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

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

Use Protocol C if sequencing TSO 500 ctDNA libraries in S2 or S4 mode. You can sequence up to eight libraries per S2 flow cell and up to 16 libraries per S4 flow cell.

For Xp loading, proceed to Protocol D: TruSight Oncology 500 ctDNA Library Denaturation and Dilution Method (Xp Loading).

Prepare PhiX Control (Optional)

1. Remove RSB from 2°C to 8°C or -25°C to -15°C storage, and bring to room temperature.
2. Thaw a tube of 10 nM PhiX (10 μl/tube).
3. Label a microcentrifuge tube dHP3 (diluted HP3).
4. Label a microcentrifuge tube dTris (diluted Tris-HCl).
5. Label a microcentrifuge tube dPhiX (diluted PhiX).
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.
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.
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.
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.

Pool and Denature Libraries

Visit the TruSight Oncology 500 ctDNA support page on the Illumina website for more information on the supported number of samples per pool per flow cell.

1. If the Normalized Library (NL) plate was stored, bring to room temperature.
2. Centrifuge the plate at 280 × g for 1 minute.
3. Preheat the heat block to 96°C.
4. Prepare an ice bucket.
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 1.5 ml screw‑top microcentrifuge 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 of each library that has been normalized to the same concentration.

4. Vortex the PDL tube to mix.
5. Centrifuge the PDL tube briefly.
1. Incubate PDL tube in a heat block at 96°C for 2 minutes.
2. Immediately place on ice for 5 minutes.
3. Vortex PDL tube to mix, and then centrifuge briefly.
4. Place PDL tube on ice.

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

1. Remove RSB from 2°C to 8°C or -25°C to -15°C storage, and bring to room temperature.
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.
1. Label a new 1.5 ml microcentrifuge tube DIL1 (Dilution 1).
2. Vortex the PDL tube to mix.
3. Centrifuge the PDL tube briefly.
4. Add the appropriate volume of PDL and RSB to the DIL1 tube as follows.

Mode

PDL (µl)

RSB (µl)

Resulting Volume (µl)

S2

65

160

225

S4

134

331

465
5. [Optional] Add the appropriate volume of denatured 2.5 nM PhiX to the DIL1 tube as follows.

Mode

2.5 nM PhiX (µl)

Resulting Volume (µl)

S2

0.9

225.9

S4

1.9

466.9
6. Vortex the DIL1 tube to mix.
7. Centrifuge the DIL1 tube briefly.
8. Transfer the entire volume of DIL1 to the library tube provided with the NovaSeq 6000 reagent kit.
9. Immediately 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.