The Illumina NextSeq 500/550 Integration v2.5.0 includes the following features and functionality:
New workflow that maps to lab protocols and instrument runs. In NextSeq 500/550 Sequencing v1.3, the forward-orientation samplesheet generated in Dilute and Denature step is now compatible with NextSeq 500/550 Control Software Version 4.2 and Local Run Manager v4.0 for the following modules:
Assembly Analysis
DNA Enrichment Analysis
GenerateFASTQ Analysis
Library QC Analysis
16S Metagenomics Analysis
PCR Amplicon
Resequencing Analysis
DNA Amplicon Analysis
RNA Fusion Analysis
Small RNA Analysis
Automated generation of the sample sheet. The reverse-complement sample sheet is used with the bcl2fastq2 v2.20.0 analysis software.
Automated tracking of the NextSeq sequencing runs and parsing of run statistics into Clarity LIMS, which includes the following information:
Sequencing run metrics
Sequencing run parameters
Real-Time Analysis v2 (RTA2) run directory location and other run-specific information
Preconfigured Library Prep Validation workflow used for validation purposes. This workflow contains a single-step protocol that models the library prep required to produce normalized libraries that are ready for the NextSeq 500/550 Sequencing workflow. The versions of the Library Prep Validation workflow NextSeq 500/550 Sequencing workflow depends on the version of IPP installed (see table above). For more information, refer to NextSeq 500/550 Integration v2.5.0 User Interaction, Validation and Troubleshooting.
Last Updated: December 2024
Release Date: December 2024
These release notes describe the key changes to software components for the Clarity LIMS NextSeq 500/550 Integration Package v2.5.0.
Refer to Compatibility under Instruments & Integrations.
The NextSeq 500/550 v1.3 protocol now also generates forward orientation samplesheet that is compatible with NextSeq 500/550 Control Software Version 4.2 and Local Run Manager v4.0 for the following modules:
Assembly Analysis
DNA Enrichment Analysis
GenerateFASTQ Analysis
Library QC Analysis
16S Metagenomics Analysis
PCR Amplicon
Resequencing Analysis
DNA Amplicon Analysis
RNA Fusion Analysis
Small RNA Analysis.
Note that sample sheet generated by the updated workflow is incompatible with NextSeq 500/550 Control Software version prior to v4.2 and Local Run Manager version prior to v4.0.
Reverse-complement sample sheet generated by NextSeq 500/550 v1.3 protocol is compatible with bcl2fastq2 v2.20.0 analysis software.
Removed unused fields Reagent Cartridge Part # and Flow Cell Version from run report.
In the run report, Genus LIMS Run ID is replaced with Clarity LIMS Run ID.
This guide explains how to validate the installation of the Illumina NextSeq 500/550 Integration Package v2.5.0.
The version of Library Prep Validation and NextSeq 500/550 Sequencing workflows depends on the IPP version installed.
For IPP v2.10, the versions of Library Prep Validation and NextSeq 500/550 Sequencing are v2.3.5 and v1.3, respectively.
The validation process involves:
Validating the creation of event files.
Running samples through the Library Prep Validation workflow. This workflow is installed by Illumina Preset Protocols (IPP) and contains a single-step protocol that models the library prep required to produce normalized libraries. At the end of the step, the normalized libraries are automatically advanced to the NextSeq 500/550 Sequencing workflow.
Running normalized libraries through the NextSeq 500/550 Sequencing workflow. This validates the automated generation of a reverse-complement sample sheet for use with bcl2fastq2 v2.20.0 analysis software. It also validates the 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
ℹ The NextSeq 500/550 Sequencing v1.3 workflow also supports generation of both reverse-complement sample sheet and forward-orientation sample sheet. The latter is compatible with NextSeq Control Software v4.2 and Local Run Manager v4.0. Refer to NextSeq 500/550 Integration v2.5.0 Release Notes for the list of supported applications.
The following steps set up Clarity LIMS in preparation for running samples through the Library Prep Validation and NextSeq 500/550 Sequencing workflows.
In the Clarity LIMS configuration area, activate the Library Prep Validation and NextSeq 500/550 Sequencing workflows.
On the Projects and Samples screen, create a project and add samples to it.
Assign your samples to the Library Prep Validation workflow.
This single-step protocol models the library prep required to produce normalized libraries that are ready for the NextSeq 500/550 Sequencing workflow.
Follow the steps in Library Prep Validation Protocol to run the Library Prep Validation workflow with the following:
Label Group = TruSeq HT Adapters v2 (D7-D5)
Sequencing Instrument = NextSeq
On exit from the step, the Routing Script automation is triggered. This automation assigns samples to the first step of the NextSeq 500/550 Sequencing workflow, which is Library Pooling (NextSeq 500/550) step.
In Lab View, locate the NextSeq 500/550 Sequencing protocol. You will see your samples queued for the Library Pooling (NextSeq 500/550) step.
Select the step to proceed to the Queue screen.
On the Queue screen, add samples as follows.
Add the samples to the Ice Bucket. In the Add Control Samples panel, add the PhiX v3 control sample to the Ice Bucket.
Select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
On the Pool Samples screen, create a pool of samples as follows.
Drag samples into the Pool Creator.
Name the pool. You can also accept the default name Pool #1.
Select Place Samples.
On the Placement screen, move the pool to the container as follows.
Select the pool in the Samples to be Placed area on the left. Drag it over to the container on the right.
Select Record Details.
On the Record Details screen, select Next Steps.
On the Assign Next Steps screen, the next step is already set to Denature and Dilute (NextSeq 500/550 v1.2).
Select Finish Step.
In Lab View, locate the NextSeq 500/550 Sequencing protocol. You will see your pooled samples queued for the Denature and Dilute (NextSeq 500/550) step.
Select the step to proceed to the Queue screen.
On the Queue screen, add the pool to the Ice Bucket. Select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
On the Placement screen, move the pool to the reagent cartridge as follows.
Scan the NextSeq reagent cartridge barcode into the NextSeq Reagent Cartridge field.
Place the pool of samples into the reagent cartridge.
Select Record Details.
On the Record Details screen, create the sample sheet as follows.
Under Reagent Lot Tracking, select the reagent lot used in the step. You may need to add/activate the lot on the Reagents and Controls screen.
In the Step Details table, populate the fields as appropriate.
The Workflow, Experiment Name and Read 1 Cycles are required fields.
In the Samplesheet Template drop-down list, select the template type required.
NextSeq_Samplesheet.csv for forward-orientiation sample sheet.
NextSeq_Reverse_Complement_Samplesheet.csv for reverse-complement sample sheet.
In the Sample Details table, enter the Final Loading Concentration. You may select preset 225 (for PCR-free workflows) or preset 400 (for Nano workflows). You can also enter a different value.
Select Generate NextSeq SampleSheet. Clarity LIMS generates the sample sheet 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.
Select Next Steps.
On the Assign Next Steps screen, make sure samples are already assigned to the NextSeq 500/550 Run (NextSeq 500/550) step.
Select Finish Step.
When the sequencing run is finished, the integration generates a run report file. The Instrument Details section is left blank by default, unless the NextSeq 500/500 Run master step is configured to track instruments created in Clarity LIMS. For more information, refer to Add and Configure Instruments from the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
In Lab View, locate the NextSeq 500/550 Sequencing protocol. You will see your pool of samples queued for the NextSeq 500/550 Run (NextSeq 500/550) step.
Select the step to proceed to the Queue screen.
Add the pool to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
On the Record Details screen, the fields in the table are read-only. Once the run completes, the integration will automatically populate the fields.
The integration also attaches files to the Illumina Run Report, Link to Run Folder, Run Parameters, and Run Info placeholders. The Log File is attached once the next step for samples has been assigned by the Next Step - Advance automation. The integration also populates the fields in the Sample Details table. For details, refer to NextSeq 500/550 v2.5.0 Configuration.
⚠ Do not continue to step 5 and complete the step until the Illumina Run Report has been attached.
If the NextSeq 500/550 Run master step is configured to track instruments, select the appropriate instrument from the Instrument Used drop-down on Record Details screen, then select Save.
For more detailed steps on adding instruments, refer to the Add and Configure Instruments section of the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
Select Next Steps.
On the Assign Next Steps screen, make sure the next step is already set to Mark protocol as complete.
Select Finish Step.
Follow the steps below to confirm that event files are created by the batch file in the destination path. The steps assume that the default configuration has been successfully imported, and support for NextSeq Control Software (NCS) v4.0 or later has been configured.
This test validates that:
Your DESTINATION_PATH is correctly configured.
The instrument computer can access and write to the DESTINATION_PATH.
There are no syntax errors in the Clarity LIMS batch file.
You can validate the gls_event_ncs_rta.bat batch file by executing it manually as follows.
Edit gls_event_ncs_rta.bat using any text editor (Notepad++ recommended).
By default, this file is installed to C:\Illumina\gls.
In the code line set DESTINATION_PATH=C:\Illumina\gls\Events, change C:\Illumina\gls\Events\ to the network path of the event files directory.
Remember to include the trailing "" at the end of the file path.
Save your changes.
Manually executing the batch file will create a dummy EndRun event file in the directory that you defined in the previous step.
If an automation trigger does not appear to run its corresponding scripts, refer to the following topics:
Troubleshooting Automation Worker in the Administration section of the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
Troubleshooting Automation in the Automation section of the Clarity LIMS (API & Database) documentation.
If an error occurs that does not provide direction on how to proceed, confirm the version of the installed Illumina NextSeq Integration Package by running the following command from the server console
If the error is related to data parsing, retrieving run results data, or report values not appearing as expected, review the NextSeqIntegrator.log file. It is located at
Additional troubleshooting information for this integration is provided on the Illumina Instrument Integrations FAQ page.
If you are unable to resolve the issue, contact the Clarity LIMS Support team. Make sure you supply the relevant information from the troubleshooting that was already performed.
The Illumina NextSeq 500/550 Integration v2.5.0 supports the integration between Clarity LIMS and the NextSeq 500/550 instrument. This document provides instructions for installing NextSeq 500/550 Integration v2.5.0. It also describes the components that are installed in the default configuration.
Refer to Compatibility under Instruments & Integrations.
This integration is not fully compatible with NextSeq 500/550 Integration v1.
For details on installed protocols and steps, automations, generated and captured files, and rules and constraints, refer to NextSeq 500/550 v2.5.0 Configuration.
For information on user interaction for each step, validating and troubleshooting the integration, refer to NextSeq 500/550 Integration v2.5.0 User Interaction, Validation and Troubleshooting.
This integration has the following prerequisites:
Mount run data network-attached storage (NAS) share
Secret Util is installed
IPP is installed
This integration is distributed as the ClarityLIMS-Illumina-NextSeq-Package-v2 RPM package that must be installed on the Clarity LIMS server. This package installs the following items:
Bash scripts used to run the service
The nextseq-sequencing.jar file
The configure_extensions_nextseq_sequencingservice.sh script
Smoke test directories
When upgrading from an existing NextSeq 500/550 workflow, the following warning messages can display when the illumina-preset-protocls-installer.sh script is running:
These messages are due to configuration changes that have increased the precision of the listed fields from 1 to 2 so that they show more decimal places. It is safe to override these warnings.
The following installation steps are required for the installation of this integration.
The NextSeq RPM must be installed on the Clarity LIMS server. This is where the AI node or Automation Worker is installed. The automations and sequencing service use the existing Automation Worker.
On the Clarity LIMS server, log in as the root user.
Run the following yum command to install the RPM:
ℹ You must use the --enablerepo command line argument to enable the repo. For the repo file and the correct name to use, contact Illumina Support.
Enter y to confirm that you want to proceed with the RPM installation. After confirmation, you are prompted to install the NextSeq 500/550 workflow from the IPP RPM.
ℹ If you have not installed the SecretUtil RPM before, this RPM installation also installs it.
For hosted installations, the Secret Util installation and configuration is handled by tooling.
For on-premise installations, if the Secret Util package is not already installed or configured, you must configure the Secret Util first. For more information, refer to Prerequisite 2: Secret Util Installation in Prerequisites.
Install the NextSeq 500/550 workflow as follows.
As the glsjboss user, run the following command to view the complete list of IPP workflows:
Run the following command to install the NextSeq 500/550 Sequencing workflow:
[Optional] Run the following command to install the Library Prep Validation workflow that is used to validate the NextSeq 500/550 Sequencing workflow:
Run the following script to configure the service properties:
The integration properties can be configured to enable capture and generation of files associated with the sequencing run. Refer to Integration Properties Details for details.
The property configuration for NextSeq 500/550 v2 has the suffix .v2 and is different from the setting for v1.
Run the following command to start the sequencing service:
In NextSeq Control Software (NCS) v4.0 the location of NextSeq.Configuration.xml RTA configuration file is changed. The new location is
A new gls_events_ncs_rta.bat batch file is required to generate a valid EndRun event file. This file is available in the NextSeq 500/550 RPM at
Configuration
Download the latest batch file from the NextSeq 500/550 RPM and place it in a folder (e.g., C:\Illumina\gls). If necessary, remove the old batch file.
Edit the NextSeq.Configuration.xml configuration file at
If you cannot modify the file, then open it as an administrator. Insert the following text inside the <Processing> tag. If the batch file is not at C:\Illumina\gls, then change that text to the applicable path.
Save the configuration file.
Validate the file by running RTA manually. In the command prompt, run the following command:
The following error message displays:
Validate the batch file by executing it manually as follows.
Using a text editor, edit the gls_events_ncs_rta.bat file.
In the "set DESTINATION_PATH=C:\Illumina\gls\Events" code line, change C:\Illumina\gls\Events\ to the network path of the event files directory. Make sure to include the backslash at the end of the file path.
After manually executing the batch file, a dummy EndRun event file is created in the event files directory.
The NextSeq 500/550 Integration works with the NextSeq 500/550 Sequencing workflow, which contains a single protocol with the same name. This protocol includes the following steps:
Library Pooling (NextSeq 500/550)
Denature & Dilute (NextSeq 500/550)
NextSeq 500/550 Run (NextSeq 500/550)
For descriptions of the protocol and the steps, refer to NextSeq 500/550 v2.5.0 Configuration. For instructions on using the Library Prep Validation workflow to validate the automated sample sheet generation, refer to NextSeq 500/550 Integration v2.5.0 User Interaction, Validation and Troubleshooting.
The NextSeq Control Software (NCS) is divided into the following modules:
NCS — Controls the instrument operation, including various configuration settings. This software is installed and runs on the instrument.
Real-Time Analysis 2 (RTA2) — Performs image processing and base calling (primary analysis). The software makes sure that data files are created and copied to the final destination folder and is installed and runs on the instrument.
For more information on NCS, refer to the NextSeq 500/550 documentation at support.illumina.com.
The instrument integration must be performed and maintained by Illumina Support. Illumina Support requires remote access to the instrument while it is idle.
Illumina Support has created batch files that use custom scripts during the key events of a sequencing run. When these batch files are used, they read the event information and write it in a TXT event file at the same network share location that the instrument uses to write the run data. Another process running on the Clarity LIMS server receives the event files and takes the appropriate actions.
The sequencing service monitors the end of the run event. This event is used to capture key step data and files and parse run statistics for output custom fields.
For NextSeq Control Software (NCS) v4.0 and later, refer to the configuration steps in NextSeq Control Software v4.0 Configuration. For NCS versions before v4.0, perform the following steps.
The Illumina NextSeq 500/550 Integration Package v2.5.0 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. It can be used with bcl2fastq2 v2.20.0 analysis software or with Local Run Manager (LRM) v4.0 depending on the format of the sample sheet generated. Refer to NextSeq 500/550 Integration v2.5.0 Release Notes for list of compatible LRM applications.
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 v2.5.0 User Interaction, Validation and Troubleshooting.
It is assumed that samples enter the NextSeq 500/550 Sequencing 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.
The NextSeq Integration Package includes the NextSeq 500/550 Sequencing workflow, which contains a single protocol of the same name. Refer to Compatibility under Instruments & Integrations for compatible version(s) of the workflow.
The NextSeq 500/550 Sequencing protocol includes the following steps:
Library Pooling (NextSeq 500/550)
Denature & Dilute (NextSeq 500/550)
NextSeq 500/550 Run (NextSeq 500/550)
The Library Pooling (NextSeq 500/550) 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) step and displays on the Record Details screen at run time.
The following table shows field configuration details.
Library Pooling (NextSeq 500/550) Master Step Field Configuration
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-down Items
Comment
Multiline Text
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) 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
In this step, pooled libraries are denatured and diluted and placed into the reagent cartridge that is loaded into the NextSeq instrument.
At run time, the master step 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 tables list the field configuration details.
Denature and Dilute (NextSeq 500/550) Master Step Field Configuration
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-Down Items
Comment
Multiline Text
Experiment Name
Text
Required Field
Genome Folder
Text
Manifest
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
151
101
76
51
Range = 0–1000
Decimal places displayed = 0
Samplesheet Template
Text Dropdown
Required Field
Presets
NextSeq_Samplesheet.csv
NextSeq_ReverseComplement_Samplesheet.csv
Taxonomy Database
Text
Workflow
Text Dropdown
Required Field
Presets
Assembly
DNA Amplicon
DNA Enrichment
GenerateFASTQ
Library QC
16S Metagenomics
PCR Amplicon
Resequencing
RNA Fusion
Small RNA
Groups of Defaults (NextSeq 500/550)
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 step.
Global Custom Field Configuration (Submitted Sample)
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-Down Items
Read Length
ℹ Displays on Queue & Ice Bucket screens
Text
Sequencing Method
ℹ 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
ℹ Displays on Record Details screen
Numeric Dropdown
Required Field
Custom Entries
Presets
225
400
Decimal places displayed = 0
Placeholders for the following files are configured on the Record Details screen of the Denature and Dilute step.
In this step, pooled samples are sequenced on the NextSeq 500/550 instrument and the run metrics are recorded in Clarity LIMS.
In the Record Details screen of the NextSeq 500/550 Run (NextSeq 500/550) step, some of the master step field values must be completed manually, whereas others are automatically populated at the end of the run.
The following table lists field configuration details.
NextSeq 500/550 Run (NextSeq 500/550) 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
Status
Text
Read Only
Workflow
Text
Read Only
The value of Status master step field configuration reflects the completed cycles (Read 1 Cycles + Read 2 Cycles) out of the total number of planned cycles (Read 1 Cycles + Read 2 Cycles) whereas Read 1 Cycles master step field reflects number of planned cycles for Read 1.
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) step. These fields are populated at the end of the sequencing run.
For more information, see the Sequencing Results Parsing section below.
Placeholders for the following files are configured on the Record Details screen of the NextSeq 500/550 Run (NextSeq 500/550) 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 Denature and Dilute (NextSeq 500/550) step. Samples are placed on the container to be loaded in the instrument. The Generate NextSeq Samplesheet automation uses the Template File Generator (DriverFileGenerator.jar) and a template file to generate the sample sheet. The template file used can be configured using the Samplesheet Template master step field. 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) 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) 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.
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
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) 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.
This integration requires installation of the Illumina Preset Protocols (IPP). For details, refer to NextSeq 500/550 Integration v2.5.0 Release Notes.
The following table lists the scripts and files installed in the Illumina NextSeq 500/550 Integration Package v2.5.0 RPM.
Illumina NextSeq 500/550 Integration Package v2.5.0 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 v2.10
NextSeq_Reverse_Complement_Samplesheet.csv
NextSeq_Samplesheet.csv
/opt/gls/clarity/extensions/conf/driverfiletemplates
Template file used for file generation. In this integration, there is no defaut template.
Refer to Integration Properties Details for the properties installed with Illumina NextSeq 500/550 Integration Package v2.5.0.
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) step.
The NextSeq 500/550 Integration v2.5.0 integration package requires the NextSeq 500/550 v1.3 workflow available from IPP v2.10. If you do not want to upgrade through the IPP, you can manually upgrade the workflow configuration from NextSeq 500/550 v1.2 workflow to v1.3 workflow by following instructions in this guide to generate samplesheet compatible with LRM v4.0.
To support the generation of LRM v4.0-compatible samplesheet, you must modify the Denature and Dilute step, and replacement of template files.
Automation
From Configuration, select the Automation tab.
Rename the Generate bcl2fastq2 NextSeq Samplesheet automation to Generate NextSeq Samplesheet.
The default command line for the automation remains unchanged.
Master Step Fields
From Configuration, select the Custom Fields tab.
Select the Denature and Dilute Master Step.
Add the following master step fields:
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-Down Items
Comment
Multiline Text
Genome Folder
Text
Manifest
Text
Samplesheet Template
Text Dropdown
Required Field
Presets
NextSeq_Samplesheet.csv
NextSeq_ReverseComplement_Samplesheet.csv
Taxonomy Database
Text
Remove the following master step fields:
Adapter
Adapter Read 2
Mask Adapter
Mask Adapter Read 2
SampleSheet Template
Update the following master step fields:
Field Name
Changes Required
Screenshot
Experiment Name
Update Required Field to Yes
Workflow
Update Dropdown presets to
Assembly
DNA Amplicon
DNA Enrichment
GenerateFASTQ
Library QC
16S Metagenomics
PCR Amplicon
Resequencing
RNA Fusion
Small RNA
Group of Defaults
From Configuration, select the Custom Fields tab.
Select the Denature and Dilute Master Step.
Add the following Group of Defaults:
Group of Defaults
Details
Screenshot
RNA Fusion
Read 1 Cycles = 251
Workflow = RNA Fusion
Small RNA
Read 1 Cycles = 251
Workflow = Small RNA
Update the following Group of Defaults:
Group of Defaults
Changes Required
Screenshot
Custom Amplicon
Rename to DNA Amplicon
Update Workflow field to DNA Amplicon
Enrichment
Rename to DNA Enrichment
Update Workflow field to DNA Enrichment
Generate FASTQ
Rename to GenerateFASTQ
Library QC
Update Workflow field to Library QC
Metagenomics
Rename to 16S Metagenomics
Update Workflow field to 16S Metagenomics
Step File Placeholders
From Configuration, select the Lab Work tab.
Select the Denature and Dilute Master Step.
Rename the following Step File Placeholders under Record Details milestone:
from bcl2fastq SampleSheet to Samplesheet
from bcl2fastq SampleSheet Generation Log to Samplesheet Generation Log
Replace the following template files at /opt/gls/clarity/extensions/conf/driverfiletemplates/:
BCL2FASTQ_Reverse_Complement_Samplesheet.csv with NextSeq_ReverseComplement_Samplesheet.csv
BCL2FASTQ_Samplesheet.csv with NextSeq_Samplesheet.csv
Contact Illumina Support to replace the files if required.
