Step 1: Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v1.1)

The following automations are configured on the step:

•

Calculate Volumes

•

Set Next Step

•

Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v1.1) automation configuration

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:

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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)::'

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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

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 v1.1):

nextStep = ::ADVANCE::

There are four fields defined on the Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v1.1) step.

Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v1.1) 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 v1.1) step.

Library Pooling and Dilution (NextSeq 1000/2000 Sequencing v1.1) 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

1.

On the Queue screen, add samples to the Ice Bucket and select View Ice Bucket.

2.

On the Ice Bucket screen, select Begin Work.

3.

On the Pooling screen:

•

Create a pool by dragging samples into the Pool Creator.

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Name the pool or accepts the default name (Pool #1).

•

Select Record Details.

4.

On the Record Details screen, the Reagent Lot Tracking section tracks the Resuspension Buffer (RSB) lot information used in the step. You might need to add and activate lots for RSB.

To add and activate reagent lots:

a.

Navigate to the Consumables configuration screen.

b.

Select the RSB reagent kit and select New Lot.

c.

Enter the lot details and select Save.

d.

Set the Status of Reagent Lot toggle switch to Active.

5.

In the Reagent Lot Tracking section, select from the active lots displayed in the drop-down list.

6.

The Step Details area contains two required fields:

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Final Loading Concentration (pM)—The value entered in this field is the recommended final loading concentration depending on the library type. There is a default drop-down list of 650, 750, 1000, and 2000 (custom value is acceptable). For more information, refer to the NextSeq 1000 and 2000 System Guide.

•

Final Loading Volume (µl)—The value in this field is the final loading volume of the pool into the reagent cartridge. The field is prepopulated with the configured default value, 24 µl, but can be edited if more volume is required.

7.

Select Calculate Volumes. This selection triggers the Calculate Volumes automation. This automation calculates the volume required for each library to form a pool that has the concentration and volume specified in the step details fields.

The automation also generates the Calculation File (CSV) and attaches it to the step. This file contains volume information for each of the samples and the RSB buffer to add to the pool. Select the file to download it, then open it in Excel.

8.

In the Sample Details table, select the pool icon to view details on the pool composition.

Populates the RSB Volume (UL) and LIMS ID (Container) columns of the Sample Details table.

9.

Select Next Steps. This selection 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. The next step in the protocol is Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v1.1).

10.

Select Finish Step.

At the end of this step, the pool of samples automatically advances to Load To Reagent Cartridge (NextSeq 1000/2000 Sequencing v1.1) step.