Configuration
Last updated
Last updated
The Illumina NextSeq 1000/2000 Integration Package v2.5.0 supports the integration of Clarity LIMS to Illumina NextSeq 1000/2000 sequencing systems.
For instructions on validating and troubleshooting the NextSeq 1000/2000 Integration, refer to NextSeq 1000/2000 Integration v2.5.0 User Interaction, Validation and Troubleshooting.
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 — could break the process.
The Illumina NextSeq 1000/2000 Integration Package v2.5.0 is compatible with Illumina cloud hosted deployments only.
This integration requires a Personal, Professional, or Enterprise BaseSpace Sequence Hub subscription.
It is assumed that samples entering the NextSeq 1000/2000 Sequencing v2.4 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 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 1000/2000 Integration Package v2.5.0 includes the following workflows:
NextSeq 1000/2000 Sequencing v2.4
[Optional] Library Prep Validation v2.3.3 (recommended for validation purposes)
The following protocols and steps are included in these workflows.
The Library Prep Validation v2.3.3 workflow allows for validation of the system after installation is complete. For details, refer to NextSeq 1000/2000 Integration v2.5.0 User Interaction, Validation and Troubleshooting.
In this step, the addition of RSB dilutes pooled samples. Manually create a working pool based on the final loading concentration required.
¹ These automations are required for CLPA support only.
The following fields are defined on the Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4) step.
Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4) Master Step Field Configuration
The following table lists the global fields that are configured to display on the Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4) step.
Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.4) Global Field Configuration
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 depending on the Run Mode selected.
¹ These automations are required for CLPA support only.
² These automations are required for the NextSeq 1000/2000 Sequencing v2.4 workflows and contain additional logic needed for CLPA support. If you would like to remove CLPA support, contact Illumina Support.
There are 24 fields defined on the Load to Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.4) step. These fields are required for sample sheet generation and planned run creation.
Load to Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.4) Master Step Fields Configuration
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.
¹ These automations are required for CLPA support only.
There following fields are defined on the AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.4) 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 How the NextSeq 1000/2000 Integration Works.
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
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
At the end of this step, the pool of samples is automatically advanced to (and queued for) the Demultiplexing (NextSeq 1000/2000 Sequencing v2.4) step.
This step is a semi-automated step.
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.
¹ These automations are required for CLPA support only.
The following table lists the master step fields that are configured on the Demultiplexing (NextSeq 1000/2000 Sequencing v2.4) master step.
Master Step Field Configuration for Demultiplexing (NextSeq 1000/2000 Sequencing v2.4) Step
The following table lists the global fields that are configured on the Demultiplexing (NextSeq 1000/2000 Sequencing v2.4) step. These fields are used to display the demultiplexing result metrics for individual library in the sample pool.
The demultiplexing statistics are aggregated for multi-lane flow cells. For each demultiplexing field (for example, # Reads) displayed in the Sample Details table for the multi-lane flow cell (for example, P3), the displayed value is the sum of the statistics across all lanes.
Global Field Configuration for Demultiplexing (NextSeq 1000/2000 Sequencing v2.4) Step
On the Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.4) 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 used to create a planned run. Then, the run and analysis configuration is validated. If the validation fails, an error message is sent to Clarity LIMS. If the validation passes, the sample sheet is generated (and sent back to Clarity LIMS) and/or a planned run is created depending on the Run Mode selected.
Local run mode: Only the sample sheet is generated and stored on Clarity LIMS. The sample sheet contains all the run and analysis configuration required to start the run on the instrument.
Hybrid/Cloud run mode: A planned run is created. This planned run contains all the run and analysis configuration required to start the run on the instrument. The sample sheet is also generated and stored on Clarity LIMS for reference purposes.
For details on how to start a run using different run modes, refer to Start A Sequencing Run On Instrument With Different Run Modes.
If a planned run with the same sample and project name has been created previously, the original case with the previous sample name can be reflected in the sample sheet generated from the Validate Run Setup and Create Planned Run automation. To resolve this issue, change the sample or project name in Clarity LIMS and run the automation again.
After the sequencing run is started on the instrument, NextSeq 1000/2000 Control Software (NCS) notifies BaseSpace Sequence Hub that the sequencing run has started.
BaseSpace Sequence Hub then does the following actions:
Local run mode: Creates a run on the BaseSpace Sequence Hub database with the run configuration information received from NCS.
Hybrid/Cloud run mode: Creates a run on the BaseSpace Sequence Hub database by copying the run configuration information from the planned run using a special Cloud Run ID received from NCS.
After the run is created on BaseSpace Sequence Hub, a run update event message that carries the RunStarted equivalent message is created on BaseSpace Sequence Hub and delivered to Clarity LIMS SQS queue service. The message is captured and processed by the integration.
Based on the reagent cartridge barcode information in the event message (refer to the following example), the integration service looks in the queue of AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.4) 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 service then updates all the other step fields (for example, Current Read, Output Folder) by parsing the details from the RunParameters.xml uploaded to BaseSpace Sequence Hub by NCS.
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.
Refer to the NextSeq 1000/2000 Product Documentation for the following information:
Configuring a run with the Proactive, Run Monitory and Storage option. Local runs are done in Local mode, and cloud or hybrid runs are done in Cloud or Hybrid mode.
Starting a local, cloud, or hybrid run.
The library preparation workflow of the samples must be configured to ensure unique derived sample names before routing the samples through the library preparation workflow.
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 (for example, appending LIMS ID to the default naming convention, like OutputItemLIMSID).
Select Save.
The following sections describe the various components that are installed by default as part of this integration. These components include the following items:
Reagent categories/label groups
Reagent kits
Control types
Containers
Information on installed workflows, protocols, steps, and automation points is provided in the Workflows, Protocols, and Steps section of NextSeq 1000/2000 Integration v2.5.0 User Interaction, Validation and Troubleshooting.
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-Z]{2}[0-9]{7}-[A-Z0-9]{4} (for example, EC1234567-EC03).
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.
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 v2.4) 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 v2.4) 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.
Do not add samples to the Ice Bucket or start or complete the step. The integration completes these actions automatically.
Do not add samples to the Ice Bucket or start or complete the step. The integration completes these actions automatically.
Create one pool per step. The Calculate Volumes automation supports one pool only.
Use alphanumeric, dash, or underscore characters only in the submitted sample names. Any other characters can cause sample sheet validation failure in the Load to Reagent Cartridge step. The NextSeq 1000/2000 Sequencing workflow handles these characters in the Load to Reagent Cartridge step by replacing them with an underscore in the submitted sample name. The Sample Details table in the Demultiplexing step reflects the modified submitted sample name.
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.
Do not modify or disable the Retrieve Analysis Workflow Versions automation script. Modifying or disabling the script breaks the integration.
Do not add samples to the Ice Bucket or start or complete the step. The integration completes these actions automatically.
Do not modify the master step field names. This action causes the integration to break.
Do not add samples to the Ice Bucket or start or complete the step. The integration completes these actions automatically.
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.
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 (ul)
Numeric
Read Only
Decimal places displayed: 2
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
Instrument Control Software Version
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
Master Step Field
Description
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
BaseSpace Run ID
Received from SQS message in the integration service¹
Hidden
Field Name
Field Type
Field Constraints/Options
Index Sequence
Text
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 v2.4)
Run Status field is updated to RunStarted.
All other step fields (except Run End Time) are updated based on RunParameters.xml detail obtained from BaseSpace Sequence Hub, the SQS event message, and the step field value from the Load to Reagent Cartridge (NextSeq 1000/2000 v2.4) step.
Reagent cartridge lot information received from BaseSpace Sequence Hub 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 BaseSpace Sequence Hub.
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 v2.4)
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 BaseSpace Sequence Hub.
Run End Time field is updated based on RunParameters.xml detail obtained from BaseSpace Sequence Hub.
Sequencing run result (primary metrics) is downloaded from BaseSpace Sequence Hub 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 Demultiplexing (NextSeq 1000/2000 Sequencing v2.4) step.
RunErroredOut
AUTOMATED - Sequencing Run (NextSeq 1000/2000 Sequencing v2.4)
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 BaseSpace Sequence Hub.
Run End Time field is updated based on RunParameters.xml detail obtained from BaseSpace Sequence Hub.
Sequencing run result (primary metrics) is downloaded from BaseSpace Sequence Hub 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 v2.4)
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 BaseSpace Sequence Hub.
Run End Time field is updated based on RunParameters.xml detail obtained from BaseSpace Sequence Hub.
Sequencing run result (primary metrics) is downloaded from BaseSpace Sequence Hub 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.
AnalysisStarted
Demultiplexing (NextSeq 1000/2000 Sequencing v2.4)
The integration starts the step automatically for the reagent cartridge container.
Multiline Sequencing Log field is updated.
Analysis Status is set to Started.
AnalysisCompleted
Demultiplexing (NextSeq 1000/2000 Sequencing v2.4)
The integration downloads the following metric files:
Adapter_Metrics.csv
Demultiplex_stats.csv
Index_Hopping_Counts.csv
Quality_Metrics.csv
Top_Unknown_Barcodes.csv
The metric files are placed in the Run_Metrics.zip folder that is attached to the file placeholder in the step.
The Demultiplex_stats.csv file contains the demultiplexing results from BaseSpace Sequence Hub. These results are parsed and the details are recorded in the Sample Details table for each library in the library pool. For multi-lane flow cells, the demultiplexing stats are aggregated for each stat across all lanes.
Hidden master step fields are updated with information from BaseSpace Sequence Hub. The Analysis Status field is updated to Complete.
The 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.
AnalysisFailed
Demultiplexing (NextSeq 1000/2000 Sequencing v2.4)
Hidden step fields are updated with information from BaseSpace Sequence Hub.
The Analysis Status field is updated to either TimedOut or Aborted.
The 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.
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
RSB Volume for Pool (ul)
Numeric
Read Only
Hidden
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
Paired End
Text Dropdown
Required Field
Presets
True
False
Read 1 Cycles
Numeric Dropdown
Required Field
Custom Entries
Presets
301
151
101
51
Read 2 Cycles
Numeric Dropdown
Required Field
Custom Entries
Presets
301
151
101
51
Index Reads
Text Dropdown
Required Field
Presets
No Index
Single Index
Dual Index
Index Read 1
Numeric Dropdown
Required Field
Custom Entries
Presets
0
6
8
Index Read 2
Numeric Dropdown
Required Field
Custom Entries
Presets
0
6
8
Analysis Workflow
Text
Required Field
Read Only
Default
GenerateFASTQ
Adapter Sequence Read 1
Text
Adapter Sequence Read 2
Text
Barcode Mismatches Index 1
Numeric Dropdown
Presets
0
1
2
Barcode Mismatches Index 2
Numeric Dropdown
Presets
0
1
2
Override Cycles
Text
Cloud Run ID
Text
Read Only
Hidden
Use Custom Index Read 1 Primer
Toggle Switch
Default
None Set
Hidden
Use Custom Index Read 2 Primer
Toggle Switch
Default
None Set
Hidden
Use Custom Read 1 Primer
Toggle Switch
Default
None Set
Hidden
Use Custom Read 2 Primer
Toggle Switch
Default
None Set
Hidden
Custom primer option is currently not supported. Do not enable as it will break the integration.
Instructions
Multiline Text
Read Only
Default
If GenerateFASTQ analysis is required, 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.
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
gzip
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
Criteria 2 - Source Data Field
Text Dropdown
Custom Entries
Presets
# Reads
# Perfect Index Reads
# One Mismatch Index Reads
Criteria 1 - Operator
Text Dropdown
Custom Entries
Presets
>=
<=
=
!=
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 Tim
Text
Read Only
Hidden
Analysis Started Time
Text
Read Only
Hidden
Analysis Status
Text
Read Only
BSSH Analysis ID
Text
Read Only
Hidden
Reagent Cartridge ID
Text
Read Only
Hidden
This field is used by the Calculate Volumes automation.</blockquote
This field is used by the Calculate Volumes automation.
Used in creating planned run.
Custom primer option is currently not supported. Do not enable as it will break the integration.
Custom primer option is currently not supported. Do not enable as it will break the integration.
Custom primer option is currently not supported. Do not enable as it will break the integration.
Used for parsing demultiplexing results