSeq 500/550 Integration Package v2.4.0.

Reagent Categories/Label Groups Installed

Reagent categories/label groups are installed with the IPP workflow configuration slices.

The NextSeq Reagent Kit is included in the NextSeq Integration.

Control Types Installed

The PhiX v3 control type is included in the NextSeq Integration.

Container Types Installed

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.

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 instrument while it is idle.

To configure the Illumina instrument for use with the Illumina NextSeq Integration, the Support team:

1. 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.

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

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

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

Rules and Constraints

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 v1.2) step.