NextSeq 500/550 Integration v2.1 Configuration and User Interaction

The Illumina NextSeq 500/550 Integration Package v2.1 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 Illumina NextSeq system. It includes information about protocols and automations, configuration options, installed components, and rules and constraints.

See NextSeq 500/550 v2.0.3 Validation and Troubleshooting Guide for instructions on validating and troubleshooting the NextSeq 500/550 Integration Package.

The Illumina NextSeq 500/550 Integration Package v2.1 includes the following items:

• Automated generation of a sample sheet for use with bcl2fastq2 v2.20.0 analysis software.
• Automated tracking of the NextSeq sequencing run and parsing of run statistics into Clarity LIMS, including:
– Run status and metrics of sequencing run
– Sequencing run parameters
– Real-Time Analysis v2 (RTA2) run directory location and other run-specific information

It is assumed that samples enter the NextSeq 500/550 Sequencing v1.0 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 details on sample accessioning, see the following documentation:

• Sample Accessioning
• Upload and Modify Samples

Samples can be assigned to the NextSeq 500/550 Sequencing v1.0 workflow automatically (using a routing script) or manually from the Projects and Samples screen (see Assign and Process Samples).

The Illumina NextSeq Integration includes the NextSeq 500/550 Sequencing v1.0 workflow, which contains a single protocol of the same name.

The NextSeq 500/550 Sequencing System v1.0 protocol includes the following steps:

• Library Pooling (NextSeq 500/550 v1.0)
• Denature & Dilute (NextSeq 500/550 v1.0)
• NextSeq 500/550 Run (NextSeq 500/550 v1.0)

Step Automations

The following table lists the automations configured on the NextSeq 500/550 Sequencing v1.0 steps. Automation details are provided in the configuration details sections following the table. See NextSeq 500/550 v2.0.3 Validation and Troubleshooting Guide for details on the validation workflow.

NextSeq 500/550 Sequencing System v1.0 Protocol Step Automation

Step

Automation
4. Library Pooling (NextSeq 500/550 v1.0)
• Set Next Step - Advance
5. Denature and Dilute (NextSeq 500/550 v1.0)
• Generate bcl2fastq2 NextSeq 500/550 SampleSheet
• Set Next Step - Advance
6. NextSeq 500/550 Run (NextSeq 500/550 v1.0)
• Set Next Step - Advance

The following sections describe the configuration installed with the Illumina NextSeq Integration.

The Illumina NextSeq 500/550 Integration Package v2.1 supports Clarity LIMS v4.2, v4.3 and v5.x.

As of Clarity LIMS v5, the term user-defined field (UDF) has been replaced with global custom field in the user interface (the API resource is still called UDF). There are two types of custom fields:

• Master step fields—Configured on master steps. These fields only apply to the master step on which they are configured and the steps derived from those master steps.
• Global fields—Configured on entities such as submitted sample, derived sample, measurement, etc. These fields apply to the whole Clarity LIMS system.

In Clarity LIMS v5, the Library Pooling (NextSeq 500/550 v1.0) step is derived from the Library Pooling v1.0 master step. Libraries are placed into pools manually.

Automations

The following automation is configured on the Library Pooling (NextSeq 500/550 v1.0) step:

• Set Next Step - Advance

Set Next Step - Advance

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

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}"

Step UDFs/Custom Fields

This field is configured on the Library Pooling (NextSeq 500/550 v1.0) step and displays on the Record Details screen at run time.

The following table shows field configuration details.

Library Pooling (NextSeq 500/550 v1.0) Step Custom Field Configuration

Field Name

Field Type

Field Constraints/Options

Comment

Multiline Text
• Required: No
• Read Only: No

Global Custom 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 v1.0) step. Most of these fields show in the expanded view only.

Global Custom Field Configuration for Library Pooling (NextSeq 500/550 v1.0)

Field Name

Field Name

Field Constraints/Options

Preset Values/Additional Options and Dropdown Items

Configured on Sample in Clarity LIMS v4.2 and Submitted Sample in Clarity LIMS v5

Application

Single-line text (v4.2)

Text Dropdown (v5)
• Required: No
• Read Only: No
• Custom Entries: Yes
• TruSeq mRNA sequencing
• TruSeq DNA sequencing (large genome de novo)
• TruSeq DNA sequencing (large genome reseq)
• TruSeq DNA sequencing (small genome de novo)
• TruSeq DNA sequencing (small genome reseq)
• Nextera DNA sequencing
• TruSeq Custom Amplicon sequencing
• ChIP-sequencing
• Exome sequencing
• Mate pair sequencing
• Small RNA sequencing

Pooling

Single-line text (v4.2)

Text Dropdown (v5)
• Required: No
• Read Only: No
• Custom Entries: Yes
• Yes
• No

Read Length

Single-line text (v4.2)

Text (v5)
• Required: No
• Read Only: No

Not applicable

Sequencing Coverage

Single-line text (v4.2)

Text (v5)
• Required: No
• Read Only: No

Not applicable

Sequencing Method

Single-line text (v4.2)

Text Dropdown (v5)
• Required: No
• Read Only: No
• Custom Entries: Yes
• Single Read
• Paired End Read
• Indexed Single read
• Indexed Paired End Read

Configured on Analyte in Clarity LIMS v4.2 and Derived Sample in Clarity LIMS v5

Normalized Molarity (nM)

Numeric
• Required: No
• Read Only: No
• Decimal Places Displayed: 2

User Interaction

1. On the Queue screen, add the samples to the Ice Bucket.
2. In the Add Control Samples panel, add the PhiX v3 control sample to the Ice Bucket.
3. Select View Ice Bucket.
4. On the Ice Bucket screen, select Begin Work.
5. On the Pool Samples screen, create a pool of samples by dragging samples into the Pool Creator. Name the pool or accept the default name (Pool #1).
6. Select Place Samples.
7. On the Placement screen, select the pool in the Samples to be Placed area on the left and drag it to the container on the right.
8. Select Record Details.
9. On the Record Details screen, select Next Steps.
10. On the Assign Next Steps screen, next step is set to Denature and Dilute (NextSeq 500/500 v1.0).
11. Select Finish Step.

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

Automations

The following automations are configured on the Denature and Dilute (NextSeq 500/550 v1.0) step:

• Generate bclfastq2 NextSeq SampleSheet
• Set Next Step - Advance

Generate bcl2fastq2 NextSeq SampleSheet

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 following command line is the default:

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

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

This automation advances samples to the next step in the protocol. In Clarity LIMS v4.2, the automation also invokes the setUDF script, which sets the value of the Progress UDF. Clarity LIMS updates this UDF value as samples progress through the workflow steps. The Progress UDF does not display in the Clarity LIMS user interface. However, it does display in the LabLink Collaborations Interface so that collaborators can see the status of their samples.

For Clarity LIMS v4.2, the following command line is the default:

bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar -u {username} -p {password} \

script:setUDF \

-i {processURI:v2} \

-f 'Progress' \

-t '//input/@uri->//sample/@uri' \

-v 'Library ready for sequencing' \

script:evaluateDynamicExpression \

-i {stepURI:v2:http} \

-t false \

-h false \

-exp 'nextStep = ::ADVANCE::' \

-log {compoundOutputFileLuid3}"

For Clarity LIMS v5, the following command line is the default:

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}"

Step UDFs/Custom 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 v1.0) Step Custom Field Configuration

Field Name

Field Type

Field Constraints/Options

Preset Values/Additional Options and Drop-down Items

Adapter

Single-line Text (v4.2)

Text (v5)
• Required: No
• Read Only: No

Not applicable

Adapter Read 2

Single-line Text (v4.2)

Text (v5)
• Required: No
• Read Only: No

Not applicable

Experiment Name

Single-line Text (v4.2)

Text (v5)
• Required: No
• Read Only: No

Not applicable

Mask Adapter

Single-line Text (v4.2)

Text (v5)
• Required: No
• Read Only: No

Not applicable

Mask Adapter Read 2

Single-line Text (v4.2)

Text (v5)
• Required: No
• Read Only: No

Not applicable

Read 1 Cycles

Numeric (v4.2)

Numeric Dropdown (v5)
• Required: No
• Read Only: No
• Custom Entries: Yes
• 251 (Default)
• 151
• 101
• 76
• 51
• Range From: 0 to 1000
• Decimal Places Displayed: 0

Read 2 Cycles

Numeric (v4.2)

Numeric Dropdown (v5)
• Required: No
• Read Only: No
• Custom Entries: Yes
• 251
• 151
• 101
• 76
• 51
• Range From: 0 to 1000
• Decimal Places Displayed: 0

SampleSheet Template

Single-line Text (v4.2)

Text Dropdown (v5)
• Required: No
• Read Only: No
• Custom Entries: No
• BCL2FASTQ_Reverse_Complement_Samplesheet.csv (default)
• BCL2FASTQ_Samplesheet.csv

Workflow

Single-line Text (v4.2)

Text Dropdown (v5)
• Required: No
• Read Only: No
• Custom Entries: No
• Assembly
• Custom Amplicon
• Enrichment
• GenerateFASTQ
• LibraryQC
• Metagenomics
• PCR Amplicon
• Resequencing

Groups of Defaults/Presets

The following group of defaults/presets are configured for Denature and Dilute (NextSeq 500/550 v1.0) master step custom fields:

• 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: LibraryQC
• Metagenomics
– Read 1 Cycles: 251
– Workflow: Metagenomics
• GenerateFastQ
– Read 1 Cycles: 251
– Workflow: GenerateFastQ
• Assembly
– Read 1 Cycles: 251
– Workflow: Assembly

Global Custom Fields

The following table lists the global custom fields that are configured to display on the Queue, Ice Bucket, and Record Details screens of the Denature and Dilute (NextSeq 500/550 v1.0) step.

Global Custom Field Configuration for Denature and Dilute (NextSeq 500/550 v1.0) Step

Field Name

Field Type

Field Constraints/Options

Preset Values/Additional Options and Drop-down Items

Configured on Sample in Clarity LIMS v4.2 and Submitted Sample in Clarity LIMS v5

Progress

(Clarity LIMS v4.2) Displays in LabLink Collaborations Interface only

Single-line Text (v4.2)

Text (v5)
• Required: No
• Read Only: No
• Custom Entries: Yes

Not applicable

Read Length

Displays on Queue & Ice Bucket screens

Single-line Text (v4.2)

Text (v5)
• Required: No
• Read Only: No

Not applicable

Sequencing Method

Displays on Queue & Ice Bucket screens

Single-line Text (v4.2)

Text Dropdown (v5)
• Required: No
• Read Only: No
• Custom Entries: Yes
• Single Read
• Paired End Read
• Indexed Single Read
• Indexed Paired End Read

Configured on Analyte in Clarity LIMS v4.2 and Derived Sample in Clarity LIMS v5

Final Loading Concentration

Displays on Record Details screen

Numeric (v4.2)

Numeric Dropdown (v5)
• Required: Yes
• Read Only: No
• Custom Entries: Yes
• 225
• 400

Step File Placeholders

Placeholders for the following files are configured on the Record Details screen of the Denature and Dilute (NextSeq 500/550 v1.0) step:

• Lab Tracking Form (manually generated)
• bcl2fastq2 Samplesheet (autogenerated)
• bcl2fastq2 Samplesheet Generation Log (autogenerated)
• Log File (autogenerated)
Sample Sheet Files & Placeholders Generated by Denature & Dilute (NextSeq 500/550 v1.0) Step

Item

Description

Lab Tracking Form placeholder

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

bcl2fastq2 SampleSheet

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.

bcl2fastq2 SampleSheet Generation Log

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

Log File

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.

User Interaction

1. On the Queue screen, add the pool to the Ice Bucket and select View Ice Bucket.
2. On the Ice Bucket screen, select Begin Work.
3. On the Placement screen, scan the barcode of the NextSeq reagent cartridge into the NextSeq Reagent Cartridge field, then place the pool of samples into the reagent cartridge.
4. Select Record Details.
5. On the Record Details screen under Reagent Lot Tracking, select the reagent lot used in the step. (Add/activate the lot on the Reagents and Controls screen, if necessary.)
6. Populate the fields in the Step Details table. Workflow and Read 1 Cycles are required fields.
7. In the Sample Details table, enter a Final Loading Concentration value. Select from the two preset options, 225 (for PCR-free workflows) or 400 (for Nano workflows), or enter a different value manually.
8. Select Generate bcl2fastq2 NextSeq SampleSheet.
9. Clarity LIMS generates the samplesheet and attaches it and a log file to placeholders in the Files area of the Record Details screen. Download the files and validate their format and content.
10. Select Next Steps.

On the Assign Next Steps screen, samples are assigned to the NextSeq 500/550 Run (NextSeq 500/550 v1.0) step.

11. Select Finish Step.

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

Automations

The following automation is configured on the NextSeq 500/550 Run (NextSeq 500/550 v1.0) step:

• Set Next Step - Advance

Set Next Step - Advance

This automation is automatically triggered on exit of the Record Details screen, and advances samples to the next step in the protocol. The default commend 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}"

Step UDFs/Custom Fields

There are 16 fields configured on the NextSeq 500/550 Run (NextSeq 500/550 v1.0) 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 v1.0) Step Custom Field Configuration

Field Name

Field Type

Filed Constraints/Options

Preset Value/Additional Options and Drop-down Items

Chemistry

Single-line Text (v4.2)

Text (v5)
• Required: No
• Read Only: Yes

Not applicable

Comment

Multiline Text
• Required: No
• Read Only: No

Not applicable

Experiment Name

Single-line Text (v4.2)

Text (v5)
• Required: No
• Read Only: Yes

Not applicable

Finish Date

Not applicable
• Required: No
• Read Only: Yes

Not applicable

Flow Cell ID

Not applicable
• Required: No
• Read Only: Yes

Not applicable

Index 1 Read Cycles

Numeric
• Required: No
• Read Only: Yes

Decimal Places Displayed: 0

Index 2 Read Cycles

Numeric
• Required: No
• Read Only: Yes

Decimal Places Displayed: 0

Output Folder

Single-line Text (v4.2)

Text (v5)
• Required: No
• Read Only: Yes

Not applicable

PR2 Bottle ID

Single-line Text (v4.2)

Text (v5)
• Required: No
• Read Only: Yes

Not applicable

Read 1 Cycles

Numeric
• Required: No
• Read Only: Yes

Decimal Places Displayed: 0

Read 2 Cycles

Numeric
• Required: No
• Read Only: Yes

Decimal Places Displayed: 0

Reagent Cartridge ID

Single-line Text (v4.2)

Text (v5)
• Required: No
• Read Only: Yes

Not applicable

Run ID

Single-line Text (v4.2)

Text (v5)
• Required: No
• Read Only: Yes

Not applicable

Run Type

Single-line Text (v4.2)

Text (v5)
• Required: No
• Read Only: Yes

Not applicable

Status

Single-line Text (v4.2)

Text (v5)
• Required: No
• Read Only: Yes

Not applicable

Workflow

Single-line Text (v4.2)

Text (v5)
• Required: No
• Read Only: Yes

Not applicable

Global Custom Fields

There are several sample and measurement global custom fields configured to display on the Record Details screen of the NextSeq 500/550 Run (NextSeq 500/550 v1.0) step. These fields are populated at the end of the sequencing run.

Step File Placeholders

Placeholders for the following files are configured on the Record Details screen of the NextSeq 500/550 Run (NextSeq 500/550 v1.0) step.

• Illumina Run Report (autogenerated)
• Link to Run Folder (autogenerated)
• Run Parameters (autogenerated)
• Run Info (autogenerated)
• Lab Tracking Form (manually generated)
• Log File (autogenerated)

User Interaction

1. On the Queue screen, add the pool to the Ice Bucket and select View Ice Bucket.
2. On the Ice Bucket screen, select Begin Work.

On the Record Details screen, the fields in the table are read-only. After the run completes, the integration automatically:

• Populates the fields.
• Attaches files to the Illumina Run Report, Link to Run Folder, Run Parameters, and Run Info placeholders.
• Populates the fields in the Sample Details table.
3. Select Next Steps.
4. On the Assign Next Steps screen, the next step is set to Mark protocol as complete.
5. Select Finish Step.

Sample sheet generation occurs on the step before the sequencing run Denature and Dilute (NextSeq 500/550 v1.0). Samples are placed on the container to be loaded in the instrument. The default configuration provides one automation, Generate bcl2fastq2 NextSeq SampleSheet. This 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 these fields. Templates can be customized to create the sample sheet and insert additional columns if the lab desires.

The NextSeq 500/550 Run (NextSeq 500/550 v1.0) 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 v1.0) 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 placeholder

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—Intended Index cycles
• Index 2 Read Cycles—Intended Index cycles
• Output Folder—Run folder root
• PR2 Bottle ID
• Reagent Cartridge ID
• 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

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 v1.0) 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.

User Interaction

1. Begin the step process and wait until the sequencing service has finished processing the run and the run report is generated and attached.
2. On the Record Details screen, the read-only field values are automatically populated as the instrument runs.

After the run has completed:

• The read-only fields are populated.
• The Illumina Run Report is generated and attached.
• The Link to Run Folder is generated and attached.
• The Run Parameters and Run Info files are attached.
3. [Optional] In the Files section, upload the Lab Tracking Form.

The steps complete. The Log File is attached after the next step for samples is assigned by the Next Step, Advance automation.

Do not complete the step until the Illumina Run Report is attached to the step in Clarity LIMS.

This integration requires installation of the Preconfigured Workflow Package (PCWP).

The following table lists the scripts and files installed in the Illumina NextSeq 500/550 Integration Package v2.1 RPM. Additional component details are provided in the sections following the table.

Illumina NextSeq 500/550 Integration Package v2.1 Scripts and Files Installed

Files Installed

Location and Description

configure_extensions_nextseq_sequencingservice.sh

/opt/gls/clarity/config/

Script that installs the service properties in the database.

log4j.xml

/opt/gls/clarity/extensions/nextseq/SequencingService/conf

File containing the settings for the sequencing jar logging.

nextseq-sequencing.jar

/opt/gls/clarity/extensions/nextseq/SequencingService

Jar file containing API-based Clarity LIMS extensions used for capturing run results and report generation.

InterOp libraries

/opt/gls/clarity/extensions/nextseq/lib

Illumina shared library for parsing InterOp data files.
• BCL2FASTQ_Reverse_Complement_Samplesheet.csv
• BCL2FASTQ_Samplesheet.csv

(Installed by the PCWP)

/opt/gls/clarity/extensions/nextseq/Templates

Template file used for file generation. In this integration, the reverse complement template is used by default.

It is possible to configure support for multiple, identical seqservice.netPathPrefixSearch property values.

Database Properties Installed

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.

Constraints

Sequencing runs are matched using the reagent cartridge ID and the sequencing steps base name, NextSeq 500/550 Run (NextSeq 500/550 v1.0).

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 database property. If this name is changed without the database 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 v1.0) to NextSeq 500/550 Run (NextSeq 500/550 v1.0) v2.1.

The following table lists the database properties installed with Illumina NextSeq 500/550 Integration v2.1.

Database Properties Installed with the Illumina NextSeq 500/550 Integration Package

Property

Description

Default Value

nextseq.v2.seqservice.sequenceProcessBaseName

(installed from the PCWP)

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 v1.0)

nextseq.v2.seqservice.eventFileDirectory.1

A network location monitored for event files. Eg, /mnt/illumina/gls_events/'

/mnt/gls_events

nextseq.v2.seqservice.netPathPrefixSearch.1

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. Eg. /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

Configured on install to point to 1.

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

Configured on install to point to 1.

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

Note: To prevent the gls_events file directory from becoming cluttered, it is recommended that the value of this property is set to true.

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

Configure support for multiple, identical seqservice.netPathPrefixSearch property values.

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

The NextSeq Reagent Kit is included in the Illumina NextSeq Integration.

Control types installed.

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

The NextSeq Reagent Cartridge container type is included in the Illumina NextSeq 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 database properties to match the specifics of the installation.

All one-dimensional container types with both numeric rows and numeric columns are supported.

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 v1.0) step.

NextSeq 550 information

NextSeq 500 System Support Resources

NextSeq 550 System Support Resources