BSSH subscription (personal/professional/enterprise) is required for NextSeq 1000/2000 Integration v2.4.0
The Illumina NextSeq 1000/2000 Integration Package v2.4.0 supports the integration of Clarity LIMS to Illumina NextSeq 1000/2000 sequencing systems.
For instructions on user interaction for each step, validating and troubleshooting the NextSeq 1000/2000 Integration, refer to .
The configuration provided in this integration has been established to support NextSeq 1000/2000 lab processes. Any configuration changes to protocols or workflows — including renaming protocols, steps, and fields (global or master step) — could break the process.
Prerequisites and Assumptions
The Illumina NextSeq 1000/2000 Integration Package v2.4.0 is compatible with Illumina cloud hosted deployments only.
It is assumed that samples entering the NextSeq 1000/2000 Sequencing v2.3 workflow have gone through library preparation and quantification processes. Before they are assigned to the workflow, samples have completed the following actions:
Samples have been accessioned into Clarity LIMS.
Samples have been run through QC and library prep.
Samples have the Molarity (nM) global field set to some value. The Calculate Volumes automation in the Library Pooling and Dilution step requires a value in the Molarity (nM) global field.
For more information on sample accessioning, refer to Sample Accessioning and Upload and Modify Samples in the Getting Started section of the .
You can assign samples to workflows automatically, using a routing script, or manually—from the Projects & Samples dashboard. Refer to Assign and Process Samples in the .
Workflows, Protocols, and Steps
The NextSeq 1000/2000 Integration Package v2.4.0 includes the following workflows:
[Optional] Library Prep Validation v2.3.1 (recommended for validation purposes).
NextSeq 1000/2000 Sequencing v2.3
ℹ Samples are routed to this protocol after they have gone through Library Prep Validation v2.3.1 (or any other library prep workflow). Samples are pooled, diluted to final loading concentration, and added to a reagent cartridge in preparation for the sequencing run.
ℹ The workflow configuration includes a global field named Illumina Universal Sample Identifier. This text field is reserved for Clarity LIMS Product Analytics support. The field is optional and not required for this integration.
Library Prep Validation v2.3.1 Workflow
Protocol 1: Library Prep Validation v2.3.1
Purpose:
Included for validation purposes only, this protocol models the library prep steps required to advance samples to the NextSeq 1000/2000 Sequencing v2.3 workflow.
NextSeq 1000/2000 Sequencing v2.3 Workflow
Protocol 1: NextSeq 1000/2000 Sequencing v2.3
Purpose:
This protocol models the lab processes of starting a NextSeq 1000/2000 sequencing run.
Steps:
Validation Workflow
The Library Prep Validation v2.3.1 workflow allows for validation of the system after installation is complete. For details, refer to .
Step 1: Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3)
In this step, the addition of RSB dilutes pooled samples. Manually create a working pool based on the final loading concentration required.
Create one pool per step. The Calculate Volumes automation supports one pool only.
Register Step Started¹
This automation registers the start time of the step by publishing messages to CLPA through Illumina Connected Analytics. This automation is automatically triggered on entry to the step.
Calculate Volumes Automation
This automation is triggered when you select Calculate Volumes on the Record Details screen. The automation completes the following actions:
Sets RSB Volume for Pool (µl) field value to 24 for calculation purpose of the 2 nM intermediate library pool.
Set Next Step Automation
Automatically triggered on exit of the Record Details screen, this automation sets the next step for samples to ADVANCE, advancing them to the next step in the protocol. The next step is Load to Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3):
Register Pools & Register Step Completed¹
This automation is automatically triggered on exit of the step and performs the following tasks:
Registers the pool and library information used by the Clarity LIMS workflow. Samples in the pool are assumed to have gone through library preparation.
¹ These automations are required for Clarity LIMS Product Analytics support only.
Master Step Fields
The following fields are defined on the Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) step.
Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) Master Step Field Configuration
Global Fields
The following table lists the global fields that are configured to display on the Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) step.
Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) Global Field Configuration
Step 2: Load to Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3)
In this step, scan the reagent cartridge barcode into Clarity LIMS, then manually place the working pool into the reagent cartridge for the NextSeq 1000/2000 run. This step validates the run setup and analysis information and generates the sample sheet file and/or creates a planned run on Illumina Connected Analytics, depending on the Run Mode selected.
The NextSeq 1000/2000 reagent cartridges support different read cycle numbers. Make sure that the read cycle values configured for the planned run are within the maximum allowable reads for the cartridge type.
Validate Single Input & Register Step Started Automation²
Automatically triggered at the beginning of the step, this automation does the following actions:
Checks that there is only one container input to the step. This script is required for the NextSeq 1000/2000 Sequencing v2.3 workflow.
Validate Reagent Cartridge Barcode Automation
Automatically triggered on exit of the Placement screen, the following automation validates the reagent cartridge barcode to make sure it conforms to the barcode mask [A-Za-z]{2}[0-9]{7}-[A-Za-z0-9]{4}:
Retrieve Analysis Workflow Versions Automation
Automatically triggered upon entry of the Record Details screen, this automation completes the following actions:
Fetches the available analysis workflow versions from Illumina Connected Analytics.
Validate Run Setup and Create Planned Run Automation
Automatically triggered when a button on the Record Details screen is selected, this automation does the following actions:
Validates the parameters entered on the Record Details screen. These parameters are used to set up the run and generate the sample sheet file and/or create the planned run on Illumina Connected Analytics.
Set Next Step Automation
Sets the next step for samples to ADVANCE, advancing them to the next step in the protocol—AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3). The automation is automatically triggered on exit of the Record Details screen.
Register Step Completed¹
Automatically triggered on exit of the step, this automation registers the completion time of the step by publishing messages to Clarity LIMS Product Analytics through Illumina Connected Analytics. This script is only used for Clarity LIMS Product Analytics support.
¹ These automations are required for Clarity LIMS Product Analytics support only.
² These automations are required for the NextSeq 1000/2000 Sequencing v2.3 workflows and contain additional logic needed for Clarity LIMS Product Analytics support. If you would like to remove Clarity LIMS Product Analytics support, contact Illumina Support.
Master Step Fields
There are 24 fields defined on the Load to Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3) step. These fields are required for sample sheet generation and planned run creation on Illumina Connected Analytics (ICA).
Step 3: AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3)
This step is fully automated.
The integration starts and completes the step automatically. Data from the run is parsed back to Clarity LIMS. No user interaction is required. In this step, the pooled samples in the reagent cartridge are sequenced on the NextSeq 1000/2000 instrument.
Do not add samples to the Ice Bucket or start or complete the step. The integration does this action automatically.
Register Step Started & Register NextSeq1k2k Run & Register NextSeq1k2k Run Association & Register NextSeq1k2k RunStarted¹
Automatically triggered at the beginning of the step, this automation completes the following actions:
Automatically triggered on exit of the step, this automation completes the following actions:
Registers the sequencing run completion time, run status, and run metrics. This script requires the Cloud Run ID field from the Load to Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3) step.
¹ These automations are required for Clarity LIMS Product Analytics support only.
Master Step Fields
There following fields are defined on the AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3) step. These fields are used to display the run status and sequencing run and analysis configuration parsed from the RunParameters.xml file of the sequencing run.
Run Parameters and Corresponding Clarity LIMS Step Fields The following table shows how some of the step fields map to the fields on the RunParameters.xml file, and whether the field is visible on the Record Details screen.
Additional Master Step Fields and Values The following table shows how the other step fields derive their values, and whether the step field is visible on the Record Details screen.
¹ For information on how the integration works, refer to Run Status, Primary Metrics, and Analysis Results Parsing and Recording in .
Global Fields
The following global fields are used to capture the run metrics in Clarity LIMS.
% Bases >=Q30 R1
% Bases >=Q30 R2
% Error Rate R1
% Error Rate R2
At the end of this step, the pool of samples is automatically advanced to (and queued for) the Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) step.
Do not add samples to the Ice Bucket or start or complete the step. The integration does this action automatically.
For the demultiplexing information in the Sample Details table to display correctly, the following conditions must be met:
The DRAGEN version must be earlier than v3.9.3.
The integration starts the step automatically and demultiplexing data from the GenerateFASTQ secondary analysis is parsed back to Clarity LIMS. The lab scientist reviews the demultiplexing result parsed into Clarity LIMS, assigns QC flags, and completes the step.
Register Step Started Automation
Automatically triggered at the beginning of the step, this automation registers the start time of the step by publishing messages to Clarity LIMS Product Analytics through Illumina Connected Analytics. This automation is only used for Clarity LIMS Product Analytics support.
Assign Demultiplexing QC Flags Automation
Automatically triggered when you select a button on the Record Details screen, this automation assigns QC flags based on the criteria set in the step fields.
Set Next Step Automation
Automatically triggered on exit of the Record Details screen, this automation sets the next step to Advance and the samples to complete the protocol.
Automatically triggered on exit of the step, this automation performs the following actions:
Registers the NextSeq 1000/2000 analysis status and result.
Master Step Fields
The following table lists the 12 master step fields that are configured on the Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) master step.
Master Step Field Configuration for Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) Step
Global Fields
The following table lists the global fields that are configured on the Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) step. These fields are used to display the demultiplexing result metrics for individual library in the sample pool.
Global Field Configuration for Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) Step
How the NextSeq 1000/2000 Integration Works
Sample Sheet Generation and Planned Run Creation
On the Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3) step, when the Validate Run Setup and Create Planned Run automation is triggered, the run and analysis parameters entered in the Run Details screen are sent to Illumina Connected Analytics (ICA). ICA validates the run and analysis configuration, and sends an error message to Clarity LIMS if the validation fails. If the validation passes, ICA generates the sample sheet (and sends back to Clarity LIMS) and/or creates a planned run depending on the selected. NextSeq 1000/2000 Integration supports local, hybrid and cloud run mode.
Refer to for details on how to start a run using different run mode.
Run Status, Primary Metrics, and Analysis Results Parsing and Recording
After the sequencing run is started on the instrument, NextSeq 1000/2000 Control Software (NCS) notifies BaseSpace Sequence Hub (BSSH) that the sequencing run has started.
BSSH then does the following actions:
Local run mode: Creates a run on the BSSH database with the run configuration information received from NCS.
Hybrid/Cloud run mode: Creates a run on the BSSH database by copying the run configuration information from the planned run created in ICA, using a special Cloud Run ID received from NCS.
After the run is created on BSSH, a run update event message that carries the RunStarted equivalent message is created on BSSH and sent to the integration service which processes the message.
Based on the reagent cartridge barcode information in the event message (refer to the following example), the integration looks in the queue of AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3) step for a container with the same reagent cartridge barcode. When identified, the step is started automatically and the Run Status field on Record Details screen is updated to RunStarted. The integration then updates all the other step fields (eg, Current Read, Output Folder) by parsing the details from the RunParameters.xml uploaded to BSSH by NCS.
How the Integration Service Handles Run Status
The NextSeq 1000/2000 Control Software (NCS) continues to notify BaseSpace Sequence Hub (BSSH) of any run status change throughout the course of the sequencing run. The run update event delivery process continues as described in the previous section.
The following table lists the run status details and how the integration service handles each status.
Start A Sequencing Run On Instrument With Different Run Modes
Configure a run with the Proactive, Run Monitoring and Storage option. Local runs are done in Local mode, and cloud or hybrid runs are done in Cloud or Hybrid mode.
Runs with local analysis (i.e., Local and Hybrid run mode) requires ICA Run Storage setting in BaseSpace Sequence Hub to be set to Off for automatic tracking of status and results by Clarity integration. Only Workgroup owner has the permission to change this setting.
Start the local, cloud, or hybrid run.
Refer to the for information on run configuration and starting a run.
Components Installed
This section describes the various components that are installed by default as part of this integration.
Information on installed workflows, protocols, steps, and automation points is provided in the Workflows, Protocols, and Steps section of .
Reagent Categories/Label Groups
TruSeq HT adapters v2 (D7-D5)
Reagent Kits
Resuspension Buffer (RSB)
NextSeq 1000/2000 reagent cartridge
Container Types
Tube
96-well plate
NextSeq 1000/2000 reagent cartridge
Control Types
PhiX v3
This integration supports the NextSeq 1000/2000 reagent cartridge with barcode provided in the format [A-Za-z]{2}[0-9]{7}-[A-Za-z0-9]{4} (eg, EC1234567-EC03).
Enabling Planned Run Generation for Samples Having Duplicate Name with Different Indexes
The before routing the samples through the library preparation workflow.
Re-queue Samples for Cloud Run
Clarity LIMS requires unique library names when samples are re-queued to the workflow in the NextSeq 1000/2000 integration. Assign unique names to libraries after going through library preparation.
The following steps are used to configure the library preparation workflow correctly before routing the samples for re-queuing:
From Configuration, select the Lab Work tab.
Search for the library preparation workflow used for the re-queued samples.
In the Master Step of the library preparation workflow, modify the naming convention under Step Type to generate unique library names (E.g., appending LIMS ID to the default naming convention, like OutputItemLIMSID).
Rules and Constraints
The NextSeq 1000/2000 Reagent Cartridge barcode should not be modified after a successful validation. Modifications can cause issues when Clarity LIMS tries to update the status and sample details of subsequent steps.
The workflow configuration contains several validation checks. To make sure that the calculations work properly, it is important that you do not disable any of this validation logic. The validation checks determine:
Which samples, and how many, can enter each step together.
Which samples, and how many, can be pooled together.
Revision History
The protocol contains a single step—Library Prep Validation v2.3.1. At the end of this step, a routing script sends the samples to the first step of the NextSeq 1000/2000 Sequencing v2.3 workflow. The first step is Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3).
Steps:
Library Prep Validation v2.3.1
Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3)
Samples are pooled and diluted to the final loading concentration with the help of the Calculate Volume script.
Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3)
The library pool from step 1 is then ready to be loaded to the NextSeq 1000/2000 reagent cartridge.
Run and analysis information is validated.
Sample sheet is generated and/or planned run is created.
AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3)
This step is a fully automated step that is started and completed automatically after the sequencing run is started and completed on the instrument side.
All the metadata (E.g., run configuration, primary run metrics, etc.) of the sequencing run are recorded automatically.
GenerateFastQ secondary analysis is planned by default for the run. Samples continues to the next step.
âš Do not add samples to the Ice Bucket or start or complete the step. The integration does this action automatically.
This step is a semi-automated step that is started automatically after the GenerateFastQ secondary analysis has started.
Demultiplexing result is parsed and recorded automatically.
User is required to assign QC label (Pass/Fail) to the individual library in the pool and complete the step.
âš Do not add samples to the Ice Bucket or start or complete the step. The integration does this action automatically.
Copies the Final Loading Concentration (pM) from step inputs to step outputs.
Calculate per sample volume required for each library to make the 2 nM intermediate library pool (24µl in volume).
Calculate the volume required of the 2 nM library pool to be diluted further to the Final Loading Concentration (pM) with the Final Loading Volume (µl).
Uses the NextSeq1K2K_Pool1.csv, NextSeq1K2K_Pool2.csv, and NextSeq1K2K_Pool3.csv template files to generate a single CSV file containing information about the pool and the samples it contains. The generated file is stored in the Calculation File placeholder, in the Files section, for download.
Registers the completion time of the step by publishing messages to CLPA through Illumina Connected Analytics.
This automation is only used for CLPA support.
RSB Volume for Pool (ul)
Numeric
Read Only
Hidden
âš This field is used by the Calculate Volumes automation. Removing, renaming, or modifying the field will break the automation.
Registers the start time of the step by publishing messages to Clarity LIMS Product Analytics through Illumina Connected Analytics. This script is only used for Clarity LIMS Product Analytics support.
Updates the preset values of both Local Analysis Workflow Versions & Cloud Analysis Workflow Versions fields.
âš Do not modify or disable the Retrieve Analysis Workflow Versions automation script. Modifying or disabling the script breaks the integration.
Run Name can only contain alphanumeric, dash, underscore, or period characters. Spaces are not permitted.
Run Name must not exceed 255 characters.
Checks the Index Reads, Index Read 1, and Index Read 2 field values.
If Index Reads is No Index, Index Read 1 and Index Read 2 values must be 0 (error results if it is otherwise).
If Index Reads is Single Index, Index Read 1 value must be greater than 0, and Index Read 2 values must be 0 (error results if it is otherwise).
If Index Reads is Dual Index, Index Read 1 and Index Read 2 values must be greater than 0 (error results if it is otherwise).
Checks the Paired End, Read 2 Cycles, and Index Read 2 field values.
If Paired End is set to True and Read 2 Cycles value is 0, an error is generated.
If Paired End is set to False and Read 2 Cycles or Index Read 2 values are greater than 0, an error is generated.
Checks the Adapter Sequence Read 1 and Adapter Sequence Read 2 field values.
Adapter Sequence Read 1 and Adapter Sequence Read 2 can only contain ACTG+ characters.
Checks Override Cycles field value.
Override Cycles can only contain Y, N, I, U, 0–9, and semicolon characters.
When the Run Mode is Cloud, the script does the following actions:
Sets Local Analysis Workflow Versions to None.
If the Cloud Analysis Workflow Versions is None, the script displays the following error: Invalid option selected for Cloud Analysis Workflow Versions field.
Due to automation script limitation, a validation rule resides in the backend service to perform the following action: If Cloud Analysis Workflow Versions is 3.7.4 or later, set FASTQ Compression Format to gzip.
When the Run Mode is Local or Hybrid, the script does the following actions:
Sets Cloud Analysis Workflow Versions to None.
If Local Analysis Workflow Versions is None, the script displays the following error message: Invalid option selected for Local Analysis Workflow Versions field.
Due to automation script limitation, a validation rule resides in the backend service. If Local Analysis Workflow Versions is 3.7.4 or later, an error occurs if FASTQ Compression Format is None.
Generates the sample sheet and/or creates the planned run on Illumina Connected Analytics.
Cloud Run ID
(Used in creating planned run in ICA)
Text
Read Only
Hidden
Use Custom Index Read 1 Primer
(Custom primer option is currently not supported)
Toggle Switch
Default
None Set
Hidden
Use Custom Index Read 2 Primer
(Custom primer option is currently not supported)
Toggle Switch
Default
None Set
Hidden
Use Custom Read 1 Primer
(Custom primer option is currently not supported)
Toggle Switch
Default
None Set
Hidden
Use Custom Read 2 Primer
(Custom primer option is currently not supported)
Toggle Switch
Default
None Set
Hidden
Instructions
Multiline Text
Read Only
Default
If GenerateFASTQ analysis is required, please select the DRAGEN version in Cloud Analysis Workflow Versions (for Cloud run mode) or Local Analysis Workflow Versions (for Local/Hybrid run mode) custom fields above.
Local Analysis Workflow Versions
Text Dropdown
Default
None
Cloud Analysis Workflow Versions
Text Dropdown
Default
None
FASTQ Compression Format
Text Dropdown
Required Field
Presets
gzip
DRAGEN
None
Default
Registers the NextSeq 1000/2000 sequencing run configuration.
Registers the association of the sequencing run with the actual samples by linking the Instrument Run ID with the IDs for the samples.
Registers the time that the sequencing run starts.
The automation completes these actions by publishing messages to Clarity LIMS Product Analytics through Illumina Connected Analytics. This automation is only used for Clarity LIMS Product Analytics support.
Registers the completion time of the step.
The automation completes these actions by publishing messages to Clarity LIMS Product Analytics through Illumina Connected Analytics. This automation is only used for Clarity LIMS Product Analytics support.
ApplicationVersion
Visible
Instrument ID
InstrumentSerialNumber
Visible
Output Folder
OutputFolder
Visible
Reagent Cartridge ID
CartridgeSerialNumber
Visible
Reagent Cartridge Lot Number
CartridgeLotNumber
Visible
RTA Version
RtaVersion
Visible
Run Name
ExperimentName
Visible
Secondary Analysis Workflow
SecondaryAnalysisWorkflow
Visible
Flow Cell Mode
FlowCellMode
Hidden
Instrument Run ID
Derived from OutputFolder
Hidden
Run End Time
RunEndTime
Hidden
Run Start Time
RunStartTime
Hidden
Secondary Analysis Mode
SecondaryAnalysisMode
Hidden
Secondary Analysis Platform Version
SecondaryAnalysisPlatformVersion
Hidden
SkipObdd
SkipObdd
Hidden
Received as part of the event payload¹
Hidden
Yield (Gb) R1
Yield (Gb) R2
Reads PF R1
Reads PF R2
%PF R1
%PF R2
% Aligned R1
% Aligned R2
% Phasing R1
% Phasing R2
% Prephasing R1
% Prephasing R2
Intensity Cycle 1 R1
Intensity Cycle 1 R2
Cluster Density R1
Cluster Density R2
The samples in the run must not repeat.
The run must be for the single lane flow cell (P1 or P2).
Registers the completion time of the step.
These actions are done by publishing messages to Clarity LIMS Product Analytics through Illumina Connected Analytics. This automation is only used for Clarity LIMS Product Analytics support.
Criteria 2 - Operator
Text Dropdown
Custom Entries
Presets
>=
<=
=
Criteria 1 - Threshold Value
Numeric
Valid integer value
Criteria 2 - Threshold Value
Numeric
Valid integer value
Log
Multiline Text
Read Only
Analysis Completed Time
Text
Read Only
Hidden
Analysis Started Time
Text
Read Only
Hidden
Analysis Status
Text
Read Only
Hidden
BSSH Analysis ID
Text
Read Only
Hidden
Reagent Cartridge ID
(Used for parsing demultiplexing results)
Hidden step fields are updated with information from BSSH. In particular, Analysis Status is updated to either TimedOut or Aborted.
Multiline Sequencing Log field is updated.
The integration does not automatically complete the step. Assign the QC flag to the individual library and manually complete the step.
Select Save.
The NextSeq 1000/2000 reagent cartridge barcode must be unique. There should not be multiple NextSeq 1000/2000 reagent cartridge containers in the system with the same name.
Reagent labels (indexes) must be unique.
One library pool can only contain one library or control with no label (index).
Do not manually start or complete the AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3) step. This step is a fully automated step, and the integration service does not update samples correctly if they have been manually started.
Do not manually start the Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) step. This step is semi-automated, and the integration service does not update the demultiplexing result correctly if they have been manually started.
For the automated run to start successfully, select Validate Run Setup and Create Planned Run in the Load to Reagent Cartridge step.
The NextSeq 1000/2000 Reagent Cartridge barcode should not be modified after it has been successfully validated. Modifying the barcode would result in Clarity failing to update subsequent steps and sample details properly.
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-down Items
Final Loading Concentration (pM)
Numeric Dropdown
Required Field
Custom Entries
Presets
650
750
1000
2000
Final Loading Volume (ul)
Numeric
Required Field
Default
24
Library Pool Volume (ul)
Numeric
Read Only
Hidden
âš This field is used by the Calculate Volumes automation. Removing, renaming, or modifying the field will break the automation.
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-down Items
Final Loading Concentration (pM)
Numeric Dropdown
Required Field
Custom Entries
Decimal places displayed = 0
Presets
225
400
RSB Volume (µl)
Numeric
Read Only
Decimal places displayed = 2
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-down Items
Run Name
Text
Required Field
Instrument Type
Text Dropdown
Required Field
Presets
NextSeq1000
NextSeq2000
Run Mode
Text Dropdown
Required Field
Presets
Local
Hybrid
Cloud
Master Step Field
RunParameters.xml Field
On Record Details Screen
Current Cycle
Calculated based on CompletedCycles Field
Visible
Current Read
Calculated based on CompletedCycles field against PlannedCycles
Visible
Flow Cell ID
FlowCellSerialNumber
Visible
Flow Cell Lot Number
FlowCellLotNumber
Visible
Master Step Field
RunParameters.xml Field
On Record Details Screen
Instrument Platform
NextSeq 1000/2000
Constant value
Visible
Instrument Type
One of the following options:
NextSeq1000
NextSeq2000
Determined from Instrument Type step field from previous Load to Reagent Cartridge step.
Visible
Run Status
One of the following options:
RunStarted
RunCompletedSuccessfully
RunAbortedByUser
RunErroredOut
Set by the integration service¹
Visible
Sequencing Log
Filled in by the integration service¹ as the sequencing run proceeds
Visible
Field Name
Field Type
Field Constraints/Options
Preset Values/Additional Options and Drop-down Items
Criteria 1 - Source Data Field
Text Dropdown
Custom Entries
Presets
# Reads
# Perfect Index Reads
# One Mismatch Index Reads
# of >= Q30 Bases (PF)
Criteria 2 - Source Data Field
Text Dropdown
Custom Entries
Presets
# Reads
# Perfect Index Reads
# One Mismatch Index Reads
# of >= Q30 Bases (PF)
Criteria 1 - Operator
Text Dropdown
Custom Entries
Presets
>=
<=
=
!=
Field Name
Field Type
Field Constraints/Options
# of >= Q30 Bases (PF)
Numeric
Read Only
# One Mismatch Index Reads
Numeric
Read Only
# Perfect Index Reads
Numeric
Read Only
# Reads
Numeric
Read Only
Run Status
Shown in Step
Details
RunStarted
AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3)
Run Status field is updated to RunStarted.
All other step fields (except Run End Time) are updated based on RunParameters.xml detail obtained from BSSH, the SQS event message, and the step field value from the Load to Reagent Cartridge (NextSeq 1000/2000 v2.3) step.
Reagent cartridge lot information received from BSSH is compared against the details of the pre-accessioned reagent cartridge on Clarity LIMS.
If the expiration date is different, it is updated to the date received from BSSH.
If reagent cartridge is not found, it is created and set to Active.
If reagent cartridge is not in Active status, it is set to Active.
Multiline Sequencing Log field is updated.
RunCompletedSuccessfully
AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3)
Run Status field is updated to RunCompletedSuccessfully
Current Read and Current Cycle fields are updated to the final cycle number and read number based on RunParameters.xml detail obtained from BSSH.
Run End Time field is updated based on RunParameters.xml detail obtained from BSSH.
Sequencing run result (primary metrics) is downloaded from BSSH and the fields in Sample Details table (e.g., % Error Rate R1, % Phasing, etc.) are updated with the values.
Multiline Sequencing Log field is updated.
The integration automatically completes the step and routes the samples in the reagent cartridge container to the next step (e.g., Demultiplexing (NextSeq 1000/2000 Sequencing v2.3)) step.
RunErroredOut
AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3)
Run Status field is updated to RunErroredOut.
Current Read and Current Cycle fields are updated to the final cycle number and read number that the run stopped based on RunParameters.xml detail obtained from BSSH.
Run End Time field is updated based on RunParameters.xml detail obtained from BSSH.
Sequencing run result (primary metrics) is downloaded from BSSH and the fields in Sample Details table (e.g., % Error Rate R1, % Phasing, etc.) are updated with the values.
Multiline Sequencing Log field is updated.
The integration does not automatically complete the step. Complete the step manually.
RunAbortedByUser
AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3)
Run Status field is updated to RunAbortedByUser.
Current Read and Current Cycle fields are updated to the final cycle number and read number that was aborted based on RunParameters.xml details obtained from BSSH.
Run End Time field is updated based on RunParameters.xml detail obtained from BSSH.
Sequencing run result (primary metrics) is downloaded from BSSH and the fields in Sample Details table (e.g., % Error Rate R1, % Phasing, etc.) are updated with the values.
Multiline Sequencing Log field is updated.
The integration does not automatically complete the step. Complete the step manually.
Version
Changes
2
Updated Start A Sequencing Run On Instrument With Different Run Modes section. Local and Hybrid run modes require ICA Run Storage setting in BaseSpace Sequence Hub to be set to Off.
if (!step.::Run Name::.matches(::[a-zA-Z0-9-_.]+::)){
fail(::Run Name contains prohibited characters. Allowed characters are: a-z, A-Z, 0-9, -, _, and .::)
}
if (step.::Run Name::.length() > 255){
fail(::Run Name shall not exceed 255 characters.::)
};
if (step.::Paired End::.toBoolean()){
if (step.::Read 2 Cycles:: == 0){
fail(::Read 2 Cycles must not be zero if it is Paired End read.::)
}
}
else{
if (step.::Read 2 Cycles:: != 0 || step.::Index Read 2:: != 0){
fail(::Read 2 Cycles and Index 2 Cycles must be 0 if it is not Paired End Read.::)
}
}
if (step.hasValue(::Override Cycles::) && !step.::Override Cycles::.matches(::[YNIU0-9;]+::)){
fail(::Override Cycles contains prohibited characters. Allowed characters are: Y, N, I, U, 0-9 and ;. Example: N1Y150;I8;I7N1;Y141U10.::)
}
if (step.::Run Mode:: == ::Cloud::){
step.::Local Analysis Workflow Versions:: = ::None::;
if (step.::Cloud Analysis Workflow Versions:: == ::None::){
fail(::Invalid option selected for Cloud Analysis Workflow Versions field.::);
}
};
if (step.::Run Mode:: == ::Local:: || step.::Run Mode:: == ::Hybrid::){
step.::Cloud Analysis Workflow Versions:: = ::None::;
if (step.::Local Analysis Workflow Versions:: == ::None::){
fail(::Invalid option selected for Local Analysis Workflow Versions field.::);
}
};
Automatic validation of run setup information before sample sheet generation or planned run creation.
Automated generation of a sample sheet. The sample sheet is used to start the sequencing run on NextSeq 1000/2000 Control Software (NCS) via Local run mode.
Automated creation of a planned run on Illumina Connected Analytics (ICA). The NCS retrieves and starts the planned run via Hybrid or Cloud run mode.
Automated tracking of the NextSeq 1000/2000 sequencing run and parsing of run statistics from BaseSpace Sequence Hub into Clarity LIMS using SIS integration service.
Automated tracking of the secondary analysis using DRAGEN (up to BCL Convert only) and parsing of demultiplexing metrics from BSSH into Clarity LIMS using SIS integration service.
Support for parsing the unicode Parameter.xml file generated by NCS v1.5.
[Optional] Preconfigured Library Prep Validation v2.3.1 workflow used for validation purposes only. The workflow contains a single-step protocol that models the library prep workflow required to produce libraries tagged with index sequences. At the end of the step, these libraries are routed to NextSeq 1000/2000 Sequencing v2.3 workflow. For more information, refer to NextSeq 1000/2000 Integration v2.4 User Interaction, Validation and Troubleshooting.
[Optional] An integration of Clarity LIMS Product Analytics (CLPA) that make Clarity LIMS data available to the ICA and CLPA. This integration also requires installation and configuration. For more information, refer to Clarity LIMS Product Analytics Integration v1.2 Configuration.
51
51
gzip
!=
Multiline Sequencing Log field is updated.
The integration does not automatically complete the step. Assign the QC flag to the individual library and manually complete the step.
if (step.::Index Reads:: == ::No Index::){
if (step.::Index Read 1:: != 0 || step.::Index Read 2:: != 0){
fail(::Index Read 1 and Index Read 2 must be 0 if the Index Reads is No Index.::)
}
}
else{
if (step.::Index Reads:: == ::Single Index::){
if (step.::Index Read 1:: == 0 || step.::Index Read 2:: != 0){
fail(::Index Read 1 must be greater than 0 and Index Read 2 must be 0 if the Index Reads is Single Index.::)
}
}
else{
if (step.::Index Read 1:: == 0 || step.::Index Read 2:: == 0){
fail(::Index Read 1 and Index Read 2 must be greater than 0 if the Index Reads is Dual Index.::)
}
}
}
Release Notes
Last Updated: November 2024
Release Date: September 2023
Document Version: 2
These release notes describe the key changes to software components for the Clarity LIMS NextSeq 1000/2000 Integration Package v2.4.0.
Compatibility
Refer to under Instruments & Integrations.
New Features
Updates third-party dependency libraries.
Defect Repairs
The NextSeq 1000/2000 sample sheet now reflects the submitted sample name instead of the library name.
The scope for the JWT token in the integration is restricted.
Fixed SIS core service not being properly registered in systemctl.
Known Issues
Sequencing run error triggered by disk space full on instrument causes RunParameters.xml to be empty and the run event cannot be processed on Clarity LIMS. This error is unlikely to occur as pre-run checks on the instrument check for sufficient disk space before run is started.
if any of the samples in the pools has been assigned QC flag in prior steps before entering the Load to Reagent Cartridge step.
Limitations
For the demultiplexing information in the Sample Details table to display correctly, the following conditions must be met:
The DRAGEN version must be earlier than v3.9.3.
The samples in the run must not repeat.
The run must be for the single lane flow cell (P1 or P2).
Revision History
Version
Changes
2
Updated Compatibility section to reference Compatibility matrix table.
This section explains how to validate the installation of the Illumina NextSeq 1000/2000 Integration Package v2.4.0.
The validation process involves the following items:
Running samples through the Library Prep Validation v2.3.1 workflow.
The workflow contains a single-step protocol that models the library prep workflow required to produce libraries tagged with index sequences. At the end of the step, these libraries are routed to NextSeq 1000/2000 Sequencing v2.3 workflow.
Running the libraries through the NextSeq 1000/2000 Sequencing v2.3 workflow. This process validates the following information:
Successful sequential step advancement of samples in the following steps:
Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3)
Before running the validation steps below, these steps assume that the NextSeq 1000/2000 Integration Package v2.4.0 is installed, and that you have imported the default Clarity LIMS configuration.
For information on how the integration works, refer to .
Activate Workflow, Create Project, Add and Assign Samples
The following steps set up Clarity LIMS in preparation for running samples through the Library Prep Validation v2.3.1 and NextSeq 1000/2000 Sequencing v2.3 workflows.
On the Configuration tab, under Workflows, activate both the Library Prep Validation v2.3.1 and NextSeq 1000/2000 Sequencing v2.3 workflows.
On the Projects and Samples screen, create a project and add samples to it.
âš Use only alphanumeric, dash, and underscore characters in the submitted sample names. Failing to do so causes sample sheet validation failure in Load To Reagent Cartridge step.
NextSeq 1000/2000 Sequencing v2.3 Protocol
Step 1: Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3)
In Lab View, locate the NextSeq 1000/2000 Sequencing v2.3 protocol. The samples are queued for the Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) step.
Add the samples to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
At the end of this step, the pool of samples automatically advances to Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3) step.
Step 2: Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3)
In Lab View, locate the NextSeq 1000/2000 Sequencing v2.3 protocol. The pool of samples is queued for the Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3) step.
Add the samples to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
The Validate Single Input automation is triggered. This automation checks that there is only one container input to the step.
At the end of this step, the pool of samples automatically advances to and is queued for the AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3) step. The sequencing run is ready to be started. For details on how to start the sequencing run for different run modes, refer to .
Step 3: AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3)
This step is fully automated.
Do not add samples to the Ice Bucket or start or complete the step. The integration does this action automatically.
The integration starts the step automatically and data from the run is parsed back to Clarity LIMS. No user interaction is required. However, you can open and review the various stages of the step in Clarity LIMS. Do not perform any action when reviewing the data.
Read summary metrics are recorded for the library pool in the Step Details section and the Sample Details table.
Values are populated in the following master step fields:
Run Name
Current Read
Flow Cell ID
Reagent Cartridge Lot Number
The summary metrics (per run level) populate in the following global fields.
% Bases >=Q30 R1
% Bases >=Q30 R2
% Error Rate R1
% Error Rate R2
At the end of this step, the pool of samples automatically advances to (and queues for) the Demultiplexing (NextSeq 1000/2000 Sequencing v2.3) step.
Do not add samples to the Ice Bucket or start or complete the step. The integration does this action automatically.
For the demultiplexing information in the Sample Details table to display correctly, the following conditions must be met:
The DRAGEN version must be earlier than v3.9.3.
The integration starts the step automatically and demultiplexing data from the GenerateFASTQ secondary analysis is parsed back to Clarity LIMS. Review the data parsed and assign QC, depending on the criteria set, and complete the step.
In the Record Details screen, perform the following actions:
Review demultiplexing data.
Demultiplexing metrics are recorded for the library pool in the Sample Details table. Metrics include columns for # Reads, # Perfect Index Reads, # One Mismatch Index Reads, and # of >= Q30 Bases (PF).
The Demultiplex_stats.csv file generated by the DRAGEN onboard or Cloud analysis module is stored under the Files section.
At this point, the whole NextSeq 1000/2000 Integration workflow is fully validated.
Troubleshooting
If an automation trigger does not appear to run its corresponding scripts, refer to Troubleshooting Automation in the .
If the AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3) step starts but does not finish, complete the following steps:
Log in to Clarity LIMS using the default user account and use one of the following methods to open the step in Clarity LIMS:
Method 1: In Lab View, find the step in the Recent Activities pane.
Method 2: Search for the step in Clarity LIMS using reagent cartridge barcode as the search term.
If you cannot reach the Record Details screen, or if the Sequencing Log field does not contain enough information to resolve the issue, contact the Clarity LIMS support team. Supply the relevant information from the troubleshooting steps already performed.
Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3)
AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3)
Automatic validation of run setup information before sample sheet generation or planned run creation.
Automated generation of a sample sheet. The sample sheet is used to start the sequencing run on NextSeq 1000/2000 Control Software (NCS) via Local run mode.
Automated creation of a planned run on Illumina Connected Analytics. The NextSeq 1000/2000 Control Software retrieves the planned run to start the run via Hybrid or Cloud run mode.
Automated tracking of the NextSeq 1000/2000 sequencing run and parsing of run statistics from BaseSpace Sequence Hub into Clarity LIMS using the SIS Integration Service.
Automated tracking of the secondary analysis using DRAGEN (up to BCL Convert only).
Parsing of demultiplexing metrics from BaseSpace Sequence Hub into Clarity LIMS using SIS integration service.
Assign the samples to the Library Prep Validation v2.3.1 workflow.
On exit from the step, the Routing Script automation is triggered. This automation assigns samples to the first step of the NextSeq 1000/2000 Sequencing v2.3 workflow, which is Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) step.
On the Pooling screen, perform the following actions:
Create a pool by dragging samples into the Pool Creator.
âš Do not create more than one pool for this step. The Calculate Volumes automation in this step supports one pool only.
Type a name for the pool or accept the default name (Pool #1).
Select Record Details.
On the Record Details screen, the Reagent Lot Tracking section tracks the Resuspension Buffer (RSB) lot information used in the step. If required, create new lots by referencing Add and Configure Reagent Kits and Lots in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
In the Reagent Lot Tracking section, select from the active lots displayed in the drop-down list.
The Step Details area contains two required fields:
Final Loading Concentration (pM) — The value entered in this field is the recommended final loading concentration specified in the Illumina NextSeq 1000/2000 Sequencing System Guide at support.illumina.com. The value depends on the library type. The default drop-down list contains the values 650, 750, 1000, and 2000. A custom value is acceptable.
Final Loading Volume (ul) — The value in this field is the final loading volume of the pool into the reagent cartridge. The field is prepopulated with the configured default value, 24 µl, specified in the Illumina NextSeq 1000/2000 Sequencing System Guide at support.illumina.com. The value is editable when more volume is necessary.
Select Calculate Volumes.
This selection triggers the Calculate Volumes automation. This automation calculates the volume required for each library to form a pool that has the concentration and volume specified in the step details fields.
The automation also generates the Calculation File (CSV) and attaches it to the step. This file contains volume information of each of the samples and RSB buffer to add to the pool. Select the file to download it, then open it in Excel.
In the Sample Details table, select the pool icon to view details on the pool composition.
Select Next Steps.
This selection triggers the Set Next Step automation, which sets the next step for samples to ADVANCE, advancing them to the next step in the protocol. The next step is Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3).
On the Assign Next Steps screen, the next step for samples is already set to the next step in the workflow. The next step is Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3).
Select Finish Step.
On the Placement screen, perform the following actions:
Drag the pool into the NextSeq 1000/2000 Reagent Cartridge field in the Placed Samples area.
Scan or type the barcode of the reagent cartridge into the NextSeq 1000/2000 Reagent Cartridge field.
Select Record Details.
On exit of the Placement screen, the Validate Reagent Cartridge Barcode automation checks that the reagent cartridge barcode conforms to the barcode mask [A-Za-z]{2}[0–9]{7}-[A-Za-z0-9]{4}. If not, an error message displays.
âš The NextSeq 1000/2000 Reagent Cartridge barcode should not be modified after a successful validation. Modifications can cause issues when Clarity LIMS tries to update the status and sample details of subsequent steps.
On entry to the Record Details screen, the Retrieve Analysis Workflow Versions automation fetches the available analysis workflow versions from ICA. It then updates the preset values of both Local Analysis Workflow Versions & Cloud Analysis Workflow Versions field.
On the Record Details screen, the Reagent Lot Tracking section tracks the NextSeq 1000/2000 Reagent Cartridge lot information used in the step. Follow the steps in Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) step if you must add a lot.
In the Reagent Lot Tracking section, select from the active lots displayed in the drop-down list.
The fields displayed on the Record Details screen are used to create planned run and generate the sample sheet file.
Run Name — Enter the experiment name. Only alphanumeric characters, dashes, and underscores are permitted. Spaces are not permitted.
Instrument Type — Select from preset options (NextSeq 1000 or NextSeq 2000).
Run Mode — Select from preset options: Local, Hybrid, Cloud. For more information on the different run modes, refer to .
Paired End — Select from preset options (True or False).
Read 1 Cycles — Select from preset options (301, 151, 101, 51) or type a custom value.
Read 2 Cycles — Select from preset options (301, 151, 101, 51) or type a custom value.
Index Reads — Select from preset options (No Index, Single Index, Dual Index).
Index Read 1 — Select from preset options (0, 6, 8) or type a custom value.
Index Read 2 — Select from preset options (0, 6, 8) or type a custom value.
Analysis Workflow — Read only. This field is set to GenerateFASTQ.
Local Analysis Workflow Versions — [Optional] (default is None). Only mandatory when Run Mode = Local/Hybrid and Analysis Workflow = GenerateFASTQ. Select according to the DRAGEN version installed on the instrument.
Cloud Analysis Workflow Versions — [Optional] (default is None). Only mandatory when Run Mode = Cloud and Analysis Workflow = GenerateFASTQ. Select the cloud DRAGEN version to be used.
FASTQ Compression Format:
If Run Mode = Local/Hybrid, Analysis Workflow = GenerateFASTQ, and Local Analysis Workflow Version >= 3.7.4, select gzip or DRAGEN.
If Run Mode = Cloud, Analysis Workflow = GenerateFASTQ, and Local Analysis Workflow Version >= 3.7.4, field is set to gzip automatically.
Adapter Sequence Read 1 — [Optional] Enter the Read 1 adapter sequence of the index adapter kit.
Adapter Sequence Read 2 — [Optional] Enter the Read 2 adapter sequence of the index adapter kit.
Barcode Mismatches Index 1 — [Optional] Select from preset options (0, 1, 2). Leave it blank if you are unsure.
Barcode Mismatches Index 2 — [Optional] Select from preset options (0, 1, 2). Leave it blank if you are unsure.
Override Cycles — [Optional] String used to specify UMI cycles and mask out cycles of a read (eg, N1Y150;I8;I7N1;Y141U10). Leave it blank if you are unsure.
Instructions — Read only.
On the Record Details screen, select Validate Run Setup and Create Planned Run.
This selection triggers the automation script, which does the following actions:
Validates the parameters entered on the Record Details screen.
Create the planned run and send it to ICA if Run Mode is Hybrid or Cloud.
Generates the sample sheet and attaches it to the placeholder in the Files area of the Record Details screen for all Run Mode types.
Select Next Steps.
On the Assign Next Steps screen, the next step for samples is set to the next step in the workflow. The next step is AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.3).
Select Finish Step.
Instrument ID
Run Status
Current Cycle
Flow Cell Lot Number
Instrument Platform
Instrument Control Software Version
Output Folder
Secondary Analysis Workflow
Reagent Cartridge ID
Instrument Type
RTA Version
Sequencing Log
Yield (Gb) R1
Yield (Gb) R2
Reads PF R1
Reads PF R2
%PF R1
%PF R2
% Aligned R1
% Aligned R2
% Phasing R1
% Phasing R2
% Prephasing R1
% Prephasing R2
Intensity Cycle 1 R1
Intensity Cycle 1 R2
Cluster Density R1
Cluster Density R2
The samples in the run must not repeat.
The run must be for the single lane flow cell (P1 or P2).
Assign QC flags to all the individual sample. There are two ways of doing this step.
Manually assign QC flags through the QC column in Sample Details table.
Automatically assign QC flag by running Assign QC flags automation. This option is for scenarios where a huge number of libraries are involved.
In the Step Details section, the following step fields are visible. (N is the number of criteria. You can use one or more criteria.)
Criteria N - Source Data Field — Select from preset options eg # Reads.
Criteria N - Operator — Select from preset options, for example, >= (greater than or equal to).
Criteria N - Threshold Value — Enter the desired threshold value.
After filling up the criteria fields, select Assign QC flags. This selection triggers the automation script, which loops through each library in the pool and apply QC flag base on the criteria set previously.
This automation also generates an AssignQC Result file under Files section.
Select Next Steps.
On the Assign Next Steps screen, the Next Step field for all samples is prepopulated with Mark protocol as complete.
Select Finish Step.
On the Record Details screen, locate the Sequencing Log field.
The multiline text field contains logging information.
