NovaSeq 6000 Integration v3.2.1 User and Configuration Guide

The Illumina NovaSeq 6000 Integration Package v3.2.1 supports the integration of BaseSpace Clarity LIMS to Illumina NovaSeq 6000 sequencers.

Note the following key changes for this version of the integration:

•

A move from a file-based to an API-based integration using the Sequencer API.

•

NovaSeq-specific configuration is now installed as part of the Preconfigured Workflows Package (PCWP) v1.2.  This configuration includes new template path locations and sample sheet generation with Template File Generator (TFG).

Configuration changes in v3.2.1 include the following:

•

Support for Sequencer API

•

Support for SPrime (SP) flow cell

•

Support for Long-Read sequencing (SP flow cell only)

•

Support for asymmetric read lengths

This User and Configuration Guide describes the integration and includes information about the preconfigured workflows, protocols, steps, and automations. The guide also lists installed components, and provides configuration requirements, rules, and constraints.

For instructions on validating and troubleshooting the Illumina NovaSeq 6000 Integration, see the NovaSeq 6000 Integration v3.2.1 Validation and Troubleshooting Guide.

Overview

The integration includes the following:

•

Preconfigured NovaSeq 6000 v3.0 workflow that maps to lab protocols and instrument runs.

•

Preconfigured protocols:

–

Run Format (NovaSeq 6000 v3.0)

–

NovaSeq Standard (NovaSeq 6000 v3.0) (supports the loading of pooled libraries into a library tube)

–

NovaSeq Xp (NovaSeq 6000 v3.0) (supports individual lane loading on the NovaSeq)

–

AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.0)

•

Automated generation of a sample sheet for use with bcl2fastq2 v2.20 analysis software. This file is automatically uploaded to the sequencing instrument via the Sequencer API and is used to set up the run.

•

Automatic validation of run setup information. This information is used to create the run recipe and initiate the run.

•

Automated tracking of the NovaSeq sequencing run and parsing of run statistics into the LIMS, including:

–

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 v1.0 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 NovaSeq 6000 v3.0 workflow. See the NovaSeq 6000 Integration v3.2.1 Validation and Troubleshooting Guide.

Information on these features is provided in the following sections.

Prerequisites and Assumptions

It is assumed that samples enter the NovaSeq 6000 v3.0 workflow as normalized libraries. That is, before they are assigned to the workflow:

•

Samples have been accessioned into the LIMS.

•

Samples have been run through QC and library prep.

•

Samples have been normalized, and the value is captured in a field called Normalized Molarity (nM).

For details on sample accessioning, see the following articles in the Samples section of the LIMS documentation for your version: LIMS Documentation v5.0 and v5.1 or LIMS Documentation v4.1 and v4.2:

•

Sample Accessioning

•

Using Sample Lists to Upload and Modify Samples

Samples can be assigned to the NovaSeq 6000 v3.0 workflow automatically (using a routing script), or manually from the Projects and Samples dashboard (see the Assigning Samples to Workflows article in the LIMS Documentation).

Workflows, Protocols, and Steps

The Illumina NovaSeq 6000 Integration Package v3.2.1 includes two workflows:

•

NovaSeq 6000 v3.0

•

Library Prep Validation v1.0 (optional, but recommended for validation purposes)

The following table lists and describes the protocols and steps included in these workflows.

Library Prep Validation v1.0 workflow
Protocol name	Protocol purpose	Steps included in protocol
Library Prep Validation v1.0	Included for validation purposes only, this protocol models the library prep steps required to advance samples to the Run Format (NovaSeq 6000 v3.0) protocol. The protocol contains a single step - Library Prep Validation v1.0. At the end of this step, a routing script sends the samples to the first step of the NovaSeq 6000 v3.0 workflow - Define Run Format (NovaSeq 6000 v3.0).	1. Library Prep Validation v1.0
NovaSeq 6000 v3.0 workflow
Protocol name	Protocol purpose	Steps included in protocol
Run Format (NovaSeq 6000 v3.0)	Allows for the assignment of per sample values for Loading Workflow Type, Normalized Molarity, Flowcell Type, and Final Loading Concentration (pM): • Loading Workflow Type: User selects from NovaSeq Standard or NovaSeq Xp. • Normalized Molarity: User enters a value for each sample. • Flowcell Type: User selects from options SP, S1, S2, or S4. • Final Loading Concentration (pM): User selects from options 225 (PCR-free workflows) or400 (Nano workflows), or may enter a different value. Compares each sample's Normalized Molarity value with the Minimum Molarity value. Routing script sends samples to the NovaSeq Standard or NovaSeq Xp protocol, according to the selected Loading Workflow Type. Samples with Normalized Molarity less than Minimum Molarity are removed from the workflow.	1. Define Run Format (NovaSeq 6000 v3.0)
NovaSeq Standard (NovaSeq 6000 v3.0)	Samples are pooled and added to the library tube in preparation for the NovaSeq run. Run setup information is validated and sample sheet is generated. Routing script sends library tube to the AUTOMATED - NovaSeq Run (NovaSeq 6000 v30) protocol.	1. Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.0) 2. Dilute and Denature (NovaSeq 6000 v3.0)
NovaSeq Xp (NovaSeq 6000 v3.0)	Samples are pooled and added to lanes on the NovaSeq flow cell. The flow cell type is determined by the option selected in the Define Run Format (NovaSeq 6000 v3.0) step. Run setup information is validated and sample sheet is generated. Routing script sends flow cell to the AUTOMATED - NovaSeq Run (NovaSeq 6000 v30) protocol.	1. Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.0) 2. Dilute, Denature and ExAmp (NovaSeq 6000 v3.0) 3. Load to Flowcell (NovaSeq 6000 v3.0)
AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.0)	All samples complete the workflow by going through this protocol. This protocol contains one fully-automated step. The user must not add samples to the Ice Bucket or start the step. The integration will do this automatically.	1. AUTOMATED - NovaSeq Run (NovaSeq 6000 v30)

The following image show the protocols and their steps displayed in the LIMS Lab View.

Validation Workflow

The Library Prep Validation v1.0 workflow allows for validation of the system after installation is complete. For details, see the NovaSeq 6000 Integration v3.2.1 Validation and Troubleshooting Guide.

Step Automations

The following table lists the step automations included in the NovaSeq 6000 v3.0 workflow. Automation details are provided in the sections following the table.

For details on the validation workflow, see the NovaSeq 6000 Integration v3.2.1 Validation and Troubleshooting Guide.

Protocol	Step	Automation
1. Run Format (NovaSeq 6000 v3.0)	1. Define Run Format (NovaSeq 6000 v3.0)	• Routing Script • Set Next Steps
2. NovaSeq Standard (NovaSeq 6000 v3.0)	1. Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.0)	• Calculate Volumes • Set Next Step • Validate Inputs Flowcell Type and Single Pool • Validate Unique Indexes Not used. This functionality is handled by the core LIMS configuration for pooling.
	2. Dilute and Denature (NovaSeq 6000 v3.0)	• Routing Script • Validate Library Tube Barcode • Validate Run Setup and Generate Sample Sheet • Validate Single Input
3. NovaSeq Xp (NovaSeq 6000 v3.0)	1. Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.0)	• Calculate Volumes • Set Next Step • Validate Inputs Flowcell Type and Single Pool • Validate Unique Indexes Not used. This functionality is handled by the core LIMS configuration for pooling.
	2. Dilute, Denature and ExAmp (NovaSeq 6000 v3.0)	• Calculate Volumes • Set Next Step • Validate Inputs Flowcell Type
	3. Load to Flowcell (NovaSeq 6000 v3.0)	• Validate Flowcell Barcode • Validate Inputs and Selected Container • Validate Run Setup and Generate Sample Sheet
4. AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.0)	1. AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.0)	• Set Next Steps Not used. By default, this functionality is determined by the Sequencer API.If required, you can enable the automation and use it to override the next step behavior used by the Sequencer API.

Configuration Details

The sections below describe the configuration installed with the Illumina NovaSeq 6000 Integration.

Protocol 1: Run Format (NovaSeq 6000 v3.0

This protocol sets the Loading Workflow Type and allows the user to choose the appropriate Flowcell Type and Final Loading Concentration (pM). At the end of the protocol, a routing script sends the normalized libraries to either the NovaSeq Standard (NovaSeq 6000 v3.0) or the NovaSeq Xp (NovaSeq 6000 v3.0) protocol.

This protocol contains one step: Define Run Format (NovaSeq 6000 v3.0), described below.

Step 1. Define Run Format (NovaSeq 6000 v3.0)

Step input: NTP (normalized libraries)

Step output: None

The following automations are configured on this step:

•

Routing Script

•

Set Next Steps

Define Run Format (NovaSeq 6000 v3.0) automation configuration - LIMS v4.2.x:

Define Run Format (NovaSeq 6000 v3.0) automation configuration - LIMS v5.x:

These automations are described below, in the order in which they are triggered at run time.

Set Next Steps

Automatically triggered on exit of the Record Details screen, this automation does the following:

•

Sets the next step for samples to REMOVE:

nextStep = ::REMOVE::

•

Calculates the Minimum Molarity using the following formula:

input.::Minimum Molarity (nM):: = (5 * input.::Final Loading Concentration (pM)::)/1000

•

Checks Normalized Molarity value. For samples with no Normalized Molarity value (i.e., empty value, not including 0), generates an error message informing the user that the field cannot be empty:

if (!input.hasValue(::Normalized Molarity (nM)::)) { \

  fail(::The Normalized Molarity cannot be empty.::) ;

}

•

Compares each sample's Normalized Molarity value with the Minimum Molarity value. If Normalized Molarity value is lower than the Minimum Molarity value, sets the sample's Loading Workflow Type to [Remove from workflow] and records a message in the Warning field for the sample:

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:: }

At this point the user has two options:

–

Correct the Normalized Molarity value on the Record Details screen. User will also need to 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 will be removed from the workflow.

Routing Script

Automatically triggered on exit of the step, this automation invokes the changeWorkflow script, which routes step inputs appropriately.

•

Samples with Loading Workflow Type field value = NovaSeq Standard are routed to the NovaSeq 6000 v3.0 workflow and queued for the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.0) step.

•

Samples with Loading Workflow Type field value = NovaSeq Xp are routed to the NovaSeq 6000 v3.0 workflow and queued for the Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.0) step.

Default automation command line:

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 'NovaSeq 6000 v3.0' \

--STEP 'Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.0)' \

--INPUTS_OR_OUTPUTS 'INPUTS' \\

--FIELD_NAME 'Loading Workflow Type' \

--FIELD_VALUE 'NovaSeq Xp' \--WORKFLOW 'NovaSeq 6000 v3.0' \

--STEP 'Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.0)' \

--INPUTS_OR_OUTPUTS 'INPUTS'"

Step UDFs/Custom Fields

There are four fields defined on the Define Run Format (NovaSeq 6000 v3.0) step.

These are shown below in the BaseSpace Clarity LIMS v5.x Configuration area (Custom Fields > Master Step Fields tab).

See the following table for LIMS v4.2.x and LIMS v5.x field configuration details:

Field Name	Field Type	Field Constraints	Preset Values/Additional Options and Dropdown Items
Comment	Multi-line Text	• Required: No • Read Only: No	None
Flowcell Type	Single-line Text (v4.2.x) Text Dropdown (v5.x)	• Required: Yes • Read Only: No • Custom Entries: Yes	• SP • S1 • S2 • S4
Instruction	Single-line Text (v4.2.x) Text (v5.x)	• Required: No • Read Only: Yes	• Default: Add Flowcell Type and Loading Workflow Type below
Loading Workflow Type	Single-line Text (v4.2.x) Text Dropdown (v5.x)	• Required: Yes • Read Only: No • Custom Entries: No	• NovaSeq Standard • NovaSeq Xp

Global UDFs/Custom Fields

The following table lists the global UDFs/custom fields that are configured to display on the Define Run Format (NovaSeq 6000 v3.0) step.

Field Name	Field Type	Field Constraints/Options	Preset Values/Additional Options and Dropdown Items
Configured on Analyte in LIMS v4.2.x and Derived Samplein LIMS v5.x:
Adjusted Per Sample Volume (ul)	Numeric	• Required: No • Read Only: Yes	• Decimal Places Displayed: 2
Final Loading Concentration (pM)	Numeric (v4.2.x) Numeric Dropdown (v5.x)	• Required: Yes • Read Only: No • Custom Entries: Yes	• 225 • 400 • Decimal Places Displayed: 0
Flowcell Type	Single-line Text (v4.2.x) Text Dropdown (v5.x)	• Required: Yes • Read Only: No • Custom Entries: No	• SP • S1 • S2 • S4
Loading Workflow Type	Single-line Text (v4.2.x) Text Dropdown (v5.x)	• Required: Yes • Read Only: No • Custom Entries: No	• NovaSeq Standard • NovaSeq Xp • [Remove from workflow]
Minimum Molarity (nM)	Numeric	• Required: No • Read Only: No	• Decimal Places Displayed: 2
Normalized Molarity (nM)	Numeric	• Required: No • Read Only: No	• Decimal Places Displayed: 2
Per Sample Volume (ul)	Numeric	• Required: No • Read Only: Yes	• Decimal Places Displayed: 2
Warning	Single-line Text (v4.2.x) Text Dropdown (v5.x)	• Required: No • Read Only: Yes • Custom Entries: Yes	• The Normalized Molarity (nM) is too low. • n/a

User Interaction

1.

On the Record Details screen, the Sample Details table contains the following fields that must be completed (values can vary across samples):

•

Loading Workflow Type: Users selects NovaSeq Standard or NovaSeq Xp from the drop-down list.

•

Flowcell Type: User selects SP, S1, S2, or S4 from the drop-down list.

•

Final Loading Concentration (pM): User selects from the two preset options - 225 (for PCR-free workflows) or400 (for Nano workflows), or may enter a different value.

•

Normalized Molarity (nM):These values are copied over from the previous step. If the user did not populate this column during library prep, they can enter the values here.

•

User clicks Next Steps. This triggers the Next Steps automation, which does the following:

2.

On the Assign Next Steps screen:

•

In the Sample Details table, the Next Step for all samples is pre-populated with Remove from workflow - regardless of the Loading Workflow Type.

For samples whose Normalized Molarity value was found to be lower than the Minimum Molarity value, the Loading Workflow Type is set to Remove from workflow and a message is recorded in the Warningfield for the sample:

At this point the user has two options:

–

Return to the Record Details screen and adjust the Normalized Molarity value so that it equals or exceeds the Minimum Molarity value. User will also need to set the Loading Workflow Type to NovaSeq Standard.

–

Complete the protocol without correcting the Normalized Molarity value. In this case, the samples in question will be removed from the LIMS workflow.

3.

User clicks Finish Step. The Routing Script automation is triggered:

•

Samples whose Loading Workflow Type is set to Remove from workflow (i.e., whereNormalized Molarity value is lower than the Minimum Molarity) are removed from the LIMS workflow.

•

Samples whose Loading Workflow Type is set to NovaSeq Standard are routed to the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.0) step. This is the first of two steps in the NovaSeq Standard (NovaSeq 6000 v3.0) protocol.

•

Samples whose Loading Workflow Type is set to NovaSeq Xp are routed to the Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.0) step. This is the first of three steps in the NovaSeq Xp (NovaSeq 6000 v3.0) protocol.

Protocol 2: NovaSeq Standard (NovaSeq 6000 v3.0)

Samples are routed to this protocol if their Loading Workflow Type value is set to NovaSeq Standard. Samples are pooled and added to a library tube in preparation for the NovaSeq run.

At the end of this protocol, a routing script sends the library tube to the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.0) protocol.

This protocol contains two steps, described below:

•

Step 1: Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.0)

•

Step 2: Dilute and Denature (NovaSeq 6000 v3.0)

Step 1: Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.0)

In this step, user manually places libraries into a single pool. Resuspension buffer and reagents are added.

Step input: NTP (normalized libraries)

Step output: Bulk pool

The following automations are configured on the step:

•

Calculate Volumes

•

Set Next Step

•

Validate Inputs Flowcell Type and Single Pool

•

Validate Unique Indexes(Not used. This functionality is handled by the core LIMS configuration for pooling.)

Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.0) automation configuration - LIMS v4.2.x:

Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.0) automation configuration - LIMS v5.x:

These automations are described below, in the order in which they are triggered at run time.

Validate Inputs Flowcell Type and Single Pool

Automatically triggered on exit of the Pooling screen, this automation:

•

Checks that all samples in the pool have the same Flowcell Type assigned to them.

•

Checks that only one pool has been created. Changes the value of error and logging messages to reference the type of pool passed as 'bulk' pool.

/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"

Calculate Volumes

Automatically triggered when the user clicks the Calculate Volumes button on the Record Details screen, this automation:

•

Calculates the number of samples in the pool:

-exp 'step.::Number of Samples in Pool:: = step.::Number of Samples in Pool:: + 1'

•

Sets the value of the Bulk Pool Volume (ul) field, based on the selected Flowcell Type:

-exp 'if ( input.::Flowcell Type:: == ::SP:: ) {

  step.::Bulk Pool Volume (ul):: = step.::Number of Flowcells to Sequence:: * 100 + 30

} ;

 

if ( input.::Flowcell Type:: == ::S1:: ) {   

  step.::Bulk Pool Volume (ul):: = step.::Number of Flowcells to Sequence:: * 100 + 30

} ;

if ( input.::Flowcell Type:: == ::S2:: ) {   

  step.::Bulk Pool Volume (ul):: = step.::Number of Flowcells to Sequence:: * 150 + 30

} ;

if ( input.::Flowcell Type:: == ::S4:: ) {   

  step.::Bulk Pool Volume (ul):: = step.::Number of Flowcells to Sequence:: * 310 + 30

} ;

•

Calculates the Per Sample Volume (ul) to be added to the pool:

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::

 

•

Calculates the Total Sample Volume (ul) field value:

-exp 'step.::Total Sample Volume (ul):: = step.::Total Sample Volume (ul):: + input.::Adjusted Per Sample Volume (ul)::'

•

If the Total Sample Volume is less than the Bulk Pool Volume, calculates the RSB Volume (ul) field value:

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)::} ;

•

Copies the Flowcell Type and Loading Workflow Type values from the step inputs to the step outputs:

output.::Flowcell Type:: = input.::Flowcell Type:: ;output.::Loading Workflow Type:: = input.::Loading Workflow Type:: ;

•

Sets the Volume of Pool to Denature (ul) value and calculates NaOH Volume (ul) and Tris-HCl Volume (ul) values, based on the Flowcell 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):: = 37.50 ; output.::Tris-HCl Volume (ul):: = 37.50 } ;

if ( input.::Flowcell Type:: == ::S4:: ) { output.::Volume of Pool to Denature (ul):: = 310 ; output.::NaOH Volume (ul):: = 77.50 ; output.::Tris-HCl Volume (ul):: = 77.50 }

•

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 by the user on the Step Setup screen of the following step - Dilute and Denature (NovaSeq 6000 v3.0).

&& /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}.csv

Set Next Step

Automatically triggered on exit of the Record Details screen, this automation sets the next step for samples to ADVANCE, advancing them to the next step in the protocol - Dilute and Denature (NovaSeq 6000 v3.0):

nextStep = ::ADVANCE::

Step UDFs/Custom Fields

There are five fields defined on the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.0) step.

These are shown below in the BaseSpace Clarity LIMS v5.x Configuration area (C ustom Fields > Master Step Fields tab).

 See the following table for LIMS v4.2.x and LIMS v5.x field configuration details:

Field Name	Field Type	Field Constraints/Options	Preset Values/Additional Options and Dropdown Items
Bulk Pool Volume (ul) (for calculation purposes, not displayed)	Numeric	• Required: No • Read Only: No	• Decimal Places Displayed: 2
Number of Flowcells to Sequence	Numeric	• Required: Yes • Read Only: No	• Range From:1 to 10 • Decimal Places Displayed: 0
Total Sample Volume (ul) (for calculation purposes, not displayed)	Numeric	• Required: No • Read Only: No	• Default: 0 • Decimal Places Displayed: 2
Minimum Per Sample Volume (uL)	Numeric	• Required: Yes • Read Only: No	• Default: 5 • Decimal Places Displayed: 2
Number of Samples in Pool (for calculation purposes, not displayed)	Numeric	• Required: No • Read Only: No	• Default: 0 • Decimal Places Displayed: 0

Global UDFs/Custom Fields

The following table lists the global UDFs/custom fields that are configured to display on the Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.0) step.

Field Name	Field Type	Field Constraints/Options	Preset Values/Additional Options and Dropdown Items
Configured on Analyte in LIMS v4.2.x and Derived Samplein LIMS v5.x:
Flowcell Type	Single-line Text (v4.2.x) Text Dropdown (v5.x)	• Required: Yes • Read Only: No • Custom Entries: No	• SP • S1 • S2 • S4
Loading Workflow Type	Single-line Text (v4.2.x) Text Dropdown (v5.x)	• Required: Yes • Read Only: No • Custom Entries: No	• NovaSeq Standard • NovaSeq Xp • [Remove from workflow]
NaOH Volume (ul)	Numeric	• Required: No • Read Only: Yes	• Decimal Places Displayed: 2
RSB Volume (ul)	Numeric	• Required: No • Read Only: Yes	• Decimal Places Displayed: 2
Tris-HCl Volume (ul)	Numeric	• Required: No • Read Only: Yes	• Decimal Places Displayed: 2
Volume of Pool to Denature (ul) Used in Make Bulk Pool for NovaSeq Standard (NovaSeq 6000 v3.0) step only. Displays on Record Detailsscreen and in the generated CSV file.	Numeric	• Required: No • Read Only: Yes	• Decimal Places Displayed: 0

User Interaction

1.

On the Queue screen, user adds samples of the same Flowcell Type to the Ice Bucket and clicks View Ice Bucket.

2.

On the Ice Bucket screen, user clicks Begin Work.

3.

On the Pooling screen, user:

•

Creates a pool by dragging samples into the Pool Creator.

•

Names the pool, or accepts the default name - Pool #1.

•

Clicks Record Details.

4.

On the Record Details screen, the Step Details area contains two required fields:

a.

Number of Flowcells to Sequence - The value entered in this field is used in volume calculations, to ensure that volumes are sufficient for the number of times the pool will be sequenced.

b.

Minimum Per Sample Volume (ul) - In many applications, and particularly where robotics are used, a minimum sample volume is required. The value in this field is used to calculate how much of each sample will be included in the pool. The field is pre-populated with the configured default value, 5 ul, but may be edited if required.

c.

In the Sample Details table, the user can click on the pool icon to view details on the pool composition.

d.

The user clicks Calculate Volumes. This triggers the Calculate Volumes automation, which performs volume calculations based on the selected Flowcell Type:

•

The user clicks Next Steps. This triggers the Set Next Step automation. This automation sets the next step for samples to ADVANCE, advancing them to the next step in the protocol - Dilute and Denature (NovaSeq 6000 v3.0).

e.

On the Assign Next Steps screen: The next step is already set to Dilute and Denature (NovaSeq 6000 v3.0).

•

User clicks Finish Step. At the end of this step, the pool of samples automatically advances to the Dilute and Denature (NovaSeq 6000 v3.0) step.

Step 2: Dilute and Denature (NovaSeq 6000 v3.0)

In this step, pooled samples are denatured and diluted by the addition of NaOH, Tris-HCl, and Resuspension Buffer (RSB), and are manually placed by the user into the library tube that will be used in the NovaSeq 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 automations are configured on the step:

•

Routing Script

•

Validate Library Tube Barcode

•

Validate Run Setup and Generate Sample Sheet

•

Validate Single Input

Dilute and Denature (NovaSeq 6000 v3.0) automation configuration - LIMS v4.2.x:

Dilute and Denature (NovaSeq 6000 v3.0) automation configuration - LIMS v5.x:

These automations are described below, in the order in which they are triggered at run time.

Validate Single Input

Automatically triggered at the beginning of the step, this automation checks that there is only one container input to the step.

script:validateSampleCount -min 1 -max 1

Validate Library Tube Barcode

Automatically triggered on exit of the Placement screen, this automation:

•

Validates the library tube barcode to ensure it conforms to the barcode mask [A-Za-z]{2}[0-9]{7}-[A-Za-z]{3}:

if (!output.container.name.matches(::[A-Za-z]{2}[0-9]{7}-[A-Za-z]{3}::)) { fail(::Invalid Library Tube Barcode. Please verify and try again.::) }

•

Copies Flowcell Type and Loading Workflow Type field values from step inputs to outputs:

output.::Flowcell Type:: = input.::Flowcell Type:: ; output.::Loading Workflow Type:: = input.::Loading Workflow Type::'

Validate Run Setup and Generate Sample Sheet

Automatically triggered when user clicks a button on the Record Details screen, this automation:

•

Copies the Flowcell Type from the step input to the Run Mode field (hidden):

step.::Run Mode:: = input.::Flowcell Type::

•

Validates the parameters entered on the Record Details screen, which will be used to set up the run and generate the sample sheet file (see Respond to Validation/Recipe Request Call from Sequencer, below).

–

Experiment Name may only contain alphanumeric, dash, or underscore characters. Spaces are not permitted.

–

When Workflow Type = No Index, Index Read 1 must be zero. For any other Workflow Type, Index Read 1 must be greater than zero.

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) {

    fail(::Index Read 1 must be 0 if the Workflow Type is No Index.::)      

  }

} else {

  if(step.::Index Read 1::== 0) {

    fail(::Index Read 1 must be greater than 0 if the Workflow Type is ::+ step.::Workflow Type::+ ::.::)

  }

};

•

Checks the Paired End and Read 2 Cycles field values.

–

If Paired End = True and Read 2 Cycles value is 0, generates an error.

if (step.::Paired End::.toBoolean() && step.::Read 2 Cycles:: == 0) {

  fail(::Read 2 Cycles must not be zero if it is Paired End read.::)

};

•

Checks the Workflow Type and Read 2 Cycles field values.

–

If Workflow Type is set to Dual Index and Index Read 2 value is 0, generates an error.

–

If Workflow Type is set to No Index or Single Index, sets Index Read 2 to 0.

if (step.::Workflow Type:: == ::Dual Index:: && step.::Index Read 2:: == 0) {

  fail(::Index Read 2 must not be zero if Workflow Type is Dual Index.::)

};

if (step.::Workflow Type:: == ::No Index:: || step.::Workflow Type:: == ::Single Index::) {

  step.::Index Read 2:: = 0

};

•

If Paired End = False, sets Read 2 Cycles value to 0.

if (!step.::Paired End::.toBoolean()) { step.::Read 2 Cycles:: = 0 };

•

Checks the Flowcell Type 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 value is greater than 151, generates an error message.

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::)

•

Sets the next step for samples to REMOVE:

nextStep = ::REMOVE::

•

Generates the sample sheet and attaches it to the step (see Sample sheet generation section, below).

&& /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/NovaSeq_BCL2FASTQ_Samplesheet.csv \   

  -o {compoundOutputFileLuid0}.csv \   

  -q true \   

  -destLIMSID {compoundOutputFileLuid0} \   

  -l {compoundOutputFileLuid1}"

Routing Script

Automatically triggered on exit of the step, this automation invokes the changeWorkflow script, which routes step outputs to the NovaSeq 6000 v3.0 workflow, and queues them for the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.0) step.

Default automation command line:

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 {compoundOutputFileLuid2} script:changeWorkflow \ \

  --FIELD_NAME 'N/A' \

  --FIELD_VALUE 'N/A' \

  --WORKFLOW 'NovaSeq 6000 v3.0' \

  --STEP 'AUTOMATED - NovaSeq Run (NovaSeq 6000 v30)' \

  --INPUTS_OR_OUTPUTS 'OUTPUTS'"

Step UDFs/Custom Fields

There are 16 fields defined on the Dilute and Denature (NovaSeq 6000 v3.0) step. These fields are required for sample sheet and JSON file generation.

The fields are shown below in the BaseSpace Clarity LIMS v5.x Configuration area (Custom Fields > Master Step Fields tab).

See the following table for LIMS v4.2.x and LIMS v5.x field configuration details:

Field Name	Field Type	Field Contraints/Options	Preset Values/Additional Options and Dropdown Items
BaseSpace Sequence Hub Configuration	Single-line Text (v4.2.x) Text Dropdown (v5.x)	• Required: Yes • Read Only: No • Custom Entries: No	• Not Used • Run Monitoring Only • Run Monitoring and Storage
Custom Recipe Path	Single-line Text (v4.2.x) Text (v5.x)	• Required: No • Read Only: No
Experiment Name	Single-line Text (v4.2.x) Text (v5.x)	• Required: Yes • Read Only: No
Index Read 1	Numeric (v4.2.x) Numeric Dropdown (v5.x)	• Required: Yes • Read Only: No • Custom Entries: Yes	• 0 • 6 • 8 • Range From: 0 to 20 • Decimal Places Displayed: 0
Index Read 2	Numeric (v4.2.x) Numeric Dropdown (v5.x)	• Required: Yes • Read Only: No • Custom Entries: Yes	• 0 • 6 • 8 • Range From: 0 to 20 • Decimal Places Displayed: 0
Output Folder	Single-line Text (v4.2.x) Text (v5.x)	• Required: Yes • Read Only: No
Paired End	Single-line Text (v4.2.x) Text Dropdown (v5.x)	• Required: Yes • Read Only: No • Custom Entries: No	• True • False
Read 1 Cycles	Numeric (v4.2.x) Numeric Dropdown (v5.x)	• Required: Yes • Read Only: No • Custom Entries: Yes	• 251* • 151 • 101 • 51 • Range From: 1 to 251 • Decimal Places Displayed: 0 *Value of 251 is only supported for SPflow cell type. For all other flow cell types, maximum value is 151.
Read 2 Cycles	Numeric (v4.2.x) Numeric Dropdown (v5.x)	• Required: Yes • Read Only: No • Custom Entries: Yes	• 251* • 151 • 101 • 51 • Range From: 1 to 251 • Decimal Places Displayed: 0 *Value of 251 is only supported for SPflow cell type. For all other flow cell types, maximum value is 151.
Run Mode (not displayed in user interface)	Single-line Text (v4.2.x) Text Dropdown (v5.x)	• Required: No • Read Only: Yes • Custom Entries: No	• SP • S1 • S2 • S4
Use Custom Index Read 1 Primer LIMS v5.0.5 and later: See Configuration Update for NovaSeq Integration with Clarity LIMS v5.0.5 and Later	Check Box (v4.2.x) Toggle Switch (v5.x)	• Required: No • Read Only: No	• None Set
Use Custom Read 1 Primer LIMS v5.0.5 and later: See Configuration Update for NovaSeq Integration with Clarity LIMS v5.0.5 and Later	Check Box (v4.2.x) Toggle Switch (v5.x)	• Required: No • Read Only: No	• None Set
Use Custom Read 2 Primer LIMS v5.0.5 and later: See Configuration Update for NovaSeq Integration with Clarity LIMS v5.0.5 and Later	Check Box (v4.2.x) Toggle Switch (v5.x)	• Required: No • Read Only: No	• None Set
Use Custom Recipe	Check Box (v4.2.x) Toggle Switch (v5.x)	• Required: Yes • Read Only: No	• Default: No
Workflow	Single-line Text (v4.2.x) Text Dropdown (v5.x)	• Required: No • Read Only: Yes	• GenerateFASTQ
Workflow Type	Single-line Text (v4.2.x) Text Dropdown (v5.x)	• Required: Yes • Read Only: No • Custom Entries: Yes	• No Index • Single Index • Dual Index • Custom

Global UDFs/Custom Fields

The following table lists the global UDFs/custom fields that are configured to display on the Dilute and Denature (NovaSeq 6000 v3.0) step.

Field Name	Field Type	Field Constraints/Options	Preset Values/Additional Options and Dropdown Items
Configured on Analyte in LIMS v4.2.x and Derived Samplein LIMS v5.x:
Flowcell Type	Single-line Text (v4.2.x) Text Dropdown (v5.x)	• Required: Yes • Read Only: No • Custom Entries: No	• SP • S1 • S2 • S4
Loading Workflow Type	Single-line Text (v4.2.x) Text Dropdown (v5.x)	• Required: Yes • Read Only: No • Custom Entries: No	• NovaSeq Standard • NovaSeq Xp • [Remove from workflow]

User Interaction

1.

On the Placement screen, user does the following:

•

Drags the pool into the library tube in the Placed Samples area.

•

Scans or types the barcode of the library tube into the Library Tube field.

•

Clicks Record Details.

2.

On the Record Details screen:

•

The Reagent Lot Tracking section lets you track the NaOH, Resuspension Buffer, and Tris-HCI reagents used in the step. The user selects 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 file.

Some of these fields are auto-populated and some must be completed by the user (see the following table for details):

Field	Value
Experiment Name	User enters the experiment name. Only alphanumeric characters, dashes, and underscores are permitted. Spaces are not permitted.
BaseSpace Sequence Hub Configuration	User selects from preset options: Not Used, Run Monitoring Only, Run Monitoring and Storage
Workflow	Automatically populated with preset - GenerateFASTQ
Workflow Type	User selects from preset options: No Index, Single Index, Dual Index, Custom
Index Read 1	User selects from preset options: 0, 6, 8 - or type a value between 0 and 20
Index Read 2	User selects from preset options: 0, 6, 8 - or type a value between 0 and 20
Paired End	User selects from preset options: True or False
Read 1 Cycles	User selects from preset options: 251, 151, 101, 51 - or 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	User selects from preset options: 251, 151, 101, 51 - or 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.
Use Custom Read 1 Primer	User selects if applicable.
Use Custom Read 2 Primer	User selects if applicable.
Use Custom Index Read 1 Primer	User selects if applicable.
Output Folder	User enters the network path for sequencing run folder. For example: \\networkshare\run_data
Use Custom Recipe	User selects if applicable.
Custom Recipe Path	If Use Custom Recipe selected, user enters the path to the custom recipe file to be used.

3.

On the Record Details screen, user clicks Validate Run Setup and Generate Sample Sheet.

This triggers the automation script, which does the following:

•

Validates the information provided to set up the run.

•

Generates the sample sheet file and attaches it to the placeholder in the Files area of the Record Detailsscreen.

•

Sets the value of the next step to Remove from workflow. The Routing Script automation expects this value, and requires it in order to successfully advance samples to the next step.

•

User clicks Next Steps.

4.

On the Assign Next Steps screen: The Next Step field for all samples is pre-populated with Remove from workflow.

•

User clicks Finish Step.

5.

On exit from the step:

•

The Routing Script automation is triggered and samples are routed to the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.0) protocol.

•

In Lab View, the pool of samples is queued for the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.0) step.

At this point in the workflow, the user interaction ends. The integration automatically starts the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.0) step and data from the run is parsed back into the LIMS.

Protocol 3: NovaSeq Xp (NovaSeq 6000 v3.0)

Samples are routed to this protocol if their Loading Workflow Type value is set to NovaSeq Xp.

Samples are pooled and added to lanes on the NovaSeq flow cell type selected in the Define Run Format (NovaSeq 6000 v3.0) step. At the end of this protocol, the flow cell is sent to the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.0) protocol.

This protocol contains three steps, described below:

•

Step 1: Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.0)

•

Step 2: Dilute, Denature and ExAmp (NovaSeq 6000 v3.0)

•

Step 3: Load to Flowcell (NovaSeq 6000 v3.0)

Step 1: Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.0)

In this step, user manually places libraries into a pool.

Step input: NTP (normalized libraries)

Step output: Bulk pool

The following automations are configured on the step:

•

Calculate Volumes

•

Set Next Step

•

Validate Inputs Flowcell Type and Single Pool

•

Validate Unique Indexes (Not used. This functionality is handled by the core LIMS configuration for pooling.)

Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.0) automation configuration - LIMS v4.2.x:

Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.0) automation configuration - LIMS v5.x:

These automations are described below, in the order in which they are triggered at run time.

Validate Inputs Flowcell Type and Single Pool

Automatically triggered on exit of the Pooling screen, this automation:

•

Checks that all samples in the pool have the same Flowcell Type assigned to them.

•

Checks that only one pool has been created.

•

Changes the value of error and logging messages to reference the type of pool passed as 'bulk' pool.

/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"

Calculate Volumes

Automatically triggered when the user clicks the Calculate Volumes button on the Record Details screen, this automation:

•

Calculates the number of samples in the pool:

step.::Number of Samples in Pool:: = step.::Number of Samples in Pool:: + 1

•

Sets the value of the Bulk Pool Volume (ul) field,based on the selected Flowcell Type:

if ( input.::Flowcell Type:: == ::SP:: ) {

  step.::Bulk Pool Volume (ul):: = step.::Number of Lanes to Sequence:: * 18 + 30

} ;

if ( input.::Flowcell Type:: == ::S1:: ) {  

  step.::Bulk Pool Volume (ul):: = step.::Number of Lanes to Sequence:: * 18 + 30

} ;

if ( input.::Flowcell Type:: == ::S2:: ) {    

  step.::Bulk Pool Volume (ul):: = step.::Number of Lanes to Sequence:: * 22 + 30

} ;

if ( input.::Flowcell Type:: == ::S4:: ) {  

  step.::Bulk Pool Volume (ul):: = step.::Number of Lanes to Sequence:: * 30 + 30

} ;

•

Calculates the Per Sample Volume (ul) to be added to the pool:

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::

•

Calculates the Total Sample Volume (ul) field value:

step.::Total Sample Volume (ul):: = step.::Total Sample Volume (ul):: + input.::Adjusted Per Sample Volume (ul)::

•

If the Total Sample Volume is less than the Bulk Pool Volum e, calculates the RSB Volume (ul) field value:

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)::} ;

•

Copies the Flowcell Type and Loading Workflow Type values from the step inputs to the step outputs:

output.::Flowcell Type::= input.::Flowcell Type::;output.::Loading Workflow Type::= input.::Loading Workflow Type::;

•

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 by the user on the Step Setup screen of the following step - Dilute, Denature and ExAmp (NovaSeq 6000 v3.0).

&& /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"

Set Next Step

Automatically triggered on exit of the Record Details screen, this automation:

•

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:

output.::Flowcell Type:: = input.::Flowcell Type:: ; nextStep = ::ADVANCE::

Step UDFs/Custom Fields

There are five fields defined on the Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.0) step.

These are shown below in the BaseSpace Clarity LIMS v5.x Configuration area (Custom Fields > Master Step Fields tab).

See the following table for LIMS v4.2.x and LIMS v5.x field configuration details:

Field Name	Field Type	Field Constraints/Options	Preset Values/Additional Options and Dropdown Items
Bulk Pool Volume (ul) (for calculation purposes, not displayed)	Numeric	• Required: No • Read Only: No	• Decimal Places Displayed: 2
Number of Lanes to Sequence	Numeric	• Required: Yes • Read Only: No	• Decimal Places Displayed: 0
Total Sample Volume (ul) (for calculation purposes, not displayed)	Numeric	• Required: No • Read Only: No	• Default: 0 • Decimal Places Displayed: 0
Minimum Per Sample Volume (uL)	Numeric	• Required: Yes • Read Only: No	• Default: 5 • Decimal Places Displayed: 2
Number of Samples in Pool (for calculation purposes, not displayed)	Numeric	• Required: No • Read Only: No	• Default: 0 • Decimal Places Displayed: 0

Global UDFs/Custom Fields

The following table lists the global UDFs/custom fields that are configured to display on the Make Bulk Pool for NovaSeq Xp (NovaSeq 6000 v3.0) step:

Field Name	Field Type	Field Constraints/Options	Preset Values/Additional Options and Dropdown Items
Configured on Analyte in LIMS v4.2.x and Derived Samplein LIMS v5.x:
RSB Volume (ul)	Numeric	• Required: No • Read Only: Yes	• Decimal Places Displayed: 2
Flowcell Type	Single-line Text (v4.2.x) Text Dropdown (v5.x)	• Required: Yes • Read Only: No • Custom Entries: No	• SP • S1 • S2 • S4
Loading Workflow Type	Single-line Text (v4.2.x) Text Dropdown (v5.x)	• Required: Yes • Read Only: No • Custom Entries: No	• NovaSeq Standard • NovaSeq Xp • [Remove from workflow]

User Interaction

1.

On the Queue screen, user adds samples of the same Flowcell Type to the Ice Bucket and clicks View Ice Bucket.

2.

On the Ice Bucket screen, user clicks Begin Work.

3.

On the Pooling screen, user:

•

Drags samples over into the Pool Creator to create a pool.

•

Names the pool, or accepts the default name - Pool #1.

•

Clicks Record Details.

On exit from the Pooling screen, the Validate Inputs Flowcell Type and Single Pool is triggered. The automation checks that all samples in the pool have been assigned the same Flowcell Type, and only one pool has been created.

4.

On the Record Details screen, the Step Details area contains two required fields:

•

Number of Lanes to Sequence: The value entered in this field is used in volume calculations, to ensure that volumes are sufficient for the number of times the pool will be sequenced.

•

Minimum Per Sample Volume (ul): In many applications, and particularly where robotics are used, a minimum sample volume is required. The value in this field is used to calculate how much of each sample will be included in the pool.

The field is pre-populated with the configured default value, 5 ul, but may be edited if required.

•

In the Sample Details table, the user can click on the pool icon to view details on the pool composition.

•

The user clicks Calculate Volumes. This triggers the Calculate Volumes automation, which performs volume calculations based on the selected Flowcell Type: Calculates Bulk Pool Volume (ul) value.

5.

On the Assign Next Steps screen:

•

The next step is already set to Dilute, Denature and ExAmp (NovaSeq 6000 v3.0).

•

User clicks Finish Step. At the end of this step, the pool of samples automatically advances to the Dilute, Denature and ExAmp (NovaSeq 6000 v3.0) step.

Step 2: Dilute, Denature and ExAmp (NovaSeq 6000 v3.0)

In this step, pooled samples are denatured and diluted by the addition of DPX, NaOH, Tris-HCl, and RSB. The user manually creates working pools based on the number of lanes they want to sequence.

Step input: Bulk pool

Step output: Working pool - variable number, the user chooses how many working pools to create per bulk pool

The following automations are configured on the step:

•

Calculate Volumes

•

Set Next Step

•

Validate Inputs Flowcell Type

Dilute, Denature and ExAmp (NovaSeq 6000 v3.0) automation configuration - LIMS v4.2.x:

Dilute, Denature and ExAmp (NovaSeq 6000 v3.0) automation configuration - LIMS v5.x:

These automations are described below, in the order in which they are triggered at run time.

Validate Inputs Flowcell Type

Automatically triggered at the beginning of the step, this automation invokes the validate_flowcell_for_input_pools script. This script does the following:

•

Checks the inputs to the step and validates that the Flowcell Type field has been set to a valid value (SP, S1, S2, or S4), and that all inputs have the same value for this field.

•

The container type selected matches the value in the Flowcell Type field.

•

Validates that the number of outputs matches the number of lanes on the selected flow cell type. If validation fails, an error message informs the user that the number of working pools does not match the number of lanes available on the flow cell.

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}"

Calculate Volumes

Automatically triggered on entry to of the Record Details screen, this automation sets the value of the following fields, based on the Flowcell Type:

•

BP Aliquot Volume (ul)

•

NaOH Volume (ul)

•

Tris-HCl Volume (ul)

•

DPX1 Volume (ul)

•

DPX2 Volume (ul)

•

DPX3 Volume (ul)

•

Mastermix per Lane (ul)

'if ( input.::Flowcell Type:: == ::SP:: ) {

  output.::BP Aliquot Volume (ul):: = 18 ;

  output.::NaOH Volume (ul):: = 4.50 ;

  output.::Tris-HCl Volume (ul):: = 4.50 ;

  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):: = 4.50;   

  output.::Tris-HCl Volume (ul):: = 4.50;   

  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):: = 5.50;   

  output.::Tris-HCl Volume (ul):: = 5.50;   

  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):: = 7.50;   

  output.::Tris-HCl Volume (ul):: = 7.50;   

  step.::DPX1 Volume (ul):: = 315;   

  step.::DPX2 Volume (ul):: = 45;   

  step.::DPX3 Volume (ul):: = 165;   

  output.::Mastermix per Lane (ul):: = 105

};

The automation also:

•

Copies the Flowcell Type and Loading Workflow Type values from the step inputs to the step outputs:

output.::Flowcell Type::= input.::Flowcell Type::;

output.::Loading Workflow Type::= input.::Loading Workflow Type::;

•

Uses the NovaSeq_Xp_Working_Pool.csv and NovaSeq_Xp_Working_Pool2.csv template files to generate a single CSV file containing 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 by the user on the Step Setup screen of the following step - Load to Flowcell (NovaSeq 6000 v3.0).

and /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"

Set Next Step

Automatically triggered on exit of the Record Details screen, this automation:

•

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:

output.::Flowcell Type:: = input.::Flowcell Type:: ; nextStep = ::ADVANCE::

Step UDFs/custom fields

There are 3 fields defined on the Dilute, Denature and ExAmp (NovaSeq 6000 v3.0) step. (These field values are set by a script and are not editable by the user running the step.)

The fields are shown below in the BaseSpace Clarity LIMS v5.x Configuration area (Custom Fields > Master Step Fields tab).

See the following table for LIMS v4.2.x and LIMS v5.x field configuration details:

Field Name	Field Type	Field Constraints/Options	Preset Values/Additional Options and Dropdown Items
DPX1 Volume (ul)	Numeric	• Required: No • Read Only: Yes	• Decimal Places Displayed: 0
DPX2 Volume (ul)	Numeric	• Required: No • Read Only: Yes	• Decimal Places Displayed: 0
DPX3 Volume (ul)	Numeric	• Required: No • Read Only: Yes	• Decimal Places Displayed: 0

Global UDFs/Custom Fields

The following table lists the global UDFs/custom fields that are configured to display on the Dilute, Denature and ExAmp (NovaSeq 6000 v3.0) step:

Field Name	Field Type	Field Constraints/Options	Preset Values/Additional Options and Dropdown Items
Configured on Analyte in LIMS v4.2.x and Derived Sample in LIMS v5.x:
BP Aliquot Volume (ul)	Numeric	• Required: No • Read Only: Yes	• Decimal Places Displayed: 0
Flowcell Type	Single-line Text (v4.2.x) Text Dropdown (v5.x)	• Required: Yes • Read Only: No • Custom Entries: No	• SP • S1 • S2 • S4
Loading Workflow Type	Single-line Text (v4.2.x) Text Dropdown (v5.x)	• Required: Yes • Read Only: No • Custom Entries: No	• NovaSeq Standard • NovaSeq Xp • [Remove from workflow]
Mastermix per Lane (ul)	Numeric	• Required: No • Read Only: Yes	• Decimal Places Displayed: 0
NaOH Volume (ul)	Numeric	• Required: No • Read Only: Yes	• Decimal Places Displayed: 2
Tris-HCl Volume (ul)	Numeric	• Required: No • Read Only: Yes	• Decimal Places Displayed: 2

User Interaction

1.

On the Queue screen, user adds the pool to the Ice Bucket and clicks View Ice Bucket.

2.

On the Ice Bucket screen, user (optionally) sets the number of derivatives to create - these will be placed into the flow cell lanes, and clicks 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 (calculation details are provided inCalculate Volumes automation section, above):

•

BP Aliquot Volume (ul)

•

NaOH Volume (ul)

•

Tris-HCl Volume (ul)

•

DPX1 Volume (ul)

•

DPX2 Volume (ul)

•

DPX3 Volume (ul)

•

Mastermix per Lane (ul)

•

Populates the Flowcell Type and Loading Workflow Type columns of the Sample Details table.

•

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-HCI to add per working pool. The user can click on the file to download it and then open it in Excel.

3.

On the Record Details screen: The Reagent Lot Tracking section lets you track the DPX1, DPX2, DPX3, NaOH, RSB, and Tris-HCI reagent lots. The user selects from the active lots displayed in each drop-down list.

•

In the Step Details area, the DPX1, DPX2, and DPX3 reagent volume values are already populated. (These values are set by a script and are not editable by the user running the step.)

•

In the Sample Details table:

•

In the Files area, the user can click on the Calculation File (CSV) file to download it and then open it in Excel to view information about the DPX Mastermix volume, and the volume of Mastermix, NaOH. and Tris-HCI to add per working pool.

•

User clicks Next Steps, which triggers the Set Next Step automation (described above).

4.

On the Assign Next Steps screen, the next step is already set to Load to Flowcell (NovaSeq 6000 v3.0). User clicks Finish Step.

Step 3: Load to Flowcell (NovaSeq 6000 v3.0)

In this step, the user scans the flow cell barcode into the LIMS, and then manually places the working pools into the lanes of the flow cell that will be used in the NovaSeq run. In addition, 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 automations are configured on the step:

•

Validate Run Setup and Generate Sample Sheet

•

Validate Flowcell Barcode

•

Validate Inputs and Selected Container

Load to Flowcell (NovaSeq 6000 v3.0) automation configuration - LIMS v4.2.x:

Load to Flowcell (NovaSeq 6000 v3.0) automation configuration - LIMS v5.x:

These automations are described below, in the order in which they are triggered at run time.

Validate Inputs and Selected Container

Automatically triggered at the beginning of the step, this automation invokes the validate_flowcell_for_input_pools and validateSelectedContainer scripts. These scripts validate the step inputs and the container selected by the user, as follows:

•

Check that the Flowcell Type field has been set to a valid value (SP, S1, S2, or S4), and that all inputs have the same value.

•

Check that the number of outputs matches the number of lanes on the selected flow cell type. If validation fails, an error message informs the user that the number of working pools does not match the number of lanes available on the flow cell.

The following table defines the number of samples allowed:

Flow Cell Type	Number of Working Pools
SP	2
S1	2
S2	2
S4	4

•

Check that the container type selected on entry to the Placement screen matches the value in the Flowcell Type field. If validation fails, displays an error message.

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"

Validate Flowcell Barcode

Automatically triggered on exit of the Placement screen, this automation:

•

Validates the flow cell barcode scanned into the LIMS by the user, using the following logic:

if (input.::Flowcell Type:: == ::SP:: && !output.container.name.matches( ::[A-Za-z0-9]{5}DRXX:: ) ) { \

  fail ( ::Invalid Flowcell Barcode. Please verify and try again.:: ) \

} ; \

if (input.::Flowcell Type:: == ::S1:: && !output.container.name.matches( ::[A-Za-z0-9]{5}DRXX:: ) ) { \

  fail ( ::Invalid Flowcell Barcode. Please verify and try again.:: ) \

} ; \

if (input.::Flowcell Type:: == ::S2:: && !output.container.name.matches( ::[A-Za-z0-9]{5}DMXX:: ) ) { \

  fail ( ::Invalid Flowcell Barcode. Please verify and try again.:: ) \

} ; \

if (input.::Flowcell Type:: == ::S4:: && !output.container.name.matches( ::[A-Za-z0-9]{5}DSXX:: ) ) { \

  fail ( ::Invalid Flowcell Barcode. Please verify and try again.:: ) \

} ;

•

Copies the Flowcell Type and Loading Workflow Type field values from step inputs to outputs:

output.::Flowcell Type:: = input.::Flowcell Type:: ; \

output.::Loading Workflow Type:: = input.::Loading Workflow Type::'

Validate Run Setup and Generate Sample Sheet

Automatically triggered when user clicks a button on the Record Details screen, this automation:

•

Copies the Flowcell Type from the step input to the Run Mode field (hidden):

step.::Run Mode:: = input.::Flowcell Type::

•

Validates the parameters entered on the Record Details screen, which will be used to set up the run and generate the sample sheet file. (See Respond to Validation/Recipe Request Call from Sequencer section, below.)

–

Experiment Name may only contain alphanumeric, dash, or underscore characters. Spaces are not permitted.

–

When Workflow Type = No Index, Index Read 1 must be zero. For any other Workflow Type, Index Read 1 must be greater than zero.

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) { \

    fail(::Index Read 1 must be 0 if the Workflow Type is No Index.::) \

  } \

} else { \

  if(step.::Index Read 1::== 0) { \

    fail(::Index Read 1 must be greater than 0 if the Workflow Type is ::+ step.::Workflow Type::+ ::.::) \

  } \

};

•

Checks the Paired End and Read 2 Cycles field values.

–

If Paired End = False, sets Read 2 Cycles value to 0.

–

If Paired End= True and 2 Cycles value is 0, generates an error.

if (!step.::Paired End::.toBoolean()) {

  step.::Read 2 Cycles:: = 0 \

}; \

if (step.::Paired End::.toBoolean() && step.::Read 2 Cycles:: == 0) \{ \

  fail(::Read 2 Cycles must not be zero if it is Paired End read.::) \

};

•

Checks the Workflow Type and Read 2 Cycles field values.

–

If Workflow Type is set to Dual Index and Index Read 2 value is 0, generates an error.

–

If Workflow Type is set to No Index or Single Index, sets Index Read 2 to 0.

if (step.::Workflow Type:: == ::Dual Index:: && step.::Index Read 2:: == 0) {

  fail(::Index Read 2 must not be zero if Workflow Type is Dual Index.::)

};

if (step.::Workflow Type:: == ::No Index:: || step.::Workflow Type:: == ::Single Index::) {

  step.::Index Read 2:: = 0

};

•

Copies the Flowcell Type value from the step inputs to the step outputs:

output.::Flowcell Type::= input.::Flowcell Type::;

•

Sets the next step for samples to ADVANCE, advancing them to the next step in the protocol - AUTOMATED - Run (NovaSeq 6000) v3.0.

nextStep = ::ADVANCE::

•

Generates the sample sheet and attaches it to the step (see Sample Sheet Generation, below).

&& /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/NovaSeq_BCL2FASTQ_Samplesheet.csv \

  -o {compoundOutputFileLuid0}.csv \

  -q true \

  -destLIMSID {compoundOutputFileLuid0} \

-l {compoundOutputFileLuid1}"

Step UDFs/Custom Fields

There are 17 fields defined on the Load to Flowcell (NovaSeq 6000 v3.0) step.

These are shown below in the BaseSpace Clarity LIMS v5.x Configuration area (Custom Fields > Master Step Fields tab).

See the following table for LIMS v4.2.x and LIMS v5.x field configuration details:

Field Name	Field Type	Field Contraints/Options	Preset Values/Additional Options and Dropdown Items
BaseSpace Sequence Hub Configuration	Check Box (v4.2.x) Toggle Switch (v5.x)	• Required: Np • Read Only: No	• Default: None Set
Use Custom Recipe	Check Box (v4.2.x) Toggle Switch (v5.x)	• Required: Yes • Read Only: No	• Default: No
Workflow	Single-line Text (v4.2.x) Text (v5.x)	• Required: No • Read Only: Yes	• GenerateFASTQ
Workflow Type	Single-line Text (v4.2.x) Text Dropdown (v5.x)	• Required: Yes • Read Only: No • Custom Entries: No	• No Index • Single Index • Dual Index • Custom

Global UDFs/Custom Fields

The following table lists the global UDFs/custom fields that are configured to display on the Load to Flowcell (NovaSeq 6000 v3.0) step.

Field Name	Field Type	Field Constraints/Options	Preset Values/Additional Options and Dropdown Items
Configured on Analyte in LIMS v4.2.x and Derived Sample in LIMS v5.x:
Flowcell Type	Single-line Text (v4.2.x) Text Dropdown (v5.x)	• Required: Yes • Read Only: No • Custom Entries: No	• SP • S1 • S2 • S4
Loading Workflow Type	Single-line Text (v4.2.x) Text Dropdown (v5.x)	• Required: Yes • Read Only: No • Custom Entries: No	• NovaSeq Standard • NovaSeq Xp • [Remove from workflow]

User Interaction

1.

On the Ice Bucket screen, user:

•

Selects the appropriate flow cell type from the Destination Container drop-down list in the Container Options panel.

•

Clicks Begin Work.

2.

On the Placement screen, user:

•

Drags the pool(s) from the left of the screen over into the Placed Samples area on the right.

•

Scans or types the barcode of the flow cell into the flow cell field.

•

Clicks Record Details.

3.

The fields displayed on the Record Details screen are used to set up the run and generate the sample sheet file.

Some of these fields are auto-populated and some must be completed by the user (see the following table for details):

Field	Value
Experiment Name	User enters the experiment name. Only alphanumeric characters, dashes, and underscores are permitted. Spaces are not permitted.
BaseSpace Sequence Hub Configuration	User selects from preset options: Not Used, Run Monitoring Only, Run Monitoring and Storage
Workflow	Automatically populated with preset - GenerateFASTQ
Workflow Type	User selects from preset options: No Index, Single Index, Dual Index, Custom
Index Read 1	User selects from preset options: 0, 6, 8 - or type a value between 0 and 20
Index Read 2	User selects from preset options: 0, 6, 8 - or type a value between 0 and 20
Paired End	User selects from preset options: True or False
Read 1 Cycles	User selects from preset options: 251, 151, 101, 51 - or 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	User selects from preset options: 251, 151, 101, 51 - or 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.
Use Custom Read 1 Primer	User selects if applicable.
Use Custom Read 2 Primer	User selects if applicable.
Use Custom Index Read 1 Primer	User selects if applicable.
Output Folder	User enters the network path for sequencing run folder. For example: \\networkshare\run_data
Use Custom Recipe	User selects if applicable.
Custom Recipe Path	If Use Custom Recipe selected, user enters the path to the custom recipe file to be used.
Library Tube Barcode	User scans the library tube barcode.

4.

On the Record Details screen, user clicks Validate Run Setup and Generate Sample Sheet.

This triggers the automation script, which does the following:

•

Validates the information provided to set up the run.

•

Generates the sample sheet file and attaches it to the placeholder in the Files area of the Record Detailsscreen.

5.

User clicks Next Steps.

6.

On the Assign Next Steps screen, the Next Step field for all samples is pre-populated with Mark protocol as complete. User clicks Finish Step.

7.

In Lab View, the pooled samples are queued for the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.0) step.

At this point in the workflow, the user interaction ends. The integration automatically starts the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.0) step and data from the run is parsed back into the LIMS.

Protocol 4: AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.0)

This final protocol contains one fully-automated step, AUTOMATED- NovaSeq Run (NovaSeq 6000 v3.0), described below.

Step 1: AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.0)

Step input: Library tube from NovaSeq Standard or flow cell from NovaSeq Xp protocol

Step output: Result file/measurement

In this step, pooled samples are sequenced on the NovaSeq instrument and the run metrics are recorded in the LIMS.

The Set Next Steps automation is the only automation configured on this step. By default, this automation is disabled (set to Not Used) and the Sequencer API is used to set the next step for samples.

AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.0) automation configuration - LIMS v4.2.x:

AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.0) automation configuration - LIMS v5.x:

If required, you can override the default next step behavior used by the Sequencer API. To do this:

1.

Enable the automation and use it to set custom next step behavior and generate a log file. By default,  the automation will set the next step to ADVANCE and the samples complete the protocol.

2.

Configure the automation to be triggered automatically on exit of the Record Details screen.

Step UDFs/Custom Fields

The following table shows how the run parameters parsed from the RunParameter.xml file map to step UDFs/custom fields configured on the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.0) step in the LIMS.

Run Parameter	Step UDF/Custom Field Name
currentCycle	Current Cycle
currentRead	Current Read
firmwareVersion	Firmware Version
FlowCellExpirationDate	Flow Cell Expiration Date
FlowCellId	Flow Cell ID
FlowCellLotNumber	Flow Cell Lot Number
FlowCellMode	Flow Cell Mode
FlowCellPartNumber	Flow Cell Part Number
FlowCellSide	Flow Cell Side
controlSoftwareVersion	Instrument Control Software Version
instrumentId	Instrument ID
instrumentType	Instrument Type
WorkflowType	Loading Workflow Type
OutputRunFolder	Output Folder
rtaVersion	RTA Version
RunId	Run ID
runStatus	Run Status
sequencingLog	Sequencing Log

Global UDFs/Custom Fields

The following global UDFs/custom fields are used to capture the run metrics in the LIMS.

•

% Aligned R1

•

% Aligned R2

•

% Bases >=Q30 R1

•

% Bases >=Q30 R2

•

% Error Rate R1

•

% Error Rate R2

•

% Phasing R1

•

% Phasing R2

•

% Prephasing R1

•

% Prephasing R2

•

%PF R1

•

%PF R2

•

Cluster Density (K/mm^2) R1

•

Cluster Density (K/mm^2) R2

•

Intensity Cycle 1 R1

•

Intensity Cycle 1 R2

•

Reads PF (M) R1

•

Reads PF (M) R2

•

Yield PF (Gb) R1

•

Yield PF (Gb) R2

Note the following:

•

Values are aggregated across all lanes. Some values - for example Yield PF (Gb) R1 - are summed, while others are averaged.

•

The names listed above are the default UDF/global field names installed with the NovaSeq Integration v3.2.1 configuration provided in the Preconfigured Workflow Package (PCWP) v1.2 or later.

•

All UDFs/global fields are configured on the Container entity.

•

All field names are configurable via the LIMS Configuration > Custom Fields screen (Global Fields tab).

•

If you change the field names in the LIMS, you must also change them through the run.metricUdfNames properties file in the application.yml file for the Sequencer API (see application.yml Properties). All fields are configured to be visible on the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.0) step's Record Details screen, in the Sample Details table.

How the Sequencer API Integration Works

Sample Sheet Generation

The sample sheet is generated on the step prior to the run – Dilute and Denature (NovaSeq 6000 v3.0) in the NovaSeq Standard protocol or Load to Flowcell (NovaSeq 6000 v3.0) in the NovaSeq Xp protocol. This is the step where samples are placed on the library tube or flow cell that will be loaded in the NovaSeq instrument.

In the out-of-the-box configuration, the Validate Run Setup and Generate Sample Sheet automation generates one CSV format sample sheet file for use with bcl2fastq v2.20 downstream analysis.

How it Works

The sample sheet is uploaded to the NVCS via the /Illumina/Sequencer/v2/sequencing-run/files endpoint to the Sequencer API. The files endpoint allows for a file to be downloaded from the LIMS using OAuth (instead of Basic Authentication), which is required for NovaSeq.

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.

Sample Sheet Contents

The sample sheet content is controlled by the following step UDFs/master step fields that display in the Step Details area of the Record Details screen.

•

Experiment Name

•

Read 1 Cycles

•

Read 2 Cycles

•

Workflow

See the Bcl2fastq2 Sample Sheet Generation article for detailed information on the following:

•

Sample sheet generation script parameters and usage

•

Sample sheet data and configuration options

•

Enabling unique FASTQ file names per sequencing run

NovaSeq Run

The steps below outline the sequence of events that occurs when a flow cell is loaded onto the NovaSeq instrument.

User Authentication and Login

User authentication and login to the NovaSeq instrument is achieved via the getSequencerLoginURL endpoint (found at /Illumina/Sequencer/v2/sequencing-run/login). This endpoint returns the URL that the sequencer uses to get an access token.

1.

The Sequencer API service provides the NovaSeq Control Software (NVCS) with a URL for the LIMS login screen and the NVCS displays this screen to the user. When the user logs in, this automatically authorizes the NVCS to use their account to access the LIMS.

2.

User enters credentials:

•

Username and password are passed to the Clarity LIMS server, to ensure the access is valid.

•

By default, the Sequencer API service tries to contact the LIMS at http://localhost:9080/clarity/. This URL is set by the installation script, but can be configured with the clarity.url property in the application.yml file (see application.yml Properties).

3.

User is redirected to the Sequencer login page at /Illumina/Sequencer/v2/sequencer_login.

•

If authentication is successful, the screen displays a success message. The LIMS server provides the OAuth token and username by returning them in redirect to /Illumina/Sequencer/v2/sequencer_login/Success. This redirect does not reload the page, but allows the NovaSeq to continue with run setup.

•

If authentication fails, the user is left on the login screen and a warning message informs them of the failure. A background redirect to /Illumina/Sequencer/v2/sequencer_login/Error informs the NovaSeq of the failure.

Respond to Validation/Recipe Request Call from Sequencer

1.

The sequencing-run/recipe/novaseq endpoint consumes a POST request with a LibraryContainerI, FlowCellId, and reagents information.

•

LibraryContainerId, FlowCellId, and reagents information are provided by the NovaSeq. LibraryContainerId is required.

•

LibraryContainerId and FlowCellId should match either the library tube ID or flow cell ID of the corresponding container(s) in the LIMS.

•

If there are no containers or multiple containers, an error is generated.

2.

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.

3.

Once a single container has passed validation, the run setup information (run recipe request) is returned to the NVCS via the API and the run recipe is generated.

Note the following:

•

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 (NovaSeq 6000 v3.0) or Load to Flowcell (NovaSeq 6000 v3.0) step, depending on workflow.

•

The default field values match the names of the fields in the NovaSeq v3.2.1 configuration included in PCWP. The field names are configurable through the recipe.udfNames.* properties. Each of these properties corresponds to a entry in the recipe and 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 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 can be configured through the recipe.sampleSheet.outputName property (Sample Sheet by default) in the application.yml (see application.yml Properties). If no matching output is found, or no file was attached to the output, then the value of the recipe.sampleSheet.notAvailableValue property (Not Available by default) is used in the recipe.

4.

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.

Run Recipe Contents

The contents of the run recipe are controlled by the sample sheet contents and the step UDFs/master step fields, configured on the Dilute and Denature (NovaSeq 6000 v3.0) and Load to Flowcell (NovaSeq 6000 v3.0) steps:

Example of run recipe content is as follows:

{

  "run_name": "NVCS16027_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": "<path to sample sheet on LIMS server>",

  "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

}

Respond to Sequencing Started Call from Sequencer

1.

Matching container for the run (library tube or flow cell) is found, and contents are validated to be queued for the correct step.

2.

Reagent kits that are configured for the Sequencer API are checked against the configuration for the step to be run:

•

If a kit is configured for the API but does not exist in 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 any kits enabled on the step are not configured for the Sequencer API, a warning is logged and the step is unable to be completed by the API 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.

3.

The number of replicates for the step is determined by the type of flow cell being used in the run.

•

For Standard mode, this is based on the number of lanes in the flow cell (this is stored as part of the Sequencer API configuration).

•

For XP mode, this is always one replicate, since there is already one input tracked for each lane.

4.

The automated sequencing step is started.

5.

Reagent lots are attached to the step.

•

The Flow Cell and Library Tube reagents in the request are ignored - information from these is tracked in the LIMS as containers and UDFs.

•

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.

•

If any reagents were expected to be in the request but were not, these reagents are logged. This prevents the step from being completed without user intervention.

6.

Step UDFs/master step fields are updated with the run information parsed from the request. See Respond to Record InterOps Metrics Call from Sequencer, below.

7.

Any important events during this request are logged into the Sequencer API log file as well as in the Sequencing Log multiline UDF/field on the step (assuming the step was started successfully). See Respond to Record InterOps Metrics Call from Sequencer, below.

Respond to Record InterOps Metrics Call from Sequencer

1.

When the metrics endpoint receives a POST, it:

•

Looks for a matching library tube or flow cell in the LIMS.

•

Find finds the in-progress sequencing step for that container.

2.

When the step is found, the run metrics in the request are written to container UDFs/global fields in the LIMS.

Respond to Sequencing Completed Call from Sequencer

If the sequencing run was successful, after the metrics have been recorded, the API call for Sequencing Completed is received. Information is matched by Sequencing Container and the following occurs in the LIMS:

1.

The Current Cycle, Current Read, and Status UDFs/master step fields are updated.

2.

The multiline Sequencing Log UDF/master step field is updated.

3.

A success message is recorded in the log file, at the info level:

Sequencing run with Run ID {runId} of Step '{step.name}' ({step.limsid}) completed successfully.

4.

The LIMS waits for any automations that are triggered automatically as it advances the step.

5.

By default, the LIMS assigns the next steps on the outputs to move them on to the first configured next step - or, if there are no next steps, to complete the protocol.

Respond to Sequencing Errored Call from Sequencer

If the sequencing run was unsuccessful, the API call for Sequencing Error is received. Information is matched by Sequencing Container and the following occurs in the LIMS:

1.

The Current Cycle, Current Read, and Status UDFs/master step fields are updated.

2.

The multiline Sequencing Log UDF/master step field is updated.

3.

A failure message is recorded in the log file, at the error level:

Step '${Step.name}' (${Step.limsid}) failed with Run ID ${runId}.

4.

The step is left at the Record Details screen.

Respond to Sequencing RunEndedByUser Call from Sequencer

If the user ends the sequencing run, the API call for Sequencing RunEndedByUser is received. Information is matched by Sequencing Container and the following occurs in the LIMS:

1.

The Current Cycle, Current Read, and Status UDFs/master step fields are updated.

2.

The multiline Sequencing Log UDF/master step field is updated.

3.

The following message is recorded in the log file, at the warning level:

"Sequencing run with Run ID ${status.runInfo.runId} of Step '${step.configuration.name}' (${step.limsid}) aborted by user ${status.runInfo.userName}."

4.

The step is left at the Record Details screen.

Components Installed with this Integration

The following sections describe the various components - files, properties, reagent categories / label groups, reagent kits, and containers - that are installed by default as part of this integration. Information on installed workflows, protocols, steps, and automation points is provided in the Workflows, protocols, and steps section, above.

Illumina NovaSeq Integration v3.2.1 is distributed as an RPM package - BaseSpaceLIMS-sequencer-api. This RPM package must be installed on the LIMS server.

The BaseSpaceLIMS-sequencer-api RPM installs the following:

•

Sequencer API WAR file

•

Configuration file - application.yml

•

Two configuration scripts - configure_sequencer_api_proxy.sh and configure_sequencer_api_application.sh

The RPM package installs the components listed in the following table:

Files Installed	Location and Description
Illumina#Sequencer#v2.war	/opt/gls/clarity/tomcat/current/webapps War file for NovaSeq v3.2.1 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_proxy.sh	/opt/gls/clarity/config/ Script that configures the proxy to allow communication with the Sequencer API (NovaSeq v3.2.1 API) application
sequencer-api.conf	/etc/httpd/clarity/ proxy setting to communicate with the LIMS

application.yml Properties

The application.yml file is located at opt/gls/clarity/extensions/sequencing-api/.

•

All properties are configured automatically during installation, but you can also configure them by editing the application.yml file and then restarting Tomcat.

•

All custom field names, both for recipe and run information, are represented by properties in the file.

•

For all custom fields, the names (values in this file) can be changed. However, the property names cannot be changed, removed, or added to. For example, you can change Loading Workflow Type but you cannot change recipe.udfNames.sampleLoadingType.

The properties it contains are listed in the following table:

Property	Description
spring.profiles.active	Tells the application that it is running deployed in Tomcat. Default value: "tomcat" DO NOT CHANGE
clarity.url	Base URL that the Sequencer API service uses to contact the LIMS. The installation script (configure_sequencer_api_application) prompts for this.
clarity.username	Username to be used when communicating with Clarity LIMS
clarity.password	Encrypted password to be used when communicating with Clarity LIMS
security.signing-key	Private key that should be used when signing/validating OAuth tokens. Changing this will invalidate any issued tokens.
security.token-expiry	Specifies (in hours) the expiry period for login tokens issued by the Sequencer API. Default value: 88
novaseq.sequenceStepNames	List of NovaSeq sequencing run step names for which the integration may find samples queued. • Names must be an exact match to the name of the step in the LIMS (not the master step/process type). • Each line should be indented to the same point and start with a dash (-). Default value: - "AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.0)"
novaseq.flowcells	List of supported NovaSeq flow cell types. • Type names exactly match the values used by the NVCS run status requests. • To add more types, provide both type and lanes properties, indented to the same point and starting with a dash (-). Default value: - type: "SP"lanes: 2- type: "S1"lanes: 2- type: "S2"lanes: 2- type: "S4"lanes: 4
novaseq.reagents	List of supported reagent kits to be tracked on sequencing steps. - lotName: The name of the lot as it appears in the run started request. - kitName: The name of the corresponding reagent kit in LIMS that the lot should be tracked as. • The name may differ in the NVCS request vs. the LIMS configuration. Default value: - lotName: "Buffer"kitName: "Buffer Cartridge"- lotName: "Cluster"kitName: "Cluster Cartridge"- lotName: "SBS"kitName: "SBS Cartridge"
recipe.udfNames	Configures the names of the UDFs/fields that will be used in the run recipe (see Run Recipe Contents for more information). • Each value must be enclosed in quotes. Example: recipe.udfNames.runMode "Run Mode" • Value provided for sampleLoadingType must match the value provided in the run configuration. Example: recipe.udfNames.sampleLoadingType "Loading Workflow Type"
recipe.sampleSheet.outputName recipe.sampleSheet.notAvailableValue	Configures the name of the sample sheet file placeholder on the step where run recipe information is populated, and the property value to use when the file is not found.
run.stepUdfNames	Configures the step UDF/master step field names that will be used when recording run results (see Step UDFs/Custom Fields for more information). • Each value must be enclosed in quotes.  Example: run.stepUdfNames.runID "Run ID" • Value provided for sampleLoadingType must match the value provided in the run configuration. • Example: run.stepUdfNames.sampleLoadingType "Loading Workflow Type". It is expected that the run step field and the field on the input derived sample will share the same name.
run.metricUdfNames	Configures the result file UDFs/measurement global fields names that will be used when recording run results. These are the fields that capture the run data (see Global UDFs/Custom Fields for more information).
run.autoComplete	Determines whether the sequencing run step should be completed automatically after parsing the run metrics on a run (see also run.autoCompleteOnlyAtSuccess). Default value: true See Configuring autoComplete Properties, below.
run.autoCompleteOnlyAtSuccess	Determines whether the sequencing run step should be: • Autocompleted when the run status is RunCompletedSuccessfully (ifautoCompleteOnlyAtSuccess is true) - or - • Autocompleted regardless of the run status (if autoCompleteOnlyAtSuccess is false). Default value: true See Configuring autoComplete Properties, below.

Reagent Categories/Label Groups

•

TruSeq HT Adapters v2 (D7-D5)

Reagent Kits

•

Buffer Cartridge

•

Cluster Cartridge

•

DPX1

•

DPX2

•

DPX3

•

NaOH

•

Resuspension Buffer (RSB)

•

SBS Cartridge

•

Tris HCl

Container Types

•

Library Tube

•

SP

•

S1

•

S2

•

S4

This integration supports:

•

Library tube with barcode provided in the format [A-Za-z]{2}[0-9]{7}-[A-Za-z]{3}

Example: AB1234567-XYZ

•

SP, S1, S2, or S4 flow cell, with barcode provided in one of the following formats:

–

[A-Za-z0-9]{5}DRXX

–

[A-Za-z0-9]{5}DMXX

–

[A-Za-z0-9]{5}DSXX

Example: AB123DRXX

NVCS Configuration

For details on configuring NVCS for integration with BaseSpace Clarity LIMS, contact the Clarity LIMS Support team.

Routing Script Requirements

The requirements for the routing script functionality are as follows:

•

On the steps that use the routing script - Define Run Format (NovaSeq 6000 v3.0) and Dilute and Denature (NovaSeq 6000 v3.0) - the Next Step for all samples must be set to Remove from workflow. This value is set by a script, and the user running the step must not change this value in the Assign Next Steps screen.

•

LIMS v5.x: In the protocol configuration screen, the following settings are required and must not be changed:

In the Next Steps section, for the last step in the protocol, the method of assigning the next step must be set to Automatic.

•

LIMS v4.2.x: In the protocol configuration area, for all protocol steps on which the Routing Script automation is configured, the following setting is required and must not be changed:

On the Next Steps configuration tab, the Next Steps are set by a script toggle switch must be set to Yes.

Configuring autoComplete Properties

As of NovaSeq Integration v3.2.1, the application.yml configuration file contains two additional properties:

•

run.autoComplete

•

run.autoCompleteOnlyAtSuccess

These properties determine the conditions under which the last step of the NovaSeq 6000 workflow - AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.0) - is automatically completed:

•

Only when the run status is "RunCompletedSuccessfully" (default behavior)

-or -

•

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 by the user, the user must manually complete the step in the LIMS.

If you would like the step to autocomplete regardless of the result of the sequencing run, you must change the autoCompleteOnlyAtSuccess property value to 'false'.

Enabling/Disabling autoComplete Properties

1.

Open the application.yml file located at /opt/gls/clarity/extensions/sequencer-api/application.yml:

vi /opt/gls/clarity/extensions/sequencer-api/application.yml

2.

Edit the value of the autoCompleteOnlyAtSuccessproperty as required.

3.

Save the file.

The following table shows how the combined value of the run.autoComplete and run.autoCompleteOnlyAtSuccess properties affects the auto-complete behavior of the sequencing step.

run.autoComplete property value	run.autoCompleteOnlyAtSuccess property value	Outcome
true (default)	true (default)	• Step auto-completes only if sequencing run is successful. • If run is not successful, step does not auto-complete and run details are recorded in the LIMS.
true	false	• Step auto-completes, regardless of the run status.
false	true	• Step does not auto-complete. • Run details are recorded in the LIMS.
false	false

Rules and Constraints

•

The workflow configuration contains several validation checks. To ensure that the calculations work properly, it is important that you do not disable any of this validation logic. The validation checks determine:

–

Which samples, and how many, can enter each step together.

–

Which samples, and how many, can be pooled together.

•

The library tube ID must be unique. There should not be multiple library tube containers in the system with the same name.

•

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 article.

•

Users must not manually start the AUTOMATED - NovaSeq Run (NovaSeq 6000 v3.0) step. This is a fully-automated step and the sequencing service will not update samples correctly if they have been manually started.

•

For the automated run to start successfully, the user must have clicked the Validate Run Setup and Generate Sample Sheet button.

Resources

•

NovaSeq 6000 Sequencing System Guide