Last Updated: July 2025
Release Date: July 2024
Document Version: 3
These release notes describe the key changes to software components for the Clarity LIMS NovaSeqDx Integration Package v1.3.0.
Refer to Compatibility under Instruments & Integrations.
Some integration properties are moved to database and can now be accessed and updated via System Setting in Clarity v6.3.
This change requires updates to the configure_sequencer_api_application.sh script.
Refer to for configurable properties.
Fixed security vulnerabilities.
Version
Changes
3
Renamed Defects Fixed section to Defects and Security Vulnerability Fixed section, and updated section contents.
2
Updated Compatibility section to reference Compatibility matrix table.
1
Initial release.
Illumina NovaSeqDx Integration Package v1.3.0 supports the integration of Clarity LIMS to Illumina NovaSeq 6000Dx instruments.
For instructions on user interaction for each step, validating and troubleshooting Illumina NovaSeqDx Integration Package v1.3.0, refer to NovaSeq 6000Dx Integration v1.3.0 User Interaction, Validation and Troubleshooting.
The configuration provided in this integration has been established to support NovaSeq 6000Dx lab processes. Any configuration changes to protocols or workflows—including renaming protocols, steps, and fields—could break the process.
Samples enter the NovaSeqDx v1.2 workflow as normalized libraries. It is assumed that the following steps have completed before samples are assigned to the workflow:
Samples have been accessioned into the 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 the following sections of the Getting Started section of the :
Sample Accessioning
Using Sample Lists to Upload and Modify Samples
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 .
The Illumina NovaSeq 6000Dx Integration Package v1.3.0 includes the following workflows:
NovaSeqDx v1.2
Library Prep Validation v2.3.4 (optional, but recommended for validation purposes)
The following describes the protocols and steps included in these workflows.
The Library Prep Validation v2.3.4 workflow allows for validation of the system after installation is complete. For details, refer to .
This protocol sets the Loading Workflow Type and allows the choice of the appropriate Flowcell Type and Final Loading Concentration (pM). After the protocol, a routing script sends the normalized libraries to either the NovaSeqDx Standard (NovaSeqDx v1.2) or the NovaSeqDx Xp (NovaSeqDx v1.2) protocol.
This protocol contains one step: Define Run Format (NovaSeqDx v1.2).
Step input: Normalized Template Plate (NTP, normalized libraries)
Step output: None
There are four fields defined on the Define Run Format (NovaSeqDx v1.2) master step.
The following table lists field configuration details.
Define Run Format (NovaSeqDx v1.2) Master Step Field Configuration
The following table lists the global custom fields that are configured to display on the Define Run Format (NovaSeqDx v1.2) step.
Global Field Configuration (Derived Sample)
Samples are routed to this protocol when their Loading Workflow Type value is set to NovaSeq Standard. Samples are pooled and added to a library tube in preparation for the NovaSeqDx Run.
At the end of this protocol, a routing script sends the library tube to the AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2) protocol.
This protocol contains the following two steps:
Step 1: Make Bulk Pool for NovaSeqDx Standard (NovaSeqDx v1.2)
Step 2: Dilute and Denature (NovaSeqDx v1.2)
In this step, libraries are placed manually into a single pool. Resuspension buffer and reagents are added.
Create only one pool per step.
Step input: NTP (normalized libraries)
Step output: Bulk pool
The following fields are defined on the Make Bulk Pool for NovaSeqDx Standard (NovaSeqDx v1.2) step.
Make Bulk Pool for NovaSeqDx Standard (NovaSeqDx v1.2) Master Step Field Configuration
The following table lists the global custom fields that are configured to display on the Make Bulk Pool for NovaSeqDx Standard (NovaSeqDx v1.2) step.
Global Custom Fields Configuration (Derived Sample)
In this step, the addition of NaOH, Tris-HCl, and Resuspension Buffer (RSB) denatures and dilutes pooled samples. Manually place the pooled samples into the library tube for the NovaSeqDx Run.
In addition, this step validates the run setup information and generates the sample sheet file.
Step input: Bulk pool
Step output: Library tube
The following table lists field configuration details for the fields that are defined on the Dilute and Denature (NovaSeqDx v1.2) step. These fields are required for sample sheet and JSON file generation.
Dilute and Denature (NovaSeqDx v1.2) Master Step Field Configuration
The following table lists the global custom fields that are configured to display on the Dilute and Denature (NovaSeqDx v1.2) step.
Global Field Configuration (Derived Sample)
Samples are routed to this protocol when their Loading Workflow Type value is set to NovaSeq Xp.
Samples are pooled and added to lanes on the NovaSeqDx flow cell type selected in the Define Run Format (NovaSeqDx v1.2) step. At the end of this protocol, the flow cell is sent to the AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2) protocol.
This protocol contains the following three steps:
Step 1: Make Bulk Pool for NovaSeqDx Xp (NovaSeqDx v1.2)
Step 2: Dilute, Denature & ExAmp (NovaSeqDx v1.2)
Step 3: Load to Flowcell (NovaSeqDx v1.2)
Manually place libraries into a pool.
Create only one pool per step.
Step input: NTP (normalized libraries)
Step output: Bulk pool
The fields in the following table are defined on the Make Bulk Pool for NovaSeqDx Xp (NovaSeqDx v1.2) step.
Make Bulk Pool for NovaSeqDx Xp (NovaSeqDx v1.2) Master Step Field Configuration
The following table lists the global custom fields that are configured to display on the Make Bulk Pool for NovaSeqDx Xp (NovaSeqDx v1.2) step.
Global Field Configuration (Derived Sample)
In this step, the addition of DPX, NaOH, Tris-HCl, and RSB denatures and dilutes pooled samples. Manually create working pools based on the number of lanes that you want to sequence.
Step input: Bulk pool
Step output: Working pool - variable number (choose how many working pools to create per bulk pool)
The following table lists field configuration details for the fields that are defined on the Dilute, Denature & ExAmp (NovaSeqDx v1.2) step. A script sets these field values. They are not editable while running the step.
Dilute, Denature & ExAmp (NovaSeqDx v1.2) Master Step Field Configuration
The following table lists the global custom fields that are configured to display on the Dilute, Denature & ExAmp (NovaSeqDx v1.2) step.
Global Field Configuration (Derived Sample)
In this step, scan the flow cell barcode into the Clarity LIMS and manually place the working pools into the lanes of the flow cell for the NovaSeqDx run. This step validates the run setup information and generates the sample sheet file.
Step input: Working pool
Step output: Flow cell (output containers: SP, S1, and S2 with 2 lanes, and S4 with 4 lanes)
The following table lists the field configuration details for the fields defined on the Load to Flowcell (NovaSeqDx v1.2) step.
Load to Flowcell (NovaSeqDx v1.2) Master Step Field Configuration
The following table lists the global custom fields that are configured to display on the Load to Flowcell (NovaSeqDx v1.2) step.
Global Field Configuration (Derived Sample)
This final protocol contains one fully automated step, AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2).
This step is fully automated. Do not add samples to the Ice Bucket or start the step manually. If steps are manually started, the sequencing service may not update the samples correctly.
Step input: Library tube from NovaSeqDx Standard or flow cell from NovaSeqDx Xp protocol
Step output: Result file/measurement
In this step, pooled samples are sequenced on the NovaSeq 6000Dx instrument and the run metrics are recorded in Clarity LIMS.
The following table shows the master step fields that are configured on the AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2) step.
Clarity LIMS Master Step Field Configuration
The following table shows the global custom fields that capture the run metrics in Clarity LIMS.
Custom Field Configuration
Note the following details:
Values are aggregated across all lanes. Some values (eg, Yield PF (Gb) R1) are summed while others are averaged.
The names listed previously are the default global custom field names installed with the NovaSeqDx v1.2 configuration provided in the Illumina Preset Protocols (IPP) Package v2.9 or later.
All global configuration fields are configured on the Container entity.
All field names are configurable via the Clarity LIMS Configuration > Custom Fields screen (Global Fields tab).
The sample sheet is generated on the step before the run. This step is either Dilute and Denature (NovaSeqDx v1.2) in the NovaSeqDx Standard protocol or Load to Flowcell (NovaSeqDx v1.2) in the NovaSeqDx Xp protocol. This step places samples on the library tube or flow cell that is loaded in the NovaSeq 6000Dx instrument.
In the default configuration, the Validate Run Setup and Generate Sample Sheet automation generates one sample sheet file for use with bcl2fastq v2.20 downstream analysis. This file is in the comma-separated values (CSV) format.
The sample sheet is uploaded to the NovaSeq 6000Dx Operating Software (NVOS) via the /Illumina/Sequencer/v2/sequencing-run/files endpoint to the Sequencer API. The file endpoint allows for a file to be downloaded from Clarity LIMS using OAuth (instead of Basic Authentication), which is required for NovaSeq 6000Dx.
The run recipe response sets the sample sheet URL to the link to download the file from this endpoint and sets the sampleSheetRequiresOAuth value to true.
The following steps outline the sequence of events that occurs when a flow cell is loaded onto the NovaSeq 6000Dx instrument.
The following sections describe the components (files, properties, reagent categories/label groups, reagent kits, and containers) that are installed by default as part of this integration.
Illumina NovaSeq 6000Dx Integration v1.3.0 is distributed as an RPM package, BaseSpaceLIMS-sequencer-api. This RPM package must be installed on the Clarity LIMS server.
The BaseSpaceLIMS-sequencer-api RPM installs the following items:
Sequencer API WAR file
application.yml configuration file
The following configuration scripts:
configure_sequencer_api_proxy.sh
The following table lists the components installed by the RPM package.
Refer to for the properties installed with the integration package.
TruSeq HT Adapters v2 (D7-D5)
Buffer Cartridge
Cluster Cartridge
DPX1
DPX2
Library Tube
SP
S1
S2
This integration supports the following items:
Library tube with barcode provided in the following formats:
RUO mode: [A-Z]{2}[0-9]{7}-[A-Z]{3}
Example: AB1234567-LIB
DX mode: DX[0-9]{7}-[A-Z]{3}
Example: DX1234567-LIB
For details on configuring the NovaSeq 6000Dx Operating Software (NVOS) for integration with Clarity LIMS, contact the Clarity LIMS Support team.
The requirements for the routing script functionality are as follows.
On the steps that use the routing script (Define Run Format (NovaSeqDx v1.2) and Dilute and Denature (NovaSeqDx v1.2)):
The Next Step for all samples must be set to Remove from workflow. A script sets this value. The value must not change in the Assign Next Steps screen.
In the protocol configuration screen, the following settings are required and must not be changed:
The integration.sequencer_api.v2.run.autoCompleteOnlyAtSuccess and integration.sequencer_api.v2.run.autoComplete properties determine the conditions under which the last step of the NovaSeq 6000Dx workflow, AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2), is automatically completed. The two scenarios for autocompletion of the AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2) step are as follows.
The step autocompletes only when the run status is RunCompletedSuccesfully (default behavior).
The step autocompletes regardless of the run status.
By default, both properties are set to true and the step only completes if the run is successful. If the run fails or is aborted, the step must be completed manually in Clarity LIMS.
For the step to autocomplete regardless of the result of the sequencing run, change the autoCompleteOnlyAtSuccess property value to false.
Edit the value of integration.sequencer_api.v2.run.autoCompleteOnlyAtSuccess property as required using the omxProps-ConfigTool utility.
Save the file.
The following table shows how the combined value of the integration.sequencer_api.v2.run.autoComplete and integration.sequencer_api.v2.run.autoCompleteOnlyAtSuccess properties affects the autoComplete behavior of the sequencing step.
run.autoComplete and run.autoCompleteOnlyAtSuccess Value Matrix
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 the following information:
Which samples, and how many, can enter each step together.
Which samples, and how many, can be pooled together.
The library tube ID must be unique. There must not be multiple library tube containers in the system with the same name.
Library Prep Validation v2.3.4
Loading Workflow Type: Select either NovaSeq Standard or NovaSeq Xp.
Normalized Molarity (nM): Enter a value for each sample.
Flowcell Type: Select from options SP, S1, S2, or S4.
Final Loading Concentration (pM): Select from options 225 (PCR-free workflows) or 400 (Nano workflows), or enter a different value.
Compares the Normalized Molarity value of each sample with the Minimum Molarity value.
Validates user selection on NovaSeqDx Run Mode, Loading Workflow Type and Flowcell Type.
Routing script sends samples to the NovaSeqDx Standard or NovaSeqDx Xp protocol, according to the selected Loading Workflow Type.
Samples with Normalized Molarity less than Minimum Molarity are removed from the workflow.
Steps:
Define Run Format (NovaSeqDx v1.2)
Make Bulk Pool for NovaSeqDx Standard (NovaSeqDx v1.2)
Dilute and Denature (NovaSeqDx v1.2)
Make Bulk Pool for NovaSeqDx Xp (NovaSeqDx v1.2)
Dilute, Denature & ExAmp (NovaSeqDx v1.2)
Load to Flowcell (NovaSeqDx v1.2)
AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2)
Calculates the Minimum Molarity with the following formula:
Checks Normalized Molarity value. For samples with no Normalized Molarity value (e.g., empty value, not including 0), generates an error message informing that the field cannot be empty:
Compares the Normalized Molarity value of each sample with the Minimum Molarity value. If the Normalized Molarity value is lower than the Minimum Molarity value, this sets the Loading Workflow Type of the sample to [Remove from workflow]. The automation also records a message in the Warning field for the sample:
At this point, the following options are available:
Correct the NovaSeqDx Run Mode and Normalized Molarity value on the Record Details screen. Edit the Loading Workflow Type field and set it to NovaSeq Standard or NovaSeq Xp, as applicable.
Complete the protocol without correcting the Normalized Molarity value. In this case, those samples are removed from the workflow.
Default automation command line is as follows.
Loading Workflow Type
Text Dropdown
Required Field
Presets
NovaSeq Standard
NovaSeq Xp
Loading Workflow Type
Text Dropdown
Required Field
Presets
NovaSeq Standard
NovaSeq Xp
[Remove from workflow]
Minimum Molarity (nM)
Numeric
Decimal places displayed = 2
Normalized Molarity (nM)
Numeric
Decimal places displayed = 2
NovaSeqDx Run Mode
Text Dropdown
Required Field
Presets
DX
RUO
Per Sample Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
Warning
Text Dropdown
Read Only
Custom Entries
Presets
The Normalized Molarity (nM) is too low.
n/a
Changes the value of error and logging messages to reference the type of pool passed as bulk pool.
Calculates the Per Sample Volume (ul) to be added to the pool.
âš For accurate pipetting of each sample in a pool for sequencing, the Per Sample Volume (ul) value must be equal to or higher than the Minimum Per Sample Volume (ul). The default value (set at 5) can be edited.
Assuming the default Minimum Per Sample Volume (ul) value of 5, this automation completes the following steps for a given batch:
If the smallest Per Sample Volume (ul) value is less than 5, automatically assigns a value of 5 to the Adjusted Per Sample Volume (ul) field of the sample.
Adjusts the Adjusted Per Sample Volume (ul) field value for all other samples in the batch, based on the ratio used to increase the lowest value to 5.
Calculates the Total Sample Volume (ul) field value.
If the Total Sample Volume is less than the Bulk Pool Volume, this automation calculates the RSB Volume (ul) field value.
Copies the Flowcell Type and Loading Workflow Type values from the step inputs to the step outputs.
Sets the Volume of Pool to Denature (ul) value a calculates the NaOH Volume (ul) and Tris-HCl Volume (ul) values based on the Flowcell Type.
Uses the NovaSeq_Standard_Bulk_Pool1.csv, NovaSeq_Standard_Bulk_Pool2.csv, and NovaSeq_Standard_Bulk_Pool3.csv template files to generate a single CSV file containing information about the pool and the samples it contains. The generated file is available for download in the Files section of the Record Details milestone in the Make Bulk Pool for NovaSeqDx Standard (NovaSeqDx v1.2) step.
Resets the Total Sample Volume (ul) and Number of Samples in Pool field values so that the automation is idempotent.
Minimum Per Sample Volume (ul)
Numeric
Required Field
Decimal places displayed = 2
Default
5
Number of Samples in Pool
ℹ For calculation purposes, not displayed
Numeric
Decimal places displayed = 0
Default
0
PhiX Volume (ul)
ℹ For calculation purposes, not displayed
Numeric
Decimal places displayed = 2
Total Sample Volume (ul)
ℹ For calculation purposes, not displayed
Numeric
Decimal places displayed = 2
Default
0
RSB Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
Tris-HCl Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
Volume of Pool to Denature (ul)
ℹ Used in Make Bulk Pool for NovaSeqDx Standard (NovaSeqDx v1.2) step only. Displays on Record Details screen and in the generated CSV file.
Numeric
Read Only
Decimal places displayed = 0
RUO mode: [A-Z]{2}[0-9]{7}-[A-Z]{3}
Copies Flowcell Type and Loading Workflow Type field values from step inputs to outputs:
Validates the parameters entered on the Record Details screen, which is used to set up the run and generate the sample sheet file. The parameters are as follows. For more information, refer to the Respond to Validation / Recipe Request Call from Instrument section of NovaSeq 6000Dx Run.
Experiment Name only contains alphanumeric, dash, or underscore characters. Spaces are not permitted.
When Workflow Type = No Index, both Index Read 1 and Index Read 2 must be zero.
When Workflow Type = Single Index, Index Read 1 must be greater than zero, and Index Read 2 must be zero.
When Workflow Type = Dual Index, both Index Read 1 and Index Read 2 must be greater than zero.
Checks the Paired End and Read 2 Cycles field values as follows.
If Paired End = True, both Read 1 Cycles and Read 2 Cycles must be greater than 0.
If Paired End = False, Read 2 Cycles must be zero.
Checks the Flowcell field value as follows.
If Flowcell Type value is not SP, checks that the values of Read 1 Cycles and Read 2 Cycles are each 151 or less. If the value is greater than 151, an error message is generated.
Validate Analysis Software Version to ensure it is a valid version string.
Validate Override Cycles value to contain only Y, N, I, U, 0-9, and semicolon characters.
Sets the next step for samples to REMOVE:
Generates the V2 sample sheet and attaches it to the step. For more information, refer to Sample Sheet Generation.
Experiment Name
Text
Required Field
Index Read 1
Numeric Dropdown
Required Field
Custom Entries
Range = 0–20
Decimal places displayed = 0
Presets
0
Index Read 2
Numeric Dropdown
Required Field
Custom Entries
Range = 0–20
Decimal places displayed = 0
Presets
0
Output Folder
Text
Required Field
Override Cycles
Text
Paired End
Text Dropdown
Required Field
Presets
True
False
Read 1 Cycle
Numeric Dropdown
Required Field
Custom Entries
Range = 1–251
Decimal places displayed = 0
Presets
251*
Read 2 Cycle
Numeric Dropdown
Required Field
Custom Entries
Range = 0–251
Decimal places displayed = 0
Presets
251*
Run Mode
ℹ Not displayed in user interface
Text Dropdown
Read Only
Presets
SP
S1
S2
Use Custom Index Read 1 Primer
Toggle Switch
Default
None Set
Use Custom Read 1 Primer
Toggle Switch
Default
None Set
Use Custom Read 2 Primer
Toggle Switch
Default
None Set
Use Custom Recipe
Toggle Switch
Required Field
Default
No
Validation Script
Multiline Text
Required Field
Read Only
Default value is provided in the drop-down section that follows the table.
ℹ Do not remove this field as it is used by Validate Run Setup and Generate Sample Sheet automation script.
Workflow
Text
Read Only
Default
GenerateFASTQ
Workflow Type
Text Dropdown
Required Field
Presets
No Index
Single Index
Dual Index
Changes the value of error and logging messages to reference the type of pool passed as 'bulk' pool.
Calculates the Per Sample Volume (ul) to be added to the pool:
âš To ensure accurate pipetting of each sample in a pool for sequencing, the Per Sample Volume (ul) value must be equal to or higher than the Minimum Per Sample Volume (ul). The default value (set at 5) can be edited.
Assuming the default Minimum Per Sample Volume (ul) value of 5, completes the following steps for a given batch:
If the smallest Per Sample Volume (ul) value is less than 5, automatically assigns a value of 5 to the sample Adjusted Per Sample Volume (ul) field.
Adjusts the Adjusted Per Sample Volume (ul) field value for all other samples in the batch, based on the ratio used to increase the lowest value to 5.
Calculates the Total Sample Volume (ul) field value:
If the Total Sample Volume is less than the Bulk Pool Volume, this automation calculates the RSB Volume (ul) field value:
Copies the Flowcell Type and Loading Workflow Type values from the step inputs to the step outputs:
Uses the NovaSeq_Xp_Bulk_Pool.csv and NovaSeq_Xp_Bulk_Pool2.csv template files to generate a single CSV file containing information about the bulk pool and the samples it contains. The generated file is available for download in the Files segment of the Make Bulk Pool for NovaSeqDx Xp (NovaSeqDx v1.2) step.
Resets the Total Sample Volume (ul) and Number of Samples in Pool field values so that the automation is idempotent.
Number of Lanes to Sequence
Numeric
Required Field
Decimal places displayed = 0
Number of Samples in Pool
ℹ For calculation purposes, not displayed
Numeric
Default
0
Decimal places displayed = 0
PhiX Volume (ul)
ℹ For calculation purposes, not displayed
Numeric
Decimal places displayed = 2
Total Sample Volume (ul)
ℹ For calculation purposes, not displayed
Numeric
Default
0
Decimal places displayed = 0
Tris-HCl Volume (ul)
DPX1 Volume (ul)
DPX2 Volume (ul)
DPX3 Volume (ul)
Mastermix per Lane (ul)
The automation also completes the following actions:
Copies the Flowcell Type and Loading Workflow Type values from the step inputs to the step outputs:
Uses the NovaSeq_Xp_Working_Pool.csv and NovaSeq_Xp_Working_Pool2.csv template files to generate a single CSV file. This file contains information about the DPX volume and the volume of BP Aliquot, Mastermix, NaOH, and Tris-HCI to add per working pool. The generated file is available for download in the Files segment of the Dilute, Denature & ExAmp (NovaSeqDx v1.2) step.
Mastermix per Lane (ul)
Numeric
Read Only
Decimal places displayed = 0
NaOH Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
Tris-HCl Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
The following defines the number of samples allowed for different flow cell types:
SP: 2 working pools
S1: 2 working pools
S2: 2 working pools
S4: 4 working pools
Checks that the container type selected on entry to the Placement screen matches the value in the Flowcell Type field. If validation fails, an error message displays.
Automatically triggered by selecting a button on the Record Details screen, this automation:
Copies the Flowcell Type from the step input to the Run Mode field (hidden):
Validates the parameters entered on the Record Details screen, which is used to set up the run and generate the sample sheet file.
Experiment Name only contains alphanumeric, dash, or underscore characters. Spaces are not permitted.
When Workflow Type = No Index, both Index Read 1 and Index Read 2 must be 0.
When Workflow Type = Single Index, Index Read 1 must be greater than 0 and Index Read 2 must be 0.
When Workflow Type = Dual Index, both Index Read 1 and Index Read 2 must be greater than 0.
Checks the Paired End and Read 2 Cycles field values.
When Paired End = True, both Read 1 Cycles and Read 2 Cycles must be greater than 0.
When Paired End = False, Read 2 Cycles must be 0.
Checks the Flowcell field value.
If Flowcell Type value is not SP, checks that the value of both Read 1 Cycles and Read 2 Cycles is 151 or less. If the value is greater than 151, an error message displays.
Validate Analysis Software Version
Checks Override Cycles field value. This field contains only Y, N, I, U, 0–9, and semicolon characters.
Sets the next step for samples to ADVANCE, advancing them to the AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2) step.
Generates the sample sheet and attaches it to the step. For more information, refer to .
Experiment Name
Text
Required Field
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
Library Tube Barcode
Text
Required Field
Output Folder
Text
Required Field
Override Cycles
Text
Paired End
Text Dropdown
Required Field
Presets
True
False
Read 1 Cycles
Numeric Dropdown
Required Field
Custom Entries
Range = 1–251
Decimal places displayed = 0
Presets
251*
Read 2 Cycles
Numeric Dropdown
Required Field
Custom Entries
Range = 0–251
Decimal places displayed = 0
Presets
251*
Run Mode
ℹ Not displayed in user interface
Text Dropdown
Read Only
Presets
SP
S1
S2
Use Custom Index Read 1 Primer
Toggle Switch
Default
None Set
Use Custom Read 1 Primer
Toggle Switch
Default
None Set
Use Custom Read 2 Primer
Toggle Switch
Default
None Set
Use Custom Recipe
Toggle Switch
Required Field
Default
No
Validation Script
Multiline Text
Required Field
Read Only
Default value provided in the drop-down section following the table.
ℹ Do not remove this field as it is used by Validate Run Setup and Generate Sample Sheet automation script.
Workflow
Text
Read Only
Default
GenerateFASTQ
Workflow Type
Text Dropdown
Required Field
Presets
No Index
Single Index
Dual Index
Disable or configure auto-complete behavior by setting the values of the integration.sequencer_api.v2.run.autoComplete and integration.sequencer_api.v2.run.autoCompleteOnlyAtSuccess properties. For details, refer to Configure autoComplete Properties.
Flow Cell Expiration Date
Date
Read Only
Flow Cell ID
Text
Read Only
Flow Cell Lot Number
Text
Read Only
Flow Cell Mode
Text
Read Only
Flow Cell Part Number
Text
Read Only
Flow Cell Side
Text
Read Only
Instrument Control Software Version
Text
Read Only
Instrument ID
Text
Read Only
Instrument Type
Text
Read Only
Lane Counter
Numeric
Read Only
Default
1
Hidden
Loading Workflow Type
Text
Read Only
Output Folder
Text
Read Only
RTA Version
Text
Read Only
Run Completion Date
Text
Read Only
Hidden
Run ID
Text
Read Only
Run Status
Text
Read Only
Sequencing Log
Multiline Text
Read Only
% Bases >=Q30 R2
Numeric
Read Only
Decimal places displayed = 2
% Error Rate R1
Numeric
Read Only
Decimal places displayed = 2
% Error Rate R2
Numeric
Read Only
Decimal places displayed = 2
% Phasing R1
Numeric
Read Only
Decimal places displayed = 3
% Phasing R2
Numeric
Read Only
Decimal places displayed = 3
% Prephasing R1
Numeric
Read Only
Decimal places displayed = 3
% Prephasing R2
Numeric
Read Only
Decimal places displayed = 3
%PF R1
Numeric
Read Only
Decimal places displayed = 2
%PF R2
Numeric
Read Only
Decimal places displayed = 2
Cluster Density (K/mm^2) R1
Numeric
Read Only
Cluster Density (K/mm^2) R2
Numeric
Read Only
Intensity Cycle 1 R1
Numeric
Read Only
Intensity Cycle 1 R2
Numeric
Read Only
Reads PF (M) R1
Numeric
Read Only
Decimal places displayed = 2
Reads PF (M) R2
Numeric
Read Only
Decimal places displayed = 2
Yield PF (Gb) R1
Numeric
Read Only
Decimal places displayed = 2
Yield PF (Gb) R2
Numeric
Read Only
Decimal places displayed = 2
If field names are changed in Clarity LIMS, they must also be changed in the integration.sequencer_api.v2.run.metricUdfNames properties. All fields are configured to be visible on the Record Details screen for the AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2) step (in the Sample Details table). For more information, refer to the Properties section of Components Installed.
ℹ These global custom fields are configured on the Container entity, visible on each input, and must not be confused with per-lane metrics. All run metrics tracked in Clarity LIMS are overall metrics for the run. There are currently no per-lane metrics provided by NovaSeq 6000Dx Operating Software.
Index 1 Cycles
Index 2 Cycles
Analysis Software Version
Override Cycles
For detailed information on the following items, refer to the bcl2fastq2 Sample Sheet Generation section of Illumina Instrument Sample Sheets (NGS v5.17 and later).
Sample sheet generation script parameters and usage
Sample sheet data and configuration options
Enabling unique FASTQ file names per sequencing run
Username and password are passed to the Clarity LIMS server to make sure that the access is valid.
By default, the Sequencer API service tries to contact Clarity LIMS at http://localhost:9080/clarity/. The installation script sets the URL, but it can be configured with the clarity.url property in the application.yml file. For more information, refer to the Properties section of Components Installed.
The user is redirected to the Sequencer login page at
If authentication is successful, the screen displays a success message. The Clarity LIMS server provides the OAuth token and username by redirecting to
This redirect does not reload the page, but allows the NovaSeqDx to continue with run setup.
If authentication fails, the login screen does not change and a warning message displays. A background redirect to
informs the NovaSeq 6000Dx of the failure.
LibraryContainerId and FlowCellId can match either the library tube ID or flow cell ID of the corresponding containers in Clarity LIMS.
If there are no containers or multiple containers, an error is generated.
All populated containers whose name matches either the LibraryContainerId or FlowCellId are validated as follows.
All samples in the container must be queued for a sequencing run step.
All samples must be queued for only one sequencing run step.
All samples must be queued for the same sequencing run step.
ℹ Configure the steps that are considered sequencing run steps in the integration.sequencer_api.v2.novaseq.sequenceStepNames property. The property is a list and each entry must be the exact name of a sequencing step, not the name of the underlying master step/process type. For more information, refer to the Properties section of .
After a single container has passed validation, the run setup information (run recipe request) is returned to the NVOS via the API. Then, the run receipt is generated.
Note the following details:
The run recipe information is read from fields configured on the parent process/master step of the samples in the container. The workflow must include recipe definition in that step.
By default, the parent process/master step is the Dilute and Denature (NovaSeqDx v1.2) or Load to Flowcell (NovaSeqDx v1.2) step, depending on workflow.
The default field values match the names of the fields in the NovaSeq 6000Dx v1.2 configuration included in IPP. The field names are configurable through the integration.sequencer_api.v2.recipe.udfNames.* properties. Each property corresponds to an entry in the recipe. The value of a property defines the name of the field that is used to populate that entry in the recipe.
It is assumed that this parent process/master step removes any illegal characters from the Experiment Name field.
The Clarity LIMS download link to the sample sheet created on the run setup step is used for the SampleSheet value in the recipe. The name of the output of the sample sheet on the setup step is configured through the integration.sequencer_api.v2.recipe.sampleSheet.outputName property (Sample Sheet by default). If no matching output is found, or no file was attached to the output, the value of the integration.sequencer_api.v2.recipe.sampleSheet.notAvailableValue property (Not Available by default) is used in the recipe. For more information, refer to the Properties section of .
After the recipe is generated, it is returned as a *.json file in the response, in the form defined by the Swagger definition for the API.
If a kit is configured for the API but does not exist in Clarity LIMS, it is created.
If a kit exists, but is not enabled on the step to be run, the step configuration is updated to enable it.
If a kit that is to be enabled is Archived, it is set to Active.
If a kit that is to be enabled is in Pending status, it is set to Active.
If any kits enabled on the step are not configured for the Sequencer API, a warning is logged, and the API cannot complete the step without user intervention.
If any kits provided in the recipe request are not configured for the Sequencer API, they are ignored, and a warning is logged during lot tracking.
The type of flow cell being used in the run determines the number of replicates for the step.
For Standard mode, this information is based on the number of lanes in the flow cell. This information is stored as part of the Sequencer API configuration.
For XP mode, there is always one replicate, because there is already one input tracked for each lane.
The automated sequencing step is started.
Reagent lots are attached to the step. The Flow Cell and Library Tube reagents in the request are ignored. Information from these reagents is tracked in Clarity LIMS as containers and custom fields.
Any other reagents in the request are handled as follows.
If the reagent is not configured to be tracked, a warning is logged, and the reagent is ignored.
You can look up existing reagent lots by kit name (determined by Sequencer API configuration), name (the serial number in the request), and lot number (the lot number in the request).
If one or more matching reagent lots exist in Clarity LIMS, the newest one is used.
If the expiry date does not match the expiry in the request, or if the status is not set to Active, the lot is updated to have the correct expiry date and an Active status.
Otherwise a new lot is created with the following configuration:
Reagent Kit — Matching kit as determined from Sequencer API configuration and step 2.
Name — Serial number in request.
Lot Number — Lot number in request.
Expiry — Expiry date in request.
If any reagents are expected to be in the request but are not, these reagents are logged. This action prevents the step from being completed without user intervention.
Master step custom fields are updated with the run information parsed from the request. For more information, refer to Respond to Record InterOps Metrics Call from Instrument.
Any important events during this request are logged into the Sequencer API log file and in the Sequencing Log multiline global custom field on the step (assuming the step was started successfully). For more information, refer to Respond to Record InterOps Metrics Call from Instrument.
A success message is recorded in the log file at the info level:
Clarity LIMS waits for any automations that are triggered automatically as it advances the step.
By default, Clarity LIMS assigns the next steps on the outputs to move them on to the first configured next step. If there are no next steps, the protocol is completed.
A failure message is recorded in the log file at the error level:
The step is left at the Record Details screen.
The following message is recorded in the log file, at the warning level:
The step is left at the Record Details screen.
configure_sequencer_api_env.sh
configure_sequencer_api_application.sh
/etc/httpd/clarity/
Proxy setting to communicate with Clarity LIMS.
NaOH
Resuspension Buffer (RSB)
SBS Cartridge
Tris-HCl
SP and S1 flow cell: [A-Z0-9]{5}DR[A-Z0-9]{2}
S2 flow cell: [A-Z0-9]{5}DM[A-Z0-9]{2}
S4 flow cell: [A-Z0-9]{5}DS[A-Z0-9]{2}
Example: H1991DMXX
Reagent labels (indexes) must be unique.
Only controls are permitted as unindexed samples. All other unindexed samples and pools are not permitted.
For sample sheet generation constraints, refer to the Bcl2fastq2 Sample Sheet Generation section in Illumina Instrument Sample Sheets (NGS v5.17 & later).
Do not manually start the AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2) step. This step is fully automated and the sequencing service may not update samples correctly if they have been manually started.
For the automated run to start successfully, the Validate Run Setup and Generate Sample Sheet button must be selected.
Field Name
Field Type
Options
Additional Options and Dropdown Items
Comment
Multiline Text
None
Flowcell Type
Text Dropdown
Required Field
Custom Entries
Presets
SP
S1
S2
S4
Instruction
Text
Read Only
Default
Add Flowcell Type, Loading Workflow Type, and NovaSeqDx Run Mode
Field Name
Field Type
Options
Additional Options and Dropdown Items
Adjusted Per Sample Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
Final Loading Concentration (pM)
Numeric Dropdown
Required Field
Custom Entries
Presets
225
400
Decimal places displayed = 0
Flowcell Type
Text Dropdown
Required Field
Presets
SP
S1
S2
S4
Field Name
Field Type
Options
Additional Options and Dropdown Items
% PhiX (2.5 nM) Spike-In
Numeric
Range = 0–100
Bulk Pool Volume (ul)
ℹ For calculation purposes, not displayed
Numeric
Decimal places displayed = 2
Number of Flowcells to Sequence
Numeric
Required Field
Range = 1–10
Decimal places displayed = 0
Field Name
Field Type
Options
Additional Options and Dropdown Items
Flowcell Type
Text Dropdown
Required Field
Presets
SP
S1
S2
S4
Loading Workflow Type
Text Dropdown
Required Field
Presets
NovaSeq Standard
NovaSeq Xp
[Remove from workflow]
NaOH Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
Field Name
Field Type
Options
Additional Options and Dropdown Items
Analysis Software Version
Text
BaseSpace Sequence Hub Configuration
Text Dropdown
Required Field
Presets
Not Used
Run Monitoring Only
Run Monitoring and Storage
Custom Recipe Path
Text
Field Name
Field Type
Options
Additional Options and Dropdown Items
Flowcell Type
Text Dropdown
Required Field
Presets
SP
S1
S2
S4
Loading Workflow Type
Text Dropdown
Required Field
Presets
NovaSeq Standard
NovaSeq Xp
[Remove from workflow]
Field Name
Field Type
Options
Additional Options and Dropdown Items
% PhiX (0.25nM) Spike-in
Numeric
Range = 0– 100
Bulk Pool Volume (ul)
ℹ For calculation purposes, not displayed
Numeric
Decimal places displayed = 2
Minimum Per Sample Volume (ul)
Numeric
Required Field
Default
5
Decimal places displayed = 2
Field Name
Field Type
Options
Additional Options and Dropdown Items
RSB Volume (ul)
Numeric
Read Only
Decimal places displayed = 2
Flowcell Type
Text Dropdown
Required Field
Presets
SP
S1
S2
S4
Loading Workflow Type
Text Dropdown
Required Field
Presets
NovaSeq Standard
NovaSeq Xp
[Remove from workflow]
Field Name
Field Type
Options
Additional Options and Dropdown Items
DPX1 Volume (ul)
Numeric
Read Only
Decimal places displayed = 0
DPX2 Volume (ul)
Numeric
Read Only
Decimal places displayed = 0
DPX3 Volume (ul)
Numeric
Read Only
Decimal places displayed = 0
Field Name
Field Type
Options
Additional Options and Dropdown Items
BP Aliquot Volume (ul)
Numeric
Read Only
Decimal places displayed = 0
Flowcell Type
Text Dropdown
Required Field
Presets
SP
S1
S2
S4
Loading Workflow Type
Text Dropdown
Required Field
Presets
NovaSeq Standard
NovaSeq Xp
[Remove from workflow]
Field Name
Field Type
Options
Additional Options and Dropdown Items
Analysis Software Version
Text
BaseSpace Sequence Hub Configuration
Text Dropdown
Required Field
Presets
Not Used
Run Monitoring Only
Run Monitoring and Storage
Custom Recipe Path
Text
Field Name
Field Type
Options
Additional Options and Dropdown Items
Flowcell Type
Text Dropdown
Required Field
Presets
SP
S1
S2
S4
Loading Workflow Type
Text Dropdown
Required Field
Presets
NovaSeq Standard
NovaSeq Xp
[Remove from workflow]
Field Name
Field Type
Options
Additional Options and Dropdown Items
Current Cycle
Numeric
Read Only
Decimal places displayed = 0
Current Read
Numeric
Read Only
Decimal places displayed = 0
Firmware Version
Text
Read Only
Field Name
Field Type
Options
Additional Options and Dropdown Items
% Aligned R1
Numeric
Read Only
Decimal places displayed = 2
% Aligned R2
Numeric
Read Only
Decimal places displayed = 2
% Bases >=Q30 R1
Numeric
Read Only
Decimal places displayed = 2
Files Installed
Location
Description
Illumina#Sequencer#v2.war
/opt/gls/clarity/tomcat/current/webapps
War file for Sequencer API.
configure_sequencer_api_application.sh
/opt/gls/clarity/config/
Script that configures the Sequencer API application through its external application.yml file.
configure_sequencer_api_env.sh
/opt/gls/clarity/config/
Script that configures the Clarity LIMS Tomcat configuration to include Secret Util settings.
configure_sequencer_api_proxy.sh
/opt/gls/clarity/config/
Script that configures the proxy to allow communication with the Sequencer API application.
run.autoComplete property value
run.autoCompleteOnlyAtSuccess property value
Outcome
true
true
Step automatically completes only if the sequencing run is successful.
If run is not successful, the step does not automatically complete, and the run details are recorded in Clarity LIMS.
true
false
Step automatically completes, regardless of the run status.
false
true
Step does not automatically complete.
Run details are recorded in Clarity LIMS.
false
false
Step does not automatically complete.
Run details are recorded in Clarity LIMS.
sequencer-api.conf
input.::Minimum Molarity (nM):: = (5 * input.::Final Loading Concentration (pM)::)/1000if (!input.hasValue(::Normalized Molarity (nM)::)) {
fail(::The Normalized Molarity cannot be empty.::) ;
}else if (input.::Normalized Molarity (nM):: < input.::Minimum Molarity (nM)::) {
input.::Warning:: = ::The Normalized Molarity is too low.::;
input.::Loading Workflow Type:: = ::[Remove from workflow]::;
} else {
input.::Warning:: = ::n/a::
}input.::Per Sample Volume (ul):: = ( ( (input.::Final Loading Concentration (pM):: * 5 / 1000) / input.::Normalized Molarity (nM):: ) * step.::Bulk Pool Volume (ul):: ) / step.::Number of Samples in Pool::step.::Total Sample Volume (ul):: = step.::Total Sample Volume (ul):: + input.::Adjusted Per Sample Volume (ul)::if (step.::Total Sample Volume (ul):: >= step.::Bulk Pool Volume (ul)::) {
output.::RSB Volume (ul):: = 0
}
else {
output.::RSB Volume (ul):: = step.::Bulk Pool Volume (ul):: - step.::Total Sample Volume (ul)::
} ;output.::Flowcell Type:: = input.::Flowcell Type:: ;
output.::Loading Workflow Type:: = input.::Loading Workflow Type:: ;if ( input.::Flowcell Type:: == ::SP:: ) {
output.::Volume of Pool to Denature (ul):: = 100 ;
output.::NaOH Volume (ul):: = 25 ;
output.::Tris-HCl Volume (ul):: = 25
} ;
if ( input.::Flowcell Type:: == ::S1:: ) {
output.::Volume of Pool to Denature (ul):: = 100 ;
output.::NaOH Volume (ul):: = 25 ;
output.::Tris-HCl Volume (ul):: = 25
} ;
if ( input.::Flowcell Type:: == ::S2:: ) {
output.::Volume of Pool to Denature (ul):: = 150 ;
output.::NaOH Volume (ul):: = 37 ;
output.::Tris-HCl Volume (ul):: = 38
} ;
if ( input.::Flowcell Type:: == ::S4:: ) {
output.::Volume of Pool to Denature (ul):: = 310 ;
output.::NaOH Volume (ul):: = 77;
output.::Tris-HCl Volume (ul):: = 78
}&& /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} \
script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeq_Standard_Bulk_Pool1.csv \
-o 1.csv \
script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeq_Standard_Bulk_Pool2.csv \
-o 2.csv \
script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeq_Standard_Bulk_Pool3.csv \
-o 3.csv \
&& cat 1.csv 2.csv 3.csv > {compoundOutputFileLuid0}.csvstep.::Number of Samples in Pool:: = step.::Number of Samples in Pool:: - 1;
step.::Total Sample Volume (ul):: = step.::Total Sample Volume (ul):: - input.::Adjusted Per Sample Volume (ul)::output.::Flowcell Type:: = input.::Flowcell Type:: ;
output.::Loading Workflow Type:: = input.::Loading Workflow Type::if (step.::Paired End::.toBoolean()) {
if (step.::Read 1 Cycles:: == 0 || step.::Read 2 Cycles:: == 0) {
fail(::Read 1 Cycles and Read 2 Cycles must be greater than 0 if Paired End is True.::)
}
} else {
if (step.::Read 2 Cycles:: != 0) {
fail(::Read 2 Cycles must be 0 if Paired End is False.::)
}
};if (input.::Flowcell Type::!=::SP:: && step.::Read 1 Cycles:: > 151) {
fail(::Read 1 Cycles must not be larger than 151 if it is not SPrime Flowcell::)
};
if (input.::Flowcell Type::!=::SP:: && step.::Read 2 Cycles:: > 151) {
fail(::Read 2 Cycles must not be larger than 151 if it is not SPrime Flowcell::)
};if (step.hasValue(::Analysis Software Version::) && !step.::Analysis Software Version::.matches(::^(\\d+)(\\.\\d+)*$::)) {
fail(::Analysis Software Version contains prohibited characters. Allowed characters are 0-9 and period. It shall start and end with numbers and separated by single period e.g. 3.8.4::)
};if (step.hasValue(::Override Cycles::) && !step.::Override Cycles::.matches(::[YNIU0-9;]+::)) {
fail(::Override Cycles ontains prohibited characters. Allowed characters are: Y, N, I, U, 0-9 and ;. Example: N1Y150;I8;I7N1;Y141U10.::)
};nextStep = ::REMOVE::/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} \
script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeqDx_BCL2FASTQ_Samplesheet_v2.csv \
-o {compoundOutputFileLuid0}.csv \
-q true \
-destLIMSID {compoundOutputFileLuid0} \
-l {compoundOutputFileLuid1}"input.::Per Sample Volume (ul)::= (((input.::Final Loading Concentration (pM)::* 5/ 1000) / input.::Normalized Molarity (nM)::) * step.::Bulk Pool Volume (ul)::) / step.::Number of Samples in Pool::step.::Total Sample Volume (ul):: = step.::Total Sample Volume (ul):: + input.::Adjusted Per Sample Volume (ul)::if (step.::Total Sample Volume (ul):: >= step.::Bulk Pool Volume (ul)::) {
output.::RSB Volume (ul):: = 0
}
else {
output.::RSB Volume (ul):: = step.::Bulk Pool Volume (ul):: - step.::Total Sample Volume (ul)::
} ;output.::Flowcell Type::= input.::Flowcell Type::;output.::Loading Workflow Type::= input.::Loading Workflow Type::;&& /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} \
script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeq_Xp_Bulk_Pool.csv \
-o {compoundOutputFileLuid0}.csv \
script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeq_Xp_Bulk_Pool2.csv \
-o append.csv \
&& cat append.csv >> {compoundOutputFileLuid0}.csv"step.::Number of Samples in Pool:: = step.::Number of Samples in Pool:: - 1;
step.::Total Sample Volume (ul):: = step.::Total Sample Volume (ul):: - input.::Adjusted Per Sample Volume (ul)::output.::Flowcell Type::= input.::Flowcell Type::;
output.::Loading Workflow Type::= input.::Loading Workflow Type::;/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} \
script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeq_Xp_Working_Pool.csv \
-o {compoundOutputFileLuid0}.csv \
script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeq_Xp_Working_Pool2.csv \
-o append.csv \
&& cat append.csv >> {compoundOutputFileLuid0}.csv"bash -l -c "/opt/gls/clarity/bin/java \
-jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar \script:validate_flowcell_for_input_pools \
-i {stepURI:v2} \
-u {username} \
-p {password} \
-l {compoundOutputFileLuid1} \
-validateSelectedContainer true"step.::Run Mode:: = input.::Flowcell Type::/Illumina/Sequencer/v2/sequencer_login/Illumina/Sequencer/v2/sequencer_login/Success/Illumina/Sequencer/v2/sequencer_login/ErrorSequencing run with Run ID {runId} of Step '{step.name}' ({step.limsid}) completed successfully.Step '${Step.name}' (${Step.limsid}) failed with Run ID ${runId}."Sequencing run with Run ID ${status.runInfo.runId} of Step '${step.configuration.name}' (${step.limsid}) aborted by user ${status.runInfo.userName}."nextStep = ::REMOVE::if (input.::NovaSeqDx Run Mode:: == ::DX::) {
if (input.::Loading Workflow Type:: == ::NovaSeq Xp::) {
fail(::Invalid option selected. NovaSeq Standard shall be selected for DX mode.::);
};
if (input.::Flowcell Type:: == ::SP:: || input.::Flowcell Type:: == ::S1::) {
fail(::Invalid option selected. Only S2 or S4 flowcell shall be selected for DX mode.::);
};
};bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar \
script:validate_same_analyte_udf_value_for_all_inputs \
-i {stepURI:v2} \
-u {username} \
-p {password} \
-l {compoundOutputFileLuid1} \
-f 'NovaSeqDx Run Mode'"step.::Number of Samples in Pool:: = step.::Number of Samples in Pool:: + 1step.::PhiX Volume (ul):: = !step.hasValue(::% PhiX (2.5nM) Spike-In::) ? 0 : step.::% PhiX (2.5nM) Spike-In::;
if (input.::Flowcell Type:: == ::SP::) {
step.::Bulk Pool Volume (ul):: = step.::Number of Flowcells to Sequence:: * 100;
step.::PhiX Volume (ul):: *= 0.6
};
if (input.::Flowcell Type:: == ::S1::) {
step.::Bulk Pool Volume (ul):: = step.::Number of Flowcells to Sequence:: * 100;
step.::PhiX Volume (ul):: *= 0.6
};
if (input.::Flowcell Type:: == ::S2::) {
step.::Bulk Pool Volume (ul):: = step.::Number of Flowcells to Sequence:: * 150;
step.::PhiX Volume (ul):: *= 0.9
};
if (input.::Flowcell Type:: == ::S4::) {
step.::Bulk Pool Volume (ul):: = step.::Number of Flowcells to Sequence:: * 310;
step.::PhiX Volume (ul):: *= 1.9
};
if (step.::PhiX Volume (ul):: == 0) {
step.::PhiX Volume (ul):: = ::::
};nextStep = ::ADVANCE::output.::NovaSeqDx Run Mode:: = input.::NovaSeqDx Run Mode::script:validateSampleCount -min 1 -max 1step.::Run Mode:: = input.::Flowcell Type::bash -l -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar
-u {username} -p {password} -i {stepURI:v2} -l {compoundOutputFileLuid1} script:changeWorkflow \
--FIELD_NAME 'N/A' \
--FIELD_VALUE 'N/A' \
--WORKFLOW 'NovaSeqDx v1.2' \
--STEP 'AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2)' \
--INPUTS_OR_OUTPUTS 'OUTPUTS'"step.::Run Mode:: = input.::Flowcell Type::; if (!step.::Experiment Name::.matches(::[a-zA-Z0-9-_]+::)) { fail(::Experiment Name contains prohibited characters. Allowed characters are: a-z, A-Z, 0-9, -, and _::) }; if (step.::Workflow Type:: == ::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 No Index is selected.::) } } else if (step.::Workflow Type:: == ::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 Single Index is selected.::) } } 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 Dual Index is selected.::) } }; if (step.::Paired End::.toBoolean()) { if (step.::Read 1 Cycles:: == 0 || step.::Read 2 Cycles:: == 0) { fail(::Read 1 Cycles and Read 2 Cycles must be greater than 0 if Paired End is True.::) } } else { if (step.::Read 2 Cycles:: != 0) { fail(::Read 2 Cycles must be 0 if Paired End is False.::) } }; if (input.::Flowcell Type:: != ::SP:: && step.::Read 1 Cycles:: > 151) { fail(::Read 1 Cycles must not be larger than 151 if it is not SPrime Flowcell::) }; if (input.::Flowcell Type:: != ::SP:: && step.::Read 2 Cycles:: > 151) { fail(::Read 2 Cycles must not be larger than 151 if it is not SPrime Flowcell::) }; if (step.hasValue(::Analysis Software Version::) && !step.::Analysis Software Version::.matches(::^(\\d+)(\\.\\d+)*$::)) { fail(::Analysis Software Version contains prohibited characters. Allowed characters are 0-9 and period. It shall start and end with numbers and separated by single period e.g. 3.8.4::) }; 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.::) }; nextStep = ::REMOVE::step.::Number of Samples in Pool:: = step.::Number of Samples in Pool:: + 1step.::PhiX Volume (ul):: = !step.hasValue(::% PhiX (0.25nM) Spike-In::) ? 0 : step.::% PhiX (0.25nM) Spike-In::;
if (input.::Flowcell Type:: == ::SP::) {
step.::Bulk Pool Volume (ul):: = step.'Number of Lanes to Sequence' * 18;
step.::PhiX Volume (ul):: *= 0.7;
};
if (input.::Flowcell Type:: == ::S1::) {
step.::Bulk Pool Volume (ul):: = step.'Number of Lanes to Sequence' * 18;
step.::PhiX Volume (ul):: *= 0.7;
};
if (input.::Flowcell Type:: == ::S2::) {
step.::Bulk Pool Volume (ul):: = step.'Number of Lanes to Sequence' * 22;
step.::PhiX Volume (ul):: *= 0.8;
};
if (input.::Flowcell Type:: == ::S4::) {
step.::Bulk Pool Volume (ul):: = step.'Number of Lanes to Sequence' * 30;
step.::PhiX Volume (ul):: *= 1.1;
};
if (step.::PhiX Volume (ul):: == 0) {
step.::PhiX Volume (ul):: = ::::;
};output.::Flowcell Type:: = input.::Flowcell Type:: ; nextStep = ::ADVANCE::bash -l -c "/opt/gls/clarity/bin/java \
-jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar \
script:validate_flowcell_for_input_pools \
-i {stepURI:v2} \
-u {username} \
-p {password} \
-l {compoundOutputFileLuid1}"output.::Flowcell Type:: = input.::Flowcell Type:: ; nextStep = ::ADVANCE::if (input.::Flowcell Type:: == ::SP:: && !output.container.name.matches( ::[A-Z0-9]{5}DR[A-Z0-9]{2}:: ) ) {
fail ( ::Invalid Flowcell Barcode. Please verify and try again.:: )
};
if (input.::Flowcell Type:: == ::S1:: && !output.container.name.matches( ::[A-Z0-9]{5}DR[A-Z0-9]{2}:: ) ) {
fail ( ::Invalid Flowcell Barcode. Please verify and try again.:: )
};
if (input.::Flowcell Type:: == ::S2:: && !output.container.name.matches( ::[A-Z0-9]{5}DM[A-Z0-9]{2}:: ) ) {
fail ( ::Invalid Flowcell Barcode. Please verify and try again.:: )
};
if (input.::Flowcell Type:: == ::S4:: && !output.container.name.matches( ::[A-Z0-9]{5}DS[A-Z0-9]{2}:: ) ) {
fail ( ::Invalid Flowcell Barcode. Please verify and try again.:: )
};output.::Flowcell Type:: = input.::Flowcell Type:: ;
output.::Loading Workflow Type:: = input.::Loading Workflow Type::step.::Run Mode:: = input.::Flowcell Type::; if (!step.::Experiment Name::.matches(::[a-zA-Z0-9-_]+::)) { fail(::Experiment Name contains prohibited characters. Allowed characters are: a-z, A-Z, 0-9, -, and _::) }; if (step.::Workflow Type:: == ::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 No Index is selected.::) } } else if (step.::Workflow Type:: == ::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 Single Index is selected.::) } } 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 Dual Index is selected.::) } }; if (step.::Paired End::.toBoolean()) { if (step.::Read 1 Cycles:: == 0 || step.::Read 2 Cycles:: == 0) { fail(::Read 1 Cycles and Read 2 Cycles must be greater than 0 if Paired End is True.::) } } else { if (step.::Read 2 Cycles:: != 0) { fail(::Read 2 Cycles must be 0 if Paired End is False.::) }}; if (input.::Flowcell Type:: != ::SP:: && step.::Read 1 Cycles:: > 151) { fail(::Read 1 Cycles must not be larger than 151 if it is not SPrime Flowcell::) }; if (input.::Flowcell Type:: != ::SP:: && step.::Read 2 Cycles:: > 151) { fail(::Read 2 Cycles must not be larger than 151 if it is not SPrime Flowcell::) }; if (step.hasValue(::Analysis Software Version::) && !step.::Analysis Software Version::.matches(::^(\\d+)(\\.\\d+)*$::)) { fail(::Analysis Software Version contains prohibited characters. Allowed characters are 0-9 and period. It shall start and end with numbers and separated by single period e.g. 3.8.4::) }; 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.::) }; step.::Settings Header:: = step.hasValue(::UMI - Read 1 Length::) || step.hasValue(::UMI - Read 2 Length::) ? ::[Settings]:: : ::::; nextStep = ::REMOVE::{
"run_name": "NVOS16027_SPrime_2x250",
"run_mode": "S4",
"workflow_type": "DualIndex",
"librarytube_id": "NV0025867-LIB",
"flowcell_id": "",
"sample_loading_type": "NovaSeqStandard",
"rehyb": false,
"paired_end": true,
"read1": 151,
"read2": 151,
"index_read1": 8,
"index_read2": 8,
"output_folder": "\\\\network_path\\run_data",
"samplesheet": "",
"require_samplesheet_authentication": true,
"usecustomrecipe": false,
"customRecipe": null,
"use_basespace": false,
"basespace_mode": null,
"use_custom_read1_primer": false,
"use_custom_read2_primer": false,
"use_custom_index_read1_primer": false
}bash -c "/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar
-u {username} -p {password} -i {stepURI:v2} -l {compoundOutputFileLuid0} script:changeWorkflow \
\
--FIELD_NAME 'Loading Workflow Type' \
--FIELD_VALUE 'NovaSeq Standard' \
--WORKFLOW 'NovaSeqDx v1.2' \
--STEP 'Make Bulk Pool for NovaSeqDx Standard (NovaSeqDx v1.2)' \
--INPUTS_OR_OUTPUTS 'INPUTS' \
\
--FIELD_NAME 'Loading Workflow Type' \
--FIELD_VALUE 'NovaSeq Xp' \
--WORKFLOW 'NovaSeqDx v1.2' \
--STEP 'Make Bulk Pool for NovaSeqDx Xp (NovaSeqDx v1.2)' \
--INPUTS_OR_OUTPUTS 'INPUTS'"/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar \
script:validate_flowcell_for_input_pools \
-i {stepURI:v2} \
-u {username} \
-p {password} \
-l {compoundOutputFileLuid1} \
-validateSingleOutput true \
-poolType bulk"if ((input.::NovaSeqDx Run Mode:: == ::DX:: && !output.container.name.matches(::^DX[0-9]{7}-[A-Z]{3}$::)) || (input.::NovaSeqDx Run Mode:: == ::RUO:: && !output.container.name.matches(::[A-Z]{2}[0-9]{7}-[A-Z]{3}::))) {
fail(::Invalid Library Tube Barcode. Please verify and try again.::)
}/opt/gls/clarity/bin/java \
-jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/ngs-extensions.jar \
script:validate_flowcell_for_input_pools \
-i {stepURI:v2} \
-u {username} \
-p {password} \
-l {compoundOutputFileLuid1} \
-validateSingleOutput true \
-poolType bulk"if ( input.::Flowcell Type:: == ::SP:: ) {
output.::BP Aliquot Volume (ul):: = 18 ;
output.::NaOH Volume (ul):: = 4 ;
output.::Tris-HCl Volume (ul):: = 5 ;
step.::DPX1 Volume (ul):: = 126 ;
step.::DPX2 Volume (ul):: = 18 ;
step.::DPX3 Volume (ul):: = 66 ;
output.::Mastermix per Lane (ul):: = 63
};
if (input.::Flowcell Type:: == ::S1::) {
output.::BP Aliquot Volume (ul):: = 18;
output.::NaOH Volume (ul):: = 4 ;
output.::Tris-HCl Volume (ul):: = 5 ;
step.::DPX1 Volume (ul):: = 126;
step.::DPX2 Volume (ul):: = 18;
step.::DPX3 Volume (ul):: = 66;
output.::Mastermix per Lane (ul):: = 63
};
if (input.::Flowcell Type:: == ::S2::) {
output.::BP Aliquot Volume (ul):: = 22;
output.::NaOH Volume (ul):: = 5 ;
output.::Tris-HCl Volume (ul):: = 6 ;
step.::DPX1 Volume (ul):: = 126;
step.::DPX2 Volume (ul):: = 18;
step.::DPX3 Volume (ul):: = 66;
output.::Mastermix per Lane (ul):: = 77
};
if (input.::Flowcell Type:: == ::S4::) {
output.::BP Aliquot Volume (ul):: = 30;
output.::NaOH Volume (ul):: = 7 ;
output.::Tris-HCl Volume (ul):: = 8 ;
step.::DPX1 Volume (ul):: = 315;
step.::DPX2 Volume (ul):: = 45;
step.::DPX3 Volume (ul):: = 165;
output.::Mastermix per Lane (ul):: = 105
};Status — Active.
6
8
6
8
151
101
51
* Value of 251 is only supported for SP flow cell type. For all other cell types, maximum value is 151.
151
101
51
* Value of 251 is only supported for SP flow cell type. For all other cell types, maximum value is 151.
S4
Custom
Range = 0–20
Decimal places displayed = 0
Range = 0–20
Decimal places displayed = 0
151
101
51
* Value of 251 is only supported for SP flow cell type. For all other flow cell types, maximum value is 151.
151
101
51
* Value of 251 is only supported for SP flow cell type. For all other flow cell types, maximum value is 151.
S4
Custom
if(!step.::Experiment Name::.matches(::[a-zA-Z0-9-_]+::)) {
fail(::Experiment Name contains prohibited characters. Allowed characters are: a-z, A-Z, 0-9, -, and _::)
};
if (step.::Workflow Type:: == ::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 No Index is selected.::)
}
} else if (step.::Workflow Type:: == ::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 Single Index is selected.::)
}
} 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 Dual Index is selected.::)
}
};if(!step.::Experiment Name::.matches(::[a-zA-Z0-9-_]+::)) {
fail(::Experiment Name contains prohibited characters. Allowed characters are: a-z, A-Z, 0-9, -, and _::)
};
if (step.::Workflow Type:: == ::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 No Index is selected.::)
}
} else if (step.::Workflow Type:: == ::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 Single Index is selected.::)
}
} 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 Dual Index is selected.::)
}
};if (step.::Paired End::.toBoolean()) {
if (step.::Read 1 Cycles:: == 0 || step.::Read 2 Cycles:: == 0) {
fail(::Read 1 Cycles and Read 2 Cycles must be greater than 0 if Paired End is True.::)
}
} else {
if (step.::Read 2 Cycles:: != 0) {
fail(::Read 2 Cycles must be 0 if Paired End is False.::)
}
};if (input.::Flowcell Type::!=::SP:: && step.::Read 1 Cycles:: > 151) {
fail(::Read 1 Cycles must not be larger than 151 if it is not SPrime Flowcell::)
}
if (input.::Flowcell Type::!=::SP:: && step.::Read 2 Cycles:: > 151) {
fail(::Read 2 Cycles must not be larger than 151 if it is not SPrime Flowcell::)
}if (step.hasValue(::Analysis Software Version::) && !step.::Analysis Software Version::.matches(::^(\\d+)(\\.\\d+)*$::)) {
fail(::Analysis Software Version contains prohibited characters. Allowed characters are 0-9 and period. It shall start and end with numbers and separated by single period e.g. 3.8.4::)
};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.::)
};nextStep = ::ADVANCE::/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/ngs-common/v5/EPP/DriverFileGenerator.jar -i {stepURI:v2} -u {username} -p {password} \
script:driver_file_generator \
-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NovaSeqDx_BCL2FASTQ_Samplesheet_v2.csv \
-o {compoundOutputFileLuid0}.csv \
-q true \
-destLIMSID {compoundOutputFileLuid0} \
-l {compoundOutputFileLuid1}The integration includes the following features:
Preconfigured NovaSeqDx v1.2 workflow that maps to lab protocols and instrument runs.
The following preconfigured protocols:
Run Format (NovaSeqDx v1.2)
NovaSeqDx Standard (NovaSeqDx v1.2) (supports the loading of pooled libraries into a library tube)
NovaSeqDx Xp (NovaSeqDx v1.2) (supports individual lane loading on the NovaSeq)
AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2)
Automated generation of v2 sample sheet. This file is automatically uploaded to the instrument via the API.
Automatic validation of run setup information. This information is used to create the run recipe and initiate the run.
Automated tracking of the NovaSeqDx sequencing run and parsing of run statistics into Clarity LIMS, including the following features:
Progress and metrics of sequencing run
Per-instrument sequencing runs (tracked as part of the Run ID field value)
Sequencing run parameters
Sequencing run data directory location
Real-Time Analysis v3 (RTA3) version and other run-specific information
[Optional] Preconfigured Library Prep Validation v2.3.4 workflow used for validation purposes only. The workflow contains a single-step protocol that models the library prep required to produce normalized libraries that are ready for the NovaSeqDx v1.2 workflow. For more information, refer to NovaSeq 6000Dx Integration v1.3.0 User Interaction, Validation and Troubleshooting. The Library Prep Validation v2.3.4 workflow is available in the Illumina Preset Protocols (IPP) v2.9 package.
The Illumina NovaSeq 6000Dx Integration v1.3.0 supports the integration between Clarity LIMS and the NovaSeq 6000Dx instrument. The integration includes the following changes:
Compatibility with Oracle Linux. For compatibility, refer to NovaSeq 6000Dx Integration v1.3.0 Release Notes.
Security fixes. This document provides instructions for installing NovaSeq 6000Dx Integration v1.3.0 and describes the components that are installed in the default configuration.
If the user is new to the NovaSeq 6000Dx integration, then follow the instructions provided in this section. If the user has installed NovaSeq 6000Dx Integration v1.1.0, then note that there are no user-facing changes in the configured workflow between NovaSeq 6000Dx Integration v1.1.0 and NovaSeq 6000Dx Integration v1.3.0. It is safe to use the existing NovaSeq 6000Dx v1.1 workflow installed with IPP v2.5.0 and reconfigure the integration for this workflow instead. For more information, refer to [Optional] Configure Properties in Installation Steps.
NovaSeq 6000Dx Integration v1.3.0 is distributed as the BaseSpaceLIMS-sequencer-api RPM package. This package must be installed on the Clarity LIMS server.
The BaseSpaceLIMS-sequencer-api RPM installs the following files and scripts:
Sequencer API WAR file
application.yml configuration file
configure_sequencer_api_proxy.sh configuration script
configure_sequencer_api_env.sh configuration script
The RPM installs the Sequencer API WAR file into the same Tomcat container as Clarity LIMS. The WAR file is installed to the following location:
The version number can differ.
The WAR file is deployed automatically when Clarity LIMS launches. Because the RPM installs a new WAR file, Clarity LIMS is automatically stopped during installation of the RPM. Restart Clarity LIMS before installing the NovaSeq and Library Prep Validation workflow configuration from the IPP.
The application.yml configuration file is installed to the following location:
The file properties are configured with a script during the installation process.
For a complete list of all properties included in the application.yml file and the properties installed into database, refer to .
The scripts in the following table are installed to the following location:
During installation, the RPM runs these scripts as they are needed.
If running the sequencing service and Automation Worker on the same instance, the instance must also be running a compatible version of the Oracle Linux operating system. For compatibility, refer to .
NovaSeq 6000Dx Integration v1.3.0 includes the following workflows, protocols, and steps listed below. These workflows can be found in IPP v2.9.
For detailed descriptions of the steps and automations included in each protocol, and details on other components in the configuration, refer to .
For instructions on using the Library Prep Validation v2.3.4 protocol, refer to .
The NovaSeq 6000Dx instrument software includes the following components:
NovaSeq 6000Dx Operating Software (NVOS) — Contains the user interface for setting up the sequencing run. Responsible for controlling the instrument and acquiring the images.
Real-Time Analysis v3 (RTA3) — Takes the images generated by the first module, processes, and analyzes them. Makes sure that data files are created and copied to the final destination folder.
Sequencing Analysis Viewer (SAV) — Displays the important quality metrics generated by the RTA3 software.
For the NovaSeq 6000Dx API integration to work, the instrument must be able to communicate with Clarity LIMS through the API. Complete the following steps to configure the NVOS and confirm that you can access Clarity LIMS from the instrument.
Launch NVOS and wait for the initialization process to complete.
On the NVOS Instrument Settings page, do as follows.
Select the Global Settings tab.
Under Instrument Mode, select one of the following modes:
Clarity DB Password
FTP password
LDAP manager password (if LDAP is enabled)
LabLink DB password
Reporting DB password (if installed)
RabbitMQ password
For more information on Secret Utility configuration, refer to the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
Library Prep Validation configuration (optional, but recommended)
For details on IPP v2.9 installation and configuration, refer to the Illumina Preset Protocols documentation.
configure_sequencer_api_application.sh configuration script
Enter y to confirm that you want to proceed with the RPM and package installation.
Make sure that NGS Extension Package v5.25.0 is installed.
If this package is not installed, you must install it or upgrade the existing package.
As the glsjboss user, install the QC_Protocols.qc-protocols configuration from the following location:
For information on IPP installer commands, parameters, and options, refer to the Illumina Preset Protocols documentation.
Enter y to confirm that you want to proceed with the RPM installation.
If necessary, install the Secret Utility Package.
For more information on this package, refer to Prerequisite 1: Secret Utility Configuration.
As the glsjboss user, run configure_sequencer_api_application.sh from the following location:
Enter values for the presented as follows.
You can also press Enter to accept the default values in the application.yml configuration file. For more information about the application.yml properties, refer to Integration Properties Details.
Would you like to use the apiuser user for communication with Clarity? [Y]
Enter required value for Token Expiry (hours) [88]
Update the Clarity LIMS proxy for the Sequencer API as follows.
For an on-premise system, run the following command:
For an Illumina cloud hosted system, make sure that the proxy has been updated as follows. Place the sequencer-api.include file in /etc/httpd/conf.d/clarity.
Update the Clarity LIMS Tomcat configuration as follows.
Make sure that Secret Utility is installed and configured. For more information, refer to Prerequisite 1: Secret Utility Configuration.
Run the following command to add the Secret Utility configuration to Clarity LIMS:
ℹ This script modifies /opt/gls/clarity/tomcat/current/bin/setenv.sh for Clarity LIMS Tomcat to load the Secret Utility environment variable file (/etc/profile.d/claritysecret.sh) during startup.
Use the following command to Start Clarity LIMS:
[Optional] Run the following command to install the Library Prep Validation v2.3.4 workflow:
This step is optional, but recommended. The Library Prep Validation v2.3.4 workflow allows you to validate the NovaSeqDx v1.2 workflow.
Enter sign in credentials.
Select Sign In. A Successfully Authenticated message displays.
Universal Copy Service — Copies output files to destinations such as final destination folder and/or BSSH (when the instrument is configured for use with BSSH).
Allow users to switch between IVD and RUO
IVD Only
RUO Only
If you selected IVD Only instrument mode, select the IVD Settings tab and complete the following steps:
Configure Run Setup as follows.
Select the Select runs from the LIMS server option.
Enter the Clarity LIMS URL (e.g., https://yourserver.claritylims.com).
[Optional] Under Output Location, enter the output folder location. You can also specify the output folder location when setting up a run in NVOS.
If you selected RUO Only instrument mode, select the RUO Settings tab and complete the following steps:
Configure Run Setup as follows.
Select the Select runs from the LIMS server option.
Enter the Clarity LIMS URL(e.g., https://yourserver.claritylims.com).
Configure Default Work Type as follows.
[Optional] If selection support between NovaSeq Xp or NovaSeq Standard is required for the workflow, select the Enable workflow selection checkbox.
Select the required default workflow type: NovaSeq Standard or NovaSeq Xp.
[Optional] Configure Output Location as follows.
[Optional] Enter the output folder location. You can also specify the output folder location when setting up a run in NVOS.
[Optional] To upload and store run data in the cloud server, select the BSSH checkbox. Select a Configuration option (Run Monitoring and Storage or Run Monitoring Only) and select your Hosting Location from the drop-down list.
Select Save to complete the configuration.
On the NVOS home page, you can now select Sequence to log into Clarity LIMS and start a run. nova
Script Name
User
Description
configure_sequencer_api_proxy.sh
root
Adds configuration to the proxy to allow communication with the Sequencer API after Tomcat has been started.
Run this script in the following scenarios:
When the Sequencer API is first installed.
When reconfiguring the Clarity LIMS proxy. This process happens until a version of Clarity LIMS that includes the necessary configuration files from /etc/httpd/clarity/*.conf is available.
ℹ Not required for Illumina cloud hosted installations.
configure_sequencer_api_env.sh
root
Adds Secret Management setting into the Clarity LIMS Tomcat configuration.
Run this script when the Sequencer API is first installed on a Secret Util-enabled Clarity LIMS.
configure_sequencer_api_application.sh
glsjboss
Configures the installed application.yml file.
Sets the Clarity LIMS URL and the key to use when signing access tokens.
âš Run this script when the Sequencer API is first installed. If you run the script multiple times, the script reconfigures each setting to its default. The only exception is the signing key. For this setting, you are asked if you want a reset. If the reset is done, all previously issued tokens are no longer valid.
/opt/gls/clarity/config/illumina-preset-protocols-installer.sh -o install QC_Protocols.qc-protocolsbash /opt/gls/clarity/config/configure_sequencer_api_application.shbash /opt/gls/clarity/config/configure_sequencer_api_proxy.shProxyPass ajp://localhost:9009/Illumina/Sequencer/v2
ProxyPassReverse ajp://localhost:9009/Illumina/Sequencer/v2bash /opt/gls/clarity/config/configure_sequencer_api_env.sh/opt/gls/clarity/config/illumina-preset-protocols-installer.sh -o install Illumina_Instruments.Library-Prep-Validation-v2.3/opt/gls/clarity/tomcat/current/webapps/Illumina#Sequencer#v2##2.7.0.0.war/opt/gls/clarity/extensions/sequencer-api/application.yml/opt/gls/clarity/configyum install BaseSpaceLIMS-Illumina-Preset-Protocols --enablerepo=<< repo name info from support >>yum install BaseSpaceLIMS-sequencer-api --enablerepo=< repo name info from support >bash /opt/gls/clarity/config/illumina-preset-protocols-installer.sh -o install Illumina_Instruments.novaseqdx-v1.2https://<< Clarity LIMS URL >>/Illumina/Sequencer/v2/oauth/authorize?client_id=illumina_sequencer&redirect_uri=../sequencer_login&response_type=token
/opt/gls/clarity/bin/run_clarity.sh start
This section explains how to validate the installation of the Illumina NovaSeqDx Integration Package v1.3.0.
The validation process involves the following items:
Running samples through the Library Prep Validation workflow.
The workflow contains a single-step protocol that models the library prep required to produce normalized libraries. At the end of the step, the normalized libraries advance to the workflow selected.
Running normalized libraries through the NovaSeqDx v1.2 workflow validates the following items:
Successful routing of samples from the Run Format (NovaSeqDx v1.2) step to the NovaSeqDx Standard (NovaSeqDx v1.2) or NovaSeqDx Xp (NovaSeqDx v1.2) step.
Automated generation of v2 sample sheet. This file automatically uploads to the sequencing system via the Sequencer API.
Automatic validation of run setup information. Information is uploaded to the NovaSeq 6000Dx Operating Software (NVOS) via the Sequencer API and is used to create the run recipe and initiate the run.
Automated tracking of the NovaSeq 6000Dx sequencing run and parsing of run statistics into Clarity LIMS, via the Sequencer API.
The validation steps assume that the NovaSeqDx Integration Package v1.3.0 is installed and you have imported the default Clarity LIMS configuration.
The following steps set up Clarity LIMS in preparation for running samples through the Library Prep Validation v2.3.4 and NovaSeqDx v1.2 workflows.
On the Configuration tab, under Workflows, activate both the Library Prep Validation v2.3.4 and NovaSeqDx v1.2 workflows.
On the Projects and Samples screen, create a project and add samples to it.
Assign the 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 NovaSeqDx v1.2 workflow.
Follow the steps in to run the Library Prep Validation workflow with the following:
Label Group = TruSeq HT Adapters v2 (D7-D5)
Sequencing Instrument = NovaSeqDx
On exit from the step, the Routing Script automation is triggered. This automation assigns samples to the first step of the NovaSeqDx v1.2 workflow, Define Run Format (NovaSeqDx v1.2). This is the only step in Protocol 1: Run Format (NovaSeqDx v1.2).
This protocol includes a single step, Define Run Format (NovaSeqDx v1.2). The step allows for the assignment of per sample values for the following fields:
NovaSeqDx Run Mode
Loading Workflow Type
Normalized Molarity
Flowcell Type
At the end of the step, samples are routed to the NovaSeqDx Standard or NovaSeqDx Xp protocol. This routing is done according to the selected Loading Workflow Type. The selected NovaSeqDx Run Mode must be compatible with the Loading Workflow Type and Flowcell Type. The following table shows the compatibility for the combinations of each Flowcell Type based on the NovaSeqDx Standard protocol.
NovaSeqDx Standard Protocol Compatibility
The following table shows the compatibility for the combinations of each Flowcell Type based on the NovaSeqDx Xp protocol.
NovaSeqDx Xp Protocol Compatibility
In Lab View, locate the Run Format (NovaSeqDx v1.2) protocol. The samples are queued for the Define Run Format (NovaSeqDx v1.2) step.
Add the samples to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
On the Record Details screen in the Sample Details table, populate the following fields (values can vary across samples):
In this protocol, samples are pooled and added to the library tube in preparation for the NovaSeq 6000Dx run. The protocol contains two steps:
Make Bulk Pool for NovaSeqDx Standard (NovaSeqDx v1.2)
Dilute and Denature (NovaSeqDx v1.2)
In Lab View, locate the NovaSeqDx Standard (NovaSeqDx v1.2) protocol.
Samples for the Make Bulk Pool for NovaSeqDx Standard (NovaSeqDx v1.2) step are queued.
On the Queue screen, add samples of the same Flowcell Type to the Ice Bucket and select View Ice Bucket.
On the Ice Bucket screen, select Begin Work.
When the step begins, the Validate Single NovaSeqDx Run Mode automation is triggered. This automation checks that the samples have the same NovaSeqDx Run Mode (RUO or DX).
At the end of this step, the pool of samples automatically advances to the Dilute and Denature (NovaSeqDx v1.2) step.
In Lab View, locate the NovaSeqDx Standard (NovaSeqDx v1.2) protocol. The pool of samples queued for the Dilute and Denature (NovaSeqDx v1.2) step are listed.
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 verifies that only one container is input for the step.
On the Placement screen:
At this point in the workflow, the user interaction ends. Proceed to AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2).
Do not add samples to the Ice Bucket or start the AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2) step. The integration adds samples automatically.
In this protocol, samples are pooled and added to lanes on the NovaSeq 6000Dx flow cell. The option selected in the Define Run Format (NovaSeqDx v1.2) step determines the flow cell type.
The protocol contains three steps:
Make Bulk Pool for NovaSeqDx Xp (NovaSeqDx v1.2)
Dilute, Denature & ExAmp (NovaSeqDx v1.2)
Load to Flowcell (NovaSeqDx v1.2)
In Lab View, locate the NovaSeqDx Xp (NovaSeqDx v1.2) protocol. The samples are queued for the Make Bulk Pool for NovaSeqDx Xp (NovaSeqDx v1.2) step.
On the Queue screen, add samples of the same Flowcell Type 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 the Dilute, Denature & ExAmp (NovaSeqDx v1.2) step.
In Lab View, locate the NovaSeqDx Xp (NovaSeqDx v1.2) protocol. The pool of samples queued for the Dilute, Denature & ExAmp (NovaSeqDx v1.2) step displays.
Add the pool to the Ice Bucket and select View Ice Bucket.
[Optional] On the Ice Bucket screen, set the number of derivatives to create (placed into the flow cell lanes) and select Begin Work.
On entry to the Record Details screen, the Calculate Volumes automation is triggered. This automation sets the following values based on the selected Flowcell Type:
Select Next Steps.
On the Assign Next Steps screen, the next step is already set to Load to Flowcell (NovaSeqDx v1.2).
Select Finish Step.
On the Ice Bucket screen, perform the following actions:
In the Container Options panel, select the appropriate flow cell type from the Destination Container drop-down list.
Select Begin Work.
At this point in the workflow, the user interaction ends. The flow cell is queued for the AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2) step.
Proceed to AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2).
Do not add samples to the Ice Bucket or start the AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2) step. The integration adds samples automatically.
This protocol contains a single fully automated step: AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2).
The integration starts the step automatically and data from the run is parsed back into Clarity LIMS. User interaction is not required, but you can review the stages of the step in Clarity LIMS (refer to Review Run Data).
The NovaSeqDx Run (NovaSeqDx v1.2) step contains two automations:
Set Next Steps — This automation is disabled (Set to Not Used). By default, the Sequencer API is used to set the next step for output samples.
The Illumina Preset Protocols (IPP) Package include a Set Next Steps automation configured on the AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2) step. You can use this automation to override the default next step behavior. For more information, refer to .
Update Lane Number — This automation is triggered automatically upon entry to the Record Details screen. To make sure that the Lane Number displays correctly, do not disable or modify this automation.
Read summary metrics are recorded for the library pool. After the run is complete, open the step and review these metrics displayed on the Next Steps screen in the Step Data section and the Sample Details table.
The following steps summarize how the Sequencer API integration works. For details on sample sheet generation and the sequence of events involved in the NovaSeq run, refer to .
When setting up a run on the NovaSeq 6000Dx instrument, NVOS sends a request for the run recipe.
The Sequencer API validates that samples and containers are correctly queued for the AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2) step.
Clarity LIMS sends a JSON response to NVOS. The response includes the run recipe information and a link to download the sample sheet (used with analysis software such as bcl2fastq2).
When the run starts, NVOS sends a RunStarted run status request. The Sequencer API then does the following:
If an automation trigger does not appear to run its corresponding scripts, refer to the following sections in the Clarity LIMS API documentation:
Troubleshooting Automated Worker in the .
Troubleshooting Automation in the .
If an error occurs that does not provide direction on how to proceed, (e.g., the error is related to the AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2) step) review the log file information. For more details, refer to .
If the automated run step starts, but does not complete, do as follows.
Use one of the following methods to open the in progress step in Clarity LIMS:
Log in to the default user account.
In Lab View, find the step in the Recent Activities pane.
Search for the step in Clarity LIMS. You can use the Library Tube or Flowcell barcode as the search term. The steps depend on whether the search is a NovaSeqDx Standard or NovaSeqDx Xp run.
In addition to updating the Sequencing Log multiline text field on the AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2) step, the Sequencer API writes a detailed log file to
Log messages include the Library Tube ID and Flow Cell ID whenever the messages are related to a sequencing run-related request. In the Clarity LIMS workflows, these IDs are recorded as container names.
Some log messages are not directly related to a sequencing run request, such as downloading the sample sheet. In this case, the file LIMS ID and file name are included in log messages. The sample sheet downloaded by the sequencing system has a name that includes the sequencing container ID (Library Tube for Standard or Flowcell for Xp). The relevant container name search finds most of the log messages for this request. However, the LIMS ID of the file must be known in order to retrieve all messages related to the file download. The LIMS ID is found in the Clarity LIMS API and, for sample sheets used in a run, in the recipe response (which can also be found in the log file).
Example lines from the log file:
NovaSeqDx Run Mode — Select DX or RUO.
Loading Workflow Type — Select NovaSeq Standard or NovaSeq Xp from the drop-down list.
Flowcell Type — Select SP, S1, S2, or S4.
Final Loading Concentration (pM) — Select from the two preset options: 225 (for PCR-free workflows), 400 (for Nano workflows), or enter a different value.
Normalized Molarity (nM) — These values are copied over from the previous step. If this column is not populated during library prep, enter the values here.
Select Next Steps, which triggers the Set Next Steps automation and the following actions:
Sets the value of the next step (for all samples) to Remove from workflow. The Routing Script automation expects this value and requires it to advance samples to the next step.
Checks that the NovaSeqDx Run Mode value is compatible with the Loading Workflow Type and Flowcell Type. This automation also generates an error message if the Loading Workflow Type or the Flowcell Type are incompatible.
Calculates the Minimum Molarity.
Checks Normalized Molarity value. For samples with no Normalized Molarity value (eg, empty value, not including 0), generates an error message notifying that the field cannot be empty.
Compares each sample Normalized Molarity value with the Minimum Molarity value.
On the Assign Next Steps screen:
In the Sample Details table, the Next Step for all samples is prepopulated with Remove from workflow, regardless of the Loading Workflow Type.
âš Do not change this value. If Next Step is not set to Remove from workflow, the routing script is not routed samples correctly.
For samples whose Normalized Molarity value is lower than the Minimum Molarity value, the Loading Workflow Type is set to Remove from workflow. A message is recorded in the Warning field for the sample.
To proceed, choose one of the following options:
Return to the Record Details screen and adjust the Normalized Molarity value so that it equals or exceeds the Minimum Molarity value. The Loading Workflow Type must be set to NovaSeq Standard or NovaSeq Xp, as applicable.
Complete the protocol without correcting the Normalized Molarity value. In this case, the samples in question are removed from the Clarity LIMS workflow.
Select Finish Step. The Routing Script automation is triggered:
Samples whose Loading Workflow Type is set to Remove from workflow are removed from the Clarity LIMS workflow. (Occurs when the Normalized Molarity value is lower than the Minimum Molarity.)
Samples whose Loading Workflow Type is set to NovaSeq Standard are routed to the Make Bulk Pool for NovaSeqDx Standard (NovaSeqDx v1.2) step. This step is the first step in the NovaSeqDx Standard protocol.
Samples whose Loading Workflow Type is set to NovaSeq Xp are routed to the Make Bulk Pool for NovaSeqDx Xp (NovaSeqDx v1.2) step. This step is the first step in the NovaSeqDx Xp protocol.
On the Pooling screen, perform the following actions:
Create a pool by dragging samples into the Pool Creator.
âš Only create one pool.
Enter a name for the pool or accept the default name (eg, Pool #1).
Select Record Details.
After exiting the Pooling screen, the Validate Inputs Flowcell Type and Single Pool is triggered. The automation verifies the following items:
All samples in the pool have been assigned the same Flowcell Type.
Only one pool has been created.
On the Record Details screen in the Step Details area, complete the two required fields and one optional field, as needed:
Number of Flowcells to Sequence — Used in volume calculations, to make sure that the volumes are sufficient for the number of times the pool is sequenced.
Minimum Per Sample Volume (ul) — Used to calculate how much of each sample is included in the pool. The field is prepopulated with the configured default value 5 µl, but can be edited.
% PhiX (2.5 nM) Spike-In — Used to calculate the volume of PhiX v3 control included in the pool for the given percentage of spike-in. The field is optional and can be edited.
ℹ Assuming the default Minimum Per Sample Volume (ul) value of 5, for a given batch:
If the smallest Per Sample Volume (ul) value is < 5, Clarity LIMS automatically assigns a value of 5 to the Adjusted Per Sample Volume (ul) field.
Clarity LIMS changes the Adjusted Per Sample Volume (ul) field value for all other samples in the batch, based on the ratio used to increase the lowest value to 5.
In the Sample Details table, select the pool icon to view details on the pool composition.
Select Calculate Volumes to trigger the Calculate Volumes automation. This automation performs calculations based on the selected Flowcell Type, then generates and attaches the Calculation File (CSV) to the step. This file contains volume information about the pool and the individual samples that it contains.
Select Next Steps to trigger the Set Next Step automation. This automation sets the next step for samples to ADVANCE and advances them to the next step in the protocol, Dilute, and Denature (NovaSeqDx v1.2). The automation also copies the NovaSeqDx Run Mode value from the input pool to the output pool.
On the Assign Next Steps screen, the next step for samples is set to the next step in the workflow: Dilute and Denature (NovaSeqDx v1.2).
Select Finish Step.
Drag the pool into the library tube in the Placed Samples area.
Scan or type the barcode of the library tube into the Library Tube field.
Select Record Details.
After exiting the Placement screen, the Validate Library Tube Barcode automation makes sure that the library tube barcode conforms to the barcode mask that corresponds with the NovaSeqDx Run Mode in the following table. If the barcode does not match, an error message displays. The automation also copies the Flowcell Type and Loading Workflow Type values from step inputs to outputs.
NovaSeqDx Run Mode
Valid Barcode Mask
RUO
[A-Z]{2}[0-9]{7}-[A-Z]{3}
DX
DX[0-9]{7}-[A-Z]{3}
On the Record Details screen, the Reagent Lot Tracking section lets you tracks the NaOH, Resuspension Buffer, and Tris-HCl reagents used in the step. To add and activate reagent lots, refer to 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 each drop-down list.
The fields displayed on the Record Details screen are used to set up the run and generate the sample sheet. Some of these fields are autopopulated and some must be completed manually.
Fields Displayed on Record Details Screen of Dilute and Denature (NovaSeqDx v1.2)
Field
Value
Experiment Name
Enter the experiment name. Only alphanumeric characters, dashes, and underscores are permitted. Spaces are not permitted.
BaseSpace Sequence Hub Configuration
Presets
Not Used
Run Monitoring Only
Run Monitoring and Storage
On the Record Details screen, select Validate Run Setup and Generate Sample Sheet. This triggers the automation script, which performs the following actions:
Validates the parameters entered on the Record Details screen.
Generates the v2 sample sheet and attaches it to the placeholder in the Files area of the Record Details screen.
ℹ The v2 sample sheet generated does not include Cloud and Analysis sections. Contact Illumina Support for information on generating v2 sample sheets with those sections.
Select Next Steps.
On the Assign Next Steps screen, the Next Step for samples is prepopulated with Remove from workflow. The Routing Script automation expects this value, and requires the value to advance samples to the next step.
âš Do not change this value. If Next Step is not set to Remove from workflow, the routing script is not able to route samples correctly.
Select Finish Step.
After exiting the step, the following actions occur:
The Routing Script automation is triggered and samples are routed to AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2).
In Lab View, the pool of samples is queued for the AUTOMATED - NovaSeqDx Run (NovaSeqDx v1.2) step.
Create a pool by dragging samples into the Pool Creator.
âš Create only one pool.
Enter a name for the pool or accept the default name (e.g., Pool #1).
Select Record Details.
On exiting the Pooling screen, the Validate Inputs Flowcell Type and Single Pool automation is triggered. The automation verifies the following items:
All samples in the pool are assigned the same Flowcell Type.
Only one pool was created.
On the Record Details screen, in the Step Details area, complete the two required fields and one optional field, as needed:
Number of Lanes to Sequence — Used in volume calculations to make sure that volumes are sufficient for the number of times the pool is sequenced.
Minimum Per Sample Volume (ul) — Used to calculate how much of each sample is included in the pool. The field is prepopulated with the configured default value 5 µl, but can be edited.
% PhiX (0.25nM) Spike-In — Used to calculate the volume of PhiX v3 control to be included in the pool for the given percentage of spike-in. The field is optional and can be edited.
ℹ Assuming the default Minimum Per Sample Volume (ul) value of 5, for a given batch:
If the smallest Per Sample Volume (ul) value is < 5, Clarity LIMS automatically assigns a value of 5 to the samples Adjusted Per Sample Volume (ul) field.
Clarity LIMS then adjusts the Adjusted Per Sample Volume (ul) field value for all other samples in the batch, based on the ratio used to increase the lowest value to 5.
[Optional] In the Sample Details table, select the pool icon to view details on the pool composition.
Select Calculate Volumes to trigger the Calculate Volumes automation. This automation performs calculations based on the selected Flowcell Type, then generates and attaches the Calculation File (CSV) to the step. This file contains volume information about the pool and the individual samples that it contains.
Select Next Steps to trigger the Set Next Step automation. This automation performs the following actions:
Copies the Flowcell Type values from the step inputs to the step outputs.
Sets the next step for samples to ADVANCE, advancing them to the next step in the protocol—Dilute, Denature & ExAmp (NovaSeqDx v1.2).
On the Assign Next Steps screen, the next step for samples is set to Dilute, Denature & ExAmp (NovaSeqDx v1.2).
Select Finish Step.
BP Aliquot Volume (ul)
NaOH Volume (ul)
Tris-HCl Volume (ul)
DPX1 Volume (ul)
DPX2 Volume (ul)
DPX3 Volume (ul)
Mastermix per Lane (ul)
The automation also populates the Flowcell Type and Loading Workflow Type columns of the Sample Details table.
The automation also generates the Calculation File (CSV) and attaches it to the step. This file contains information about the DPX Mastermix volume and the volume of Mastermix, NaOH, and Tris-HCl to add per working pool (refer to the next step).
On the Record Details screen, the Reagent Lot Tracking section tracks the DPX1, DPX2, DPX3, NaOH, Resuspension Buffer, and Tris-HCl reagents used in the step. These reagents must be added. To add and activate reagent lots, refer to Add and Configure Reagent Kits and Lots in the Clarity LIMS (Clarity & LabLink Reference Guide) documentation.
On the Record Details screen, perform the following actions:
In the Reagent Lot Tracking section, select from the active lots displayed in each drop-down list.
In the Step Details section, verify that the DPX1, DPX2, and DPX3 reagent volume values are populated. The script sets these values and they cannot be edited.
[Optional] In the Sample Details table, select the pool icon to view details of the working pool composition.
Make sure that the following values and columns are populated:
BP Aliquot
Mastermix per lane
NaOH
In the Files area, select the Calculation File (CSV) to open it and view details on the DPX Mastermix volume and the volume of Mastermix, NaOH, and Tris-HCl to add per working pool.
The Flowcell Type field is set to a valid value (SP, S1, S2, or S4) and each input has the same value for the field.
The container type selected matches the value in the Flowcell Type field.
The number of outputs matches the number of lanes on the selected flow cell type. If validation fails, an error message reports the number of working pools does not match the number of lanes available on the flow cell.
On the Placement screen, do as follows.
Drag pools from the left of the screen over into the Placed Samples area on the right.
Scan or type the barcode of the flow cell into the Flowcell field.
Select Record Details.
After exiting the Placement screen, the Validate Flowcell Barcode automation makes sure that the container barcode conforms to the barcode mask for the selected Flowcell Type. This automation also copies the Flowcell Type and Loading Workflow Type field values from step inputs to outputs. The following table shows the barcode mask information.
The fields displayed on the Record Details screen are used to set up the run and generate the sample sheet.
Some of these fields are autopopulated and some must be entered manually. Refer to the following table for details.
Fields Displayed on Record Details Screen of Load to Flowcell (NovaSeqDx v1.2) Step
Field
Value
Experiment Name
Enter the experiment name. Only alphanumeric characters, dashes, and underscores are permitted. Spaces are not permitted.
BaseSpace Sequence Hub Configuration
Presets
Not Used
Run Monitoring Only
Run Monitoring and Storage
Workflow
Select Validate Run Setup and Generate Sample Sheet to trigger the automation script. The script performs the following actions:
Validates the parameters entered on the Record Details screen.
Generates the sample sheet and attaches it to the placeholder in the Files area on the Record Details screen.
ℹ The v2 sample sheet generated does not include Cloud and Analysis sections. Contact Illumina Support for information on generating v2 sample sheets with these sections.
Select Next Steps.
On the Assign Next Steps screen, the Next Step field for samples is prepopulated with Mark protocol as complete.
Select Finish Step.
Current Cycle
Current Read
Loading Workflow Type
Flow Cell ID
Flow Cell Side
Flow Cell Mode
Flow Cell Part Number
Flow Cell Lot Number
Flow Cell Expiration Date
Instrument ID
Instrument Type
Instrument Control Software Version
Firmware Version
RTA Version
Sequencing Log
Reads PF (M) R1
Reads PF (M) R2
Cluster Density (K/mm^2) R1
Cluster Density (K/mm^2) R2
%PF R1
%PF R2
% Bases >=Q30 R1
% Bases >=Q30 R2
Intensity Cycle 1 R1
Intensity Cycle 1 R2
% Phasing R1
% Phasing R2
% Prephasing R1
% Prephasing R2
% Aligned R1
% Aligned R2
% Error Rate R1
% Error Rate R2
Validates reagent kit information in the RunStarted request:
Verifies that the reagent kit exists. If the reagent kit does not exist, the Sequencer API creates the kit and enables on the master step.
Verifies that the reagent kit is activated. If the reagent kit is not active, the Sequencer API activates it.
Initiates the step for the queued samples. The step produces one output file placeholder per lane of the flow cell in use, based on the flow cell type.
Records all relevant information from the RunStarted request on the step (such as reagent lots and step fields).
At the end of the run, NVOS sends one of the following run status requests:
RunCompletedSuccessfully
RunEndedByUSer
RunErroredOut
The Sequencer API updates the step with any new information, which includes only the status and cycle/read information.
After primary analysis completes, NVOS sends a request containing the parsed run metrics. NVOS sends a request only when the run completes successfully. However, the API does not make this assumption and accepts the request regardless of status. The Sequencer API then does the following:
Records the metrics into the fields on file placeholder outputs in Clarity LIMS.
Completes the step in Clarity LIMS if the status is RunCompletedSuccessfully. For all other status options, the step remains in progress. This action is the default autocomplete step behavior. For more information, refer to NovaSeq 6000Dx Integration v1.3.0 Configuration.
ℹ NovaSeq 6000Dx Integration v1.3.0 Configuration provides detailed information on how the following components work with the Sequencer API integration:
Sample sheet generation and contents
NovaSeq 6000Dx run
User authentication and login
Run recipe contents
On the Record Details screen, the Sequencing Log multiline text field contains logging information.
If unable to reach the Record Details screen, or if the Sequencing Log field does not contain enough information to resolve the issue, review the sequencer-api.log file. For details, refer to Logging.
Contact the Clarity LIMS support team. Provide the relevant information from the troubleshooting steps already performed.
Flowcell Type
Run Mode
SP
RUO
S1
RUO
S2
RUO, DX
S4
RUO, DX
Flowcell Type
Run Mode
SP
RUO
S1
RUO
S2
RUO
S4
RUO
/opt/gls/clarity/tomcat/current/logs/sequencer-api.log2021-06-19 21:32:19.705 INFO --- [] SequencerAPIApplication : Started SequencerAPIApplication in 7.026 seconds (JVM running for 15.964)
...
2021-06-19 21:34:44.564 INFO --- [Library Tube Id=NV1234567-LIB, Flow Cell Id=RM123DSXX] LimsApiLookupUtil : Searching for containers with name 'NV1234567-LIB' or 'RM123DSXX'
....
2021-06-19 21:35:27.158 INFO --- [] FileService : Successfully retrieved file 'NV1234567-LIB.csv' with LIMS ID 40-51 through the API.Tris-HCl
Flowcell Type
Loading Workflow Type
A script sets these values and columns and they cannot be edited. The working pool number is appended to the bulk pool name, so that you can identify which working pools are derived from the same bulk pool.
Workflow
Automatically populated with the preset GenerateFASTQ.
Workflow Type
Presets
No Index
Single Index
Dual Index
Custom
Index Read 1
Presets
0
6
8
Type a value between 0 and 20
Index Read 2
Presets
0
6
8
Type a value between 0 and 20
Paired End
Presets
True
False
Read 1 Cycles
Presets
251*
151
101
51
Type a value between 1 and 251
* Value of 251 is only supported for SP flow cell type. For all other flow cell types, maximum value is 151.
Read 2 Cycles
Presets
251*
151
101
51
Type a value between 0 and 251
* Value of 251 is only supported for SP flow cell type. For all other flow cell types, maximum value is 151.
Use Custom Read 1 Primer
Select if applicable.
Use Custom Read 2 Primer
Select if applicable.
Use Custom Index Read 1 Primer
Select if applicable.
Use Custom Recipe
Select if applicable.
Custom Recipe Path
If the Use Custom Recipe option is selected, enter the path to the custom recipe file to be used.
Output Folder
Enter network path for sequencing run folder. For example:
Analysis Software Version
Enter the software version for BCL Convert Analysis for v2 sample sheet. Leave blank if not applicable.
Override Cycles
Enter the Override Cycles value for v2 sample sheet. Leave blank if not applicable.
Flowcell Type
Barcode Mask
SP
[A-Z0-9]{5}DR[A-Z0-9]{2}
S1
[A-Z0-9]{5}DR[A-Z0-9]{2}
S2
[A-Z0-9]{5}DM[A-Z0-9]{2}
S4
[A-Z0-9]{5}DS[A-Z0-9]{2}
Automatically populated with the preset GenerateFASTQ.
Workflow Type
Presets
No Index
Single Index
Dual Index
Custom
Index Read 1
Presets
0
6
8
Type a value between 0 and 20
Index Read 2
Presets
0
6
8
Type a value between 0 and 20
Paired End
Presets
True
False
Read 1 Cycles
Presets
251*
151
101
51
Type a value between 1 and 251
* Value of 251 is only supported for SP flow cell type. For all other flow cell types, maximum value is 151.
Read 2 Cycles
Presets
251*
151
101
51
Type a value between 0 and 251
* Value of 251 is only supported for SP flow cell type. For all other flow cell types, maximum value is 151.
Use Custom Read 1 Primer
Select if applicable.
Use Custom Read 2 Primer
Select if applicable.
Use Custom Index Read 1 Primer
Select if applicable.
Use Custom Recipe
Select if applicable.
Custom Recipe Path
If the Use Custom Recipe option is selected, enter the path to the custom recipe file to be used.
Output Folder
Enter network path for sequencing run folder. For example:
Library Tube Barcode
Scan the library tube barcode.
Analysis Software Version
Enter the software version for BCL Convert Analysis for v2 sample sheet. Leave blank if not applicable.
Override Cycles
Enter the Override Cycles value for v2 sample sheet. Leave blank if not applicable.
\\networkshare\run_data\\networkshare\run_data