Protocol I: TruSight Oncology 500 ctDNA v2 Library Denaturation and Dilution Method (Standard Loading)

The NovaSeq Standard workflow for TruSight Oncology 500 ctDNA v2 libraries is used to denature and dilute libraries intended for loading onto the NovaSeq 6000 Sequencing System. Libraries prepared using the TruSight Oncology 500 ctDNA v2 workflow are normalized to a starting concentration that is ready for sample pooling. For addressable lane loading, refer to the NovaSeq Xp workflow in the NovaSeq 6000 System Guide (document # 1000000019358).

Use this protocol if sequencing TruSight Oncology 500 ctDNA v2 libraries in S2 or S4 mode for standard loading.

Pool and Denature Libraries

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.

Sequence up to eight libraries per S2 flow cell and up to 24 libraries per S4 flow cell.

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

Use fresh tips for each library.

2. Label a 2.0 ml LoBind PCR tube PDL (pooled DNA libraries).
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:

Mode

Recommended Pool Volume (µl)

S2

100

S4

200

For example, for an eight-plex library pool and S2 mode, combine 12.5 µl from each bead-based normalized library.

4. Vortex the PDL tube briefly to mix, and then centrifuge briefly.
1. Incubate the PDL tube in a heat block at 96°C for 2 minutes.
2. Invert two 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

1. Remove RSB from 2°C to 8°C storage, and bring to room temperature.
1. Label a 1.5 ml low DNA binding microcentrifuge tube DIL1 (Dilution 1).
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.

Mode

PDL (µl)

RSB (µl)

Resulting Volume (µl)

S2

80

145

225

S4

165

300

465
4. Vortex the DIL1 tube briefly to mix, and then centrifuge briefly.
5. Transfer the entire volume of the DIL1 tube to the library tube provided with the NovaSeq 6000 reagent kit.
6. After denaturing and diluting the libraries, proceed to Prepare SBS and Cluster Cartridges in the Standard Workflow section of the NovaSeq 6000 System Guide (document # 1000000019358).

The reagent cartridges, including the library tube, must be loaded onto the instrument within 30 minutes.