Pool and Denature Libraries

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. An optional PhiX spike-in provides additional metrics, base diversity, or a positive control.

Instructions for the 1.5B flow cell, the 10B flow cell, and the 25B flow cell are provided. Refer to Flow Cell Loading Options.

Preparation

1.

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

2.

Centrifuge the plate at 280 × g for 10 seconds.

3.

Preheat the heat block to 96°C.

4.

Prepare an ice bucket.

[Optional] Prepare PhiX Control

Preparation

1.

Remove RSB from 2°C to 8°C or -25°C to -15°C storage, and bring to room temperature.

2.

Remove HP3 (2 N NaOH) from storage, and bring to room temperature.

3.

Thaw a tube of 10 nM PhiX (10 μl/tube).

4.

Label a microcentrifuge tube dHP3 (diluted HP3).

5.

Label a microcentrifuge tube dTris (diluted Tris-HCl).

6.

Label a microcentrifuge tube dPhiX (diluted PhiX).

Prepare a Fresh Dilution of NaOH

1.

Vortex HP3 to mix, and then centrifuge briefly.

2.

Combine the following volumes in the dHP3 tube.

•

RNase/DNase-free water (32.5 μl)

•

HP3 (7.5 μl)

3.

Vortex dHP3 to mix, and then centrifuge briefly.

Prepare a Fresh Dilution of Tris-HCl

1.

Combine the following volumes in the dTris tube.

•

RNase/DNase-free water (25.0 μl)

•

1 M Tris-HCl, pH 8.0 (15.0 μl)

2.

Vortex dTris to mix, and then centrifuge briefly.

Dilute PhiX

1.

Vortex RSB to mix.

2.

Vortex PhiX control to mix, and then centrifuge briefly.

3.

Combine the following volumes in the dPhiX tube.

•

RSB (2.0 μl)

•

PhiX control (6.0 μl)

4.

Vortex dPhiX tube to mix, and then centrifuge briefly.

5.

[Optional] Store dPhiX at -25°C to -15°C for up to 3 months.

Denature PhiX

1.

Add 8 μl dHP3 to the dPhiX tube.

2.

Discard the dHP3 tube.

3.

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

4.

Incubate at room temperature for 5 minutes.

5.

Immediately add 8 μl dTris to the dPhiX tube to neutralize the reaction.

6.

Discard the dTris tube.

7.

Vortex to mix, and then centrifuge briefly.

The final concentration of PhiX is 2.5 nM.

8.

[Optional] Store denatured 2.5 nM PhiX at -25°C to -15°C for up to 2 weeks.

Flow Cell Loading Options

Refer to the following table for the appropriate volumes for your flow cell type and plexity.

Flow Cell Type	Sample Per Flow Cell	Standard Load	Lane Splitting
1.5B	4	4-plex pool over 2 lanes	N/A
10B	24	24-plex pool over 8 lanes	N/A
25B	64	N/A	Eight, 8-plex pools, loaded onto 8 lanes

Pool Normalized Libraries

1.

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.

2.

[1.5B and 10B] Label a 1.5 ml low DNA binding microcentrifuge tube PDL (pooled DNA libraries).

[25B] Label eight 1.5 ml low DNA binding microcentrifuge tubes PDL L1 through PDL L8 (Pooled DNA Libraries, for loading onto lanes one through eight).

3.

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

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

4.

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

Denature Library Pool

1.

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

2.

Invert 2 times to mix.

3.

Centrifuge briefly.

4.

Immediately place on ice for 5 minutes.

5.

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 and Add Optional PhiX Control

Prepare RSB

1.

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

[Optional] Prepare Denatured 2.5 nM PhiX

1.

If the denatured PhiX was stored, remove the denatured 2.5 nM PhiX from -25°C to -15°C storage, and bring to room temperature.

2.

Vortex to mix, and then centrifuge briefly.

Dilute Libraries

1.

[1.5B and 10B] Label a 2.0 ml low DNA binding microcentrifuge tube DIL1 (Dilution 1).

[25B] Label eight 2.0 ml low DNA binding microcentrifuge tubes DIL1 L1 through DIL1 L8 (Dilution 1, for loading libraries onto lanes one through eight).

2.

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

3.

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

4.

Vortex briefly to mix, and then centrifuge briefly.

5.

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

6.

Vortex briefly to mix, and then centrifuge briefly.

7.

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 v01	October 2025	Added 25B flow cell and optional PhiX control information.
Document # 200055975 v00	June 2024	Initial release.