Step 1: Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.0)

The following automations are configured on the step:

•

Calculate Volumes

•

Set Next Step

Calculate Volumes

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

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} \

&& 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. The next step is Load to Reagent Cartridge (NextSeq 1000/2000 Sequencing v2.0):

nextStep = ::ADVANCE::

There are four fields defined on the Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.0) step.

Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.0) 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	• 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	Not applicable
RSB Volume for Pool (µl)	Numeric	• Required: No • Read Only: Yes	Not applicable

The following table lists the global fields that are configured to display on the Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.0) step.

Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v2.0) 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	• 225 • 400 • Decimal Places Displayed: 0
RSB Volume (µl)	Numeric	• Required: No • Read Only: Yes	• Decimal Places Displayed: 2

Refer to NextSeq 1000/2000 Integration v2.1 Validation and Troubleshooting for the details.