Protocol D: TruSight Oncology 500 ctDNA Library Denaturation and Dilution Method (Xp Loading)

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

Use Protocol D if sequencing TSO 500 ctDNA libraries in S4 mode for addressable lane loading. You can sequence up to six libraries per lane.

For Standard loading, proceed to Protocol C: TruSight Oncology 500 ctDNA Library Denaturation and Dilution Method (Standard 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.
8. Add 216 μl RSB to the diluted and denatured PhiX solution.
9. Vortex to mix, and then centrifuge briefly.

The final concentration of PhiX is 0.25 nM.

10. [Optional] Store denatured 0.25 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 new 1.5 ml screw-top microcentrifuge as follows.
PDL Tube Naming Convention

Flow Cell

Lane 1

Lane 2

Lane 3

Lane 4

S4

PDL_L1

PDL_L2

PDL_L3

PDL_L4
3. Transfer 5 μl of each normalized DNA library from the NL plate to the PDL tube, and then repeat for each additional lane.
4. Vortex each PDL tube to mix.
5. Centrifuge each PDL tube briefly.
1. Incubate each PDL tube in a heat block at 96°C for 2 minutes.
2. Immediately place on ice for 5 minutes.
3. Vortex each PDL tube to mix, and then centrifuge briefly.
4. Place PDL tubes 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 0.25 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 screw-top microcentrifuge as follows.
DIL1 Tube Naming Convention

Flow Cell

Lane 1

Lane 2

Lane 3

Lane 4

S4

DIL1_L1

DIL1_L2

DIL1_L3

DIL1_L4
2. Vortex the PDL tubes to mix.
3. Centrifuge the PDL tubes briefly.
4. Transfer the appropriate volume of PDL and RSB to each DIL1 tube as follows.

Mode

PDL (µl)

RSB (µl)

Resulting Volume (µl)

S4

6.8

38.2

45
5. [Optional] Add the appropriate volume of denatured 0.25 nM PhiX to each DIL1 tube as follows.

Mode

0.25 nM PhiX (µl)

Resulting Volume (µl)

S4

1.1

46.1
6. Vortex the DIL1 tubes to mix.
7. Centrifuge the DIL1 tubes briefly.
8. After denaturing and diluting the libraries and preparing the optional PhiX control, proceed to Prepare the Flow Cell and Dock in the Xp Workflow section of the NovaSeq 6000 System Guide (document # 1000000019358).