Step 1: Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3)

In this step, the addition of RSB dilutes pooled samples. Manually create a working pool based on the final loading concentration required.

Only create one pool per step for the Calculate Volume automation script to work properly.

This automation registers the start time of the step by publishing messages to UPA through the LINK service. This automation is automatically triggered on the step. It is only used for UPA support.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -s 'com/illumina/upa/scripts/common/step_started.groovy'

Select the Calculate Volumes button on the Record Details screen to trigger this automation automatically, which does the following actions:

• Sets RSB Volume for Pool (µl) field value to 24 for calculation purpose of the 2 nM intermediate library pool.
• Copies the Final Loading Concentration (pM) from step inputs to step outputs.

-exp 'step.::RSB Volume for Pool (µl):: = 24; output.::Final Loading Concentration (pM):: = step.::Final Loading Concentration (pM)::'

• Calculate per sample volume required for each library to make the 2 nM intermediate library pool (24ul in volume).

-exp 'input.::Per Sample Volume (µl):: = ( 48/ input.::Molarity (nM):: ); step.::RSB Volume for Pool (µl):: -= input.::Per Sample Volume (µl)::'

• Calculate the volume required of the 2 nM library pool to be diluted further to the Final Loading Concentration (pM) with the Final Loading Volume (µl).

-exp 'step.::Library Pool Volume (µl):: = (step.::Final Loading Concentration (pM):: * step.::Final Loading Volume (µl)::/2000); output.::RSB Volume (µl):: = step.::Final Loading Volume (µl):: - step.::Library Pool Volume (µl)::'

• Uses the NextSeq1K2K_Pool1.csv, NextSeq1K2K_Pool2.csv, and NextSeq1K2K_Pool3.csv template files to generate a single CSV file containing information about the pool and the samples it contains. The generated file is stored in the Calculation File placeholder, in the Files section, for download.

/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/NextSeq1K2K_Pool1.csv \

-o 1.csv \

script:driver_file_generator \

-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NextSeq1K2K_Pool2.csv \

-o 2.csv \

script:driver_file_generator \

-t /opt/gls/clarity/extensions/conf/driverfiletemplates/NextSeq1K2K_Pool3.csv \

-o 3.csv \

&& cat 1.csv 2.csv 3.csv > {compoundOutputFileLuid0}.csv

Automatically triggered on exit of the Record Details screen, this automation sets the next step for samples to ADVANCE, advancing them to the next step in the protocol. The next step is Load to Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.3):

nextStep = ::ADVANCE::

Register Pools & Register Step Completed

• This automation is automatically triggered on exit of the step and performs the following tasks:
• Registers the pool and library information used by the Clarity LIMS workflow. Samples in the pool are assumed to have gone through library preparation.
• Registers the completion time of the step by publishing messages to UPA through the LINK service.

This automation is only used for UPA support.

/opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -s 'com/illumina/upa/scripts/common/pooling.groovy' && /opt/gls/clarity/bin/java -jar /opt/gls/clarity/extensions/unified-product-analytics/automation/unified-product-analytics-automation.jar script:executeUPAAutomationScript -i {stepURI:v2} -u {username} -p {password} -l {compoundOutputFileLuid1} -s 'com/illumina/upa/scripts/common/step_completed.groovy'

There are four fields defined on the Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) step.

Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) Master Step Field Configuration

Field Name

Field Type

Field Constraints/Options

Preset Values/Additional Options and Drop-down Items

Final Loading Concentration (pM)

Numeric Dropdown

Required: Yes

Read Only: No

Custom Entries: Yes

Presets

• 650
• 750
• 1000
• 2000

Final Loading Volume (µl)

Numeric

Required: Yes

Read Only: No

Default

• 24

Library Pool Volume (µl)

Numeric

Required: No

Read Only: Yes

Hidden

This field is used by the Calculate Volumes automation. Removing, renaming, or modifying the field will break the automation.

 

RSB Volume for Pool (µl)

Numeric

Required: No

Read Only: Yes

Hidden

This field is used by the Calculate Volumes automation. Removing, renaming, or modifying the field will break the automation.

 

The following table lists the global fields that are configured to display on the Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) step.

Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.3) Global Field Configuration

Field Name

Field Type

Field Constraints/Options

Preset Values/Additional Options and Drop-down Items

Final Loading Concentration (pM)

Numeric Dropdown

Required: Yes

Read Only: No

Custom Entries: Yes

Decimal Places Displayed: 0

Presets

• 225
• 400

RSB Volume (µl)

Numeric

Required: No

Read Only: Yes

Decimal Places Displayed: 2

Refer to NextSeq 1000/2000 Integration v2.3 Validation and Troubleshooting for details.