Pool and Denature Libraries

Sequencing System: NovaSeq X Plus

Library Prep: TruSight Oncology 500 ctDNA v2

Protocol J

Use this protocol to denature and dilute TruSight Oncology 500 ctDNA v2 libraries. Libraries prepared using the TSO 500 ctDNA v2 workflow are normalized to a starting concentration that is ready for sample pooling.

The NovaSeq X Plus 10B Flow Cell has eight lanes and the NovaSeq X Plus 1.5B Flow Cell has two lanes. You can sequence four libraries per 1.5B Flow Cell and up to 24 libraries per 10B Flow Cell.

This protocol requires the following consumables and equipment.

Consumables

Consumables	Supplier	Purpose
Low DNA binding microcentrifuge tube, 1.5 ml	General lab supplier	Combining volumes when pooling libraries.
Low DNA binding microcentrifuge tube, 2.0 ml	General lab supplier	Combining volumes when diluting libraries.
Pipette tips, 20 μl	General lab supplier	Pipetting reagents and libraries.
Pipette tips, 200 μl	General lab supplier	Pipetting reagents and libraries.
Pipette tips, 1000 μl	General lab supplier	Pipetting reagents and libraries.
Pre-load buffer	Illumina, provided in the NovaSeq X Series reagent kit	Diluting libraries to the required loading concentration.
Resuspension Buffer (RSB)	Illumina, provided in the library prep kit contents	Diluting libraries.

Equipment

Supplier

Heat block for 1.5 ml microcentrifuge tubes. The heat block must meet the following performance specifications:

•

Temperature range: Ambient 5°C to 99°C

•

Temperature regulation: ±0.2°C

General lab supplier

Microcentrifuge

General lab supplier

Vortexer

General lab supplier

Preparation

If the normalized library (NL) plate was stored, bring to room temperature.

Centrifuge the plate at 280 × g for 10 seconds.

Preheat the heat block to 96°C.

Prepare an ice bucket.

Pool Normalized Libraries

Set a pipette to 30 μl, and then gently pipette to mix the libraries in the NL plate 5 times.

Use fresh tips for each library.

Label a 1.5 ml low DNA binding microcentrifuge tube PDL (pooled DNA libraries).

Transfer equal volumes of each normalized DNA library from the NL plate to the PDL tube to result in one of the following volumes:

Flow Cell Type	Recommended Pool Volume (µl)
1.5B	70
10B	200

For example, for a 4-plex library pool and 1.5B flow cell, combine 17.5 µl from each bead-based normalized library.

Vortex the PDL tube briefly to mix, and then centrifuge briefly.

Denature Library Pool

Incubate the PDL tube in a heat block at 96°C for 2 minutes.

Invert 2 times to mix.

Centrifuge briefly.

Immediately place on ice for 5 minutes.

Store on ice until use.

Safe Stopping Point

If you are stopping, store denatured libraries at -25°C to -15°C for up to 30 days. To use frozen library pools, thaw the tubes and repeat Denature Library Pool before proceeding to the next step.

Dilute Libraries

Prepare RSB

Remove RSB from 2°C to 8°C storage, and bring to room temperature.

Dilute Libraries

Label a 2.0 ml low DNA binding microcentrifuge tube DIL1 (Dilution 1).

Vortex the PDL tube briefly to mix, and then centrifuge briefly.

Add the appropriate volume of PDL and RSB to the DIL1 tube as follows.

Flow Cell Type	PDL (µl)	RSB (µl)	Resulting Volume (µl)
1.5B	51	76.5	127.5
10B	153	229.5	382.5

Vortex briefly to mix, and then centrifuge briefly.

Add the appropriate volume of Pre-load buffer as follows.

Flow Cell Type	Pre-load Buffer (µl)	Resulting Volume (µl)
1.5B	382.5	510
10B	1147.5	1530

Vortex briefly to mix, and then centrifuge briefly.

Store libraries on ice until transferred to the library tube strip.

After denaturing and diluting the libraries, you are ready to load the lyo insert and library tube strip into the reagent cartridge in preparation for sequencing. Refer to the sequencing system product documentation for more information.

Revision History - Protocol for TruSight Oncology 500 ctDNA v2

Document	Date	Description of Change
Document # 200055975 v00	June 2024	Initial release.