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.

Set Next Step - Advance Automation

This automation is automatically triggered on exit of the Record Details screen. The automation advances samples to the next step in the protocol.

The default command line is as follows.

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

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 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)

Field Name	Field Type	Field Constraints/Options	Preset Values/Additional Options and Dropdown 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 Custom Field Configuration (Derived Sample)

Field Name	Field Type	Field Constraints/Options	Preset Values/Additional Options and Dropdown Items
Normalized Molarity (nM)	Numeric		Decimal places displayed = 2

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.

Generate bcl2fastq2 NextSeq SampleSheet Automation

This automation is triggered by a button on the Record Details screen.

This automation generates the sample sheet and attaches it to the step. For details, see the following section on sample sheet generation.

The default command line is as follows.

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}"

Set Next Step - Advance Automation

This automation is automatically triggered on exit of the Record Details screen.

This automation advances samples to the next step in the protocol.

The default command line is as follows.

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

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	Required Field Custom Entries	Presets 251 (Default) 151 101 76 51 Range = 0–1000 Decimal places displayed = 0
Read 2 Cycles	Numeric Dropdown	Custom Entries	Presets 251 (Default) 151 101 76 51 Range = 0–1000 Decimal places displayed = 0
SampleSheet Template	Text Dropdown		Presets BCL2FASTQ_Reverse_Complement_Samplesheet.csv (default) BCL2FASTQ_Samplesheet.csv
Workflow	Text Dropdown	Required Field	Presets Assembly Custom Amplicon Enrichment GenerateFASTQ LibraryQC Metagenomics PCR Amplicon Resequencing

Groups of Defaults

Resequencing

• Read 1 Cycles = 251

• Workflow = Resequencing

Custom Amplicon

• Read 1 Cycles = 251

• Workflow = Custom Amplicon

PCR Amplicon

• Read 1 Cycles = 251

• Workflow = PCR Amplicon

Enrichment

• Read 1 Cycles = 251

• Workflow = Enrichment

Library QC

• Read 1 Cycles = 251

• Workflow = Library QC

Metagenomics

• Read 1 Cycles = 251

• Workflow = Metagenomics

GenerateFastQ

• Read 1 Cycles = 251

• Workflow = GenerateFastQ

Assembly

• Read 1 Cycles = 251

• Workflow = Assembly

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)

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 Custom 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 (NextSeq 500/550 v1.2) step

Lab Tracking Form

• Manually uploaded

• This form in Clarity LIMS allows for manually attaching a lab-specific tracking form to the step.

bcl2fastq SampleSheet

• Automatically attached

• This CSV file is automatically generated by Clarity LIMS for use with the bcl2fastq2 v2.20.0 analysis software. It can be opened as a text file or as an MS Excel spreadsheet.

bcl2fastq SampleSheet Generation Log

• Automatically attached

• Automatically generated by Clarity LIMS, this log file captures any errors that Clarity LIMS can encounter when generating the sample sheet.

Log File

• Automatically attached

• Automatically generated by Clarity LIMS, this log file captures the status of the EvaluateDynamicExpression script that is launched by the Set Next Step - Advance automation.

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.

Set Next Step - Advance Automation

This automation is automatically triggered on exit of the Record Details screen. The automation advances samples to the next step in the protocol.

The default command line is as follows.

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

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

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

Item	Description
Run Info Run Parameters	These XML files are captured automatically by Clarity LIMS from the instrument run folder. They include the key run parameters, many of which are parsed out into key step global custom fields.
Link to Run Folder	Automatically generated by Clarity LIMS, and 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, and primary analysis metrics for the instrument, summarized per flow cell lane for the entire run, and individual reads if there are paired-end runs. These metrics are compared against the instrument per lane averages, calculated using metrics from the last 5 sequencing runs. Any values outside of 1 standard deviation are highlighted.
Lab Tracking Form	This placeholder in Clarity LIMS allows you to attach a lab-specific tracking form to the step manually.

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

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

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

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 BCL2FASTQ_Reverse_Complement_Samplesheet.csv BCL2FASTQ_Samplesheet.csv	/opt/gls/clarity/extensions/conf/driverfiletemplates	Template file used for file generation. In this integration, the reverse complement template is used by default.

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.