Protocol H: TruSight Oncology 500 HRD Library Denaturation and Dilution Method (Xp Loading)

The NovaSeq Xp workflow for TruSight Oncology 500 HRD 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 HRD workflow are normalized to a starting concentration that is ready for sample pooling. Use Protocol H to sequence TSO 500 HRD libraries in SP mode with Xp loading. You can sequence up to 16 DNA and 16 RNA libraries on an SP flow cell.

Refer to 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.

For Standard loading, use Protocol G: TruSight Oncology 500 HRD 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 dPhiX tube.
9. Vortex to mix, and then centrifuge briefly.

The final concentration of PhiX is 0.25 nM.

10. [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.

2. To pool the normalized libraries, use one of the following options:
• To sequence libraries derived from RNA samples and DNA samples simultaneously, refer to Pool RNA and DNA.
• To sequence libraries derived from DNA samples only, refer to Pool DNA Only.

In the procedure, use the naming convention tables as a guide to labeling tubes. Make sure that the tubes you transfer to have the correct labeling for the corresponding flow cell lane.

1. Label 2 1.5 ml screw cap microcentrifuge tubes for PRL (Pooled RNA Libraries) with the flow cell lane number (PRL_L1, PRL_L2).
2. Label 2 1.5 ml screw cap microcentrifuge tubes for PDL (Pooled DNA Libraries) with the flow cell lane number (PDL_L1, PDL_L2).
3. Label 2 1.5 ml screw cap microcentrifuge tubes for PHL (Pooled HRD Libraries) with the flow cell lane number (PHL_L1, PHL_L2).
4. Pool 8 normalized RNA libraries, 5 µl of each library from the NL plate to a PRL_L1 tube. Repeat with 8 normalized RNA libraries for the second lane to a PRL_L2 tube.
5. Pool 8 normalized TSO 500-enriched DNA libraries, 5 µl of each library from the NL plate to a PDL_L1 tube. Repeat with 8 normalized TSO 500-enriched DNA libraries for the second lane to a PDL_L2 tube.
6. Pool 8 normalized HRD-enriched DNA libraries, 5 µl of each library from the NL plate to a PHL_L1 tube. Repeat with 8 normalized HRD-enriched DNA libraries for the second lane to a PHL_L2 tube.
7. Vortex each tube to mix.
8. Centrifuge each tube briefly.
9. Proceed to Denature Library Pool.
1. Label 2 1.5 ml screw cap microcentrifuge tubes for PDL (Pooled DNA Libraries) with the flow cell lane number (PDL_L1, PDL_L2).
2. Label 2 1.5 ml screw cap microcentrifuge tubes for PHL (Pooled HRD Libraries) with the flow cell lane number (PHL_L1, PHL_L2).
3. Pool 8 normalized TSO 500-enriched DNA libraries, 5 μl of each library from the NL plate to a PDL_L1 tube. Repeat with 8 normalized TSO 500-enriched DNA libraries for the second lane to a PDL_L2 tube.
4. Pool 8 normalized HRD-enriched DNA libraries, 5 μl of each library from the NL plate to a PHL_L1 tube. Repeat with 8 normalized HRD-enriched DNA libraries for the second lane to a PHL_L2 tube.
5. Vortex each tube to mix.
6. Centrifuge each tube briefly.
1. Vortex and centrifuge PRL, PDL, and PHL tubes 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 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. To dilute the libraries, select one of the following options:
• To sequence libraries derived from RNA samples and DNA samples simultaneously, refer to Dilute RNA and DNA Libraries.
• To sequence libraries derived from DNA samples only, refer to Dilute DNA Libraries Only.
1. Label 2 new 1.5 ml screw cap microcentrifuge tubes to combine PRL, PDL, and PHL libraries for each flow cell lane (PRL+PDL+PHL_L1, PRL+PDL+PHL_L2).
2. Vortex and centrifuge each of the following types of tubes briefly:
• PRL (= 8 RNA libraries per lane)
• PDL (= 8 TSO 500 DNA libraries per lane)
• PHL (= 8 HRD DNA libraries per lane)
3. Transfer 5 μl of each PRL tube into the corresponding PRL+PDL+PHL tube for each lane.
4. Transfer 16.25 μl of each PDL tube into the corresponding PRL+PDL+PHL tube for each lane.
5. Transfer 3.75 μl of each PHL tube into the corresponding PRL+PDL+PHL tube for each lane.
6. Vortex the PRL+PDL+PHL tubes to mix.
7. Centrifuge the PRL+PDL+PHL tubes briefly.
8. Label 2 new 1.5 ml screw cap microcentrifuge tubes to dilute the combined PRL+PDL+PHL libraries for each lane (DIL1_L1, DIL1_L2).
9. Transfer 4 μl of each PRL + PDL + PHL tube to each of the corresponding DIL1 tubes.
10. Add 23ul of RSB to each of the corresponding DIL1 tubes.
11. [Optional] Add 0.7ul of denatured 0.25 nM PhiX to each of the corresponding DIL1 tubes.
12. Vortex the DIL1 tubes to mix.
13. Centrifuge the DIL1 tubes briefly.
14. After denaturing and diluting 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).
1. Label 2 new 1.5 ml screw cap microcentrifuge tubes to combine PDL and PHL libraries for each flow cell lane (PDL+PHL_L1, PDL+PHL_L2).
2. Vortex and centrifuge each of the following types of tubes briefly:
• PDL (= 8 TSO 500 DNA libraries per lane)
• PHL (= 8 HRD DNA libraries per lane)
3. Transfer 20 μl of each PDL tube into the corresponding PDL+PHL tube for each lane.
4. Transfer 5 μl of each PHL tube into the corresponding PDL+PHL tube for each lane.
5. Vortex the PDL+PHL tubes to mix.
6. Centrifuge the PDL+PHL tubes briefly.
7. Label 2 new 1.5 ml screw cap microcentrifuge tubes to dilute the combined PDL+PHL libraries for each lane (DIL1_L1, DIL1_L2).
8. Vortex and centrifuge the PDL+PHL tubes briefly.
9. Transfer 4 μl of each PDL+PHL tube to each of the corresponding DIL1 tubes.
10. Add 23ul of RSB to each of the corresponding DIL1 tubes.
11. [Optional] Add 0.7ul of denatured 0.25 nM PhiX to each of the corresponding DIL1 tubes.
12. Vortex the DIL1 tubes to mix.
13. Centrifuge the DIL1 tubes briefly.
14. 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).