Protocol G: TruSight Oncology 500 HRD Library Denaturation and Dilution Method (Standard Loading)

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

Use Protocol G to pool, denature, and dilute libraries prepared using a compatible TruSight Oncology 500 HRD workflow for SP flow cells. Up to 16 DNA (Standard loading or Xp loading) and 16 DNA + 16 RNA libraries (Xp loading) can be sequenced on an SP flow cell.

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

If sequencing DNA libraries only, use Protocol G.

If sequencing DNA and RNA libraries using Xp loading, proceed to Protocol H: TruSight Oncology 500 HRD Library Denaturation and Dilution Method (Xp Loading).

Prepare PhiX Control (Optional)

PhiX control is optional for TruSight Oncology 500 DNA HRD only libraries or combined DNA and RNA libraries.

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 HRD 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 for 1.5 ml microcentrifuge tubes to 96°C.
4. Prepare an ice bucket.
1. Set a pipette to 30 μl, and then gently pipette five times to mix the libraries in the NL plate.

Use fresh tips for each library. Library sequencing performance is diminished if libraries are not sufficiently mixed before pooling.

Pool DNA Only

1. Label a 1.5 ml screw cap microcentrifuge tube PDL (Pooled DNA Libraries).
2. Label a 1.5 ml screw cap microcentrifuge tube PHL (Pooled HRD Libraries).
3. Transfer 5 μl of each normalized TSO 500-enriched DNA library (up to 16 libraries) from the NL plate to the PDL tube.
4. Transfer 5 μl of each normalized HRD-enriched DNA library (up to 16 HRD libraries) from the NL plate to the PHL tube.
5. Vortex each tube to mix.
6. Centrifuge each tube briefly.
7. Proceed to Denature Library Pool.
1. Vortex and centrifuge each PDL and PHL tube briefly.
2. Incubate in a heat block at 96°C for 2 minutes.
3. Immediately place on ice for 5 minutes.
4. Vortex each tube to mix, and then centrifuge briefly.
5. Place 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 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 and PHL tubes to mix.
3. Add 41.6 μl of PDL to the DIL1 tube.
4. Add 10.4 μl of PHL to the DIL1 tube.
5. Add 98 μl of RSB to the DIL1 tube.
6. [Optional] Add 0.6 μl of denatured 2.5 nM PhiX to the DIL1 tube.
7. Vortex the DIL1 tube to mix.
8. Centrifuge the DIL1 tube briefly.
9. Transfer the full volume of DIL1 to the library tube provided with the NovaSeq 6000 reagent kit.
10. 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.