Protocol F: TruSight Oncology 500 HT Library Denaturation and Dilution Method (Xp Loading)

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

Use Protocol F if sequencing TSO 500 HT libraries in SP, S1, S2, or S4 mode with Xp loading. You can sequence up to eight samples per lane on an SP flow cell, 16 samples per lane on an S1 flow cell, 36 samples per lane on an S2 flow cell, and 48 samples per lane on an S4 flow cell.

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

For Standard loading, proceed to Protocol E: TruSight Oncology 500 HT 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 0.25 nM PhiX at -25°C to -15°C for up to 2 weeks.

Pool Normalized Libraries

Visit the TruSight Oncology 500 HT 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 five times to mix the libraries in the NL plate.
2. To pool the normalized libraries, use one of the following options:
• To sequence libraries derived from RNA samples and DNA samples simultaneously, see Pool RNA and DNA.
• To sequence libraries derived from DNA samples only, see 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 a 1.5 ml screw cap microcentrifuge tube PRL with the flow cell lane number. Repeat for any additional lanes. Use as a guide.
• If pooling more than 40 RNA (cDNA) libraries, label an additional 1.5 ml screw cap microcentrifuge tube TPRL with the flow cell lane number. Repeat for any additional lanes.
Naming Convention for RNA Tubes

Flow Cell

Lane 1

Lane 2

Lane 3

Lane 4

SP/S1

PRL_L1

PRL_L2

N/A

N/A

S2

PRL_L1

PRL_L2

N/A

N/A

S4

PRL_L1

PRL_L2

PRL_L3

PRL_L4
2. Label a 1.5 ml screw cap microcentrifuge tube PDL with the flow cell lane number. Repeat for any additional lanes. Use as a guide.
• If pooling more than 40 DNA libraries, label an additional 1.5 ml screw cap microcentrifuge tube TPDL with the flow cell lane number. Repeat for any additional lanes.
Naming Convention for DNA Tubes

Flow Cell

Lane 1

Lane 2

Lane 3

Lane 4

SP/S1

PDL_L1

PDL_L2

N/A

N/A

S2

PDL_L1

PDL_L2

N/A

N/A

S4

PDL_L1

PDL_L2

PDL_L3

PDL_L4
3. Transfer 5 µl of each normalized RNA library from the NL plate to the PRL tube. Repeat for any additional lanes.
4. Transfer 5 µl of each normalized DNA library from the NL plate to the PDL tube. Repeat for any additional lanes.
5. Vortex each tube to mix.
6. Centrifuge each tube briefly.
7. If the PRL tube contains more than 40 RNA libraries, transfer 200 µl from the PRL tube to the TPRL tube, and then discard the PRL tube. Repeat for any additional lanes.
8. If the PDL tube contains more than 40 DNA libraries, transfer 200 µl from the PDL tube to the TPDL tube, and then discard the PDL tube. Repeat for any additional lanes.
9. Proceed to Denature Library Pool.
1. Label a 1.5 ml screw cap microcentrifuge tube PDL with the flow cell lane number. Repeat for any additional lanes.

Use the following table as a guide.

Naming Convention for DNA Tubes

Flow Cell

Lane 1

Lane 2

Lane 3

Lane 4

SP/S1

PDL_L1

PDL_L2

N/A

N/A

S2

PDL_L1

PDL_L2

N/A

N/A

S4

PDL_L1

PDL_L2

PDL_L3

PDL_L4
• If pooling more than 40 DNA libraries, label an additional 1.5 ml screw‑top microcentrifuge tube TPRL (Transferred Pooled DNA Libraries) with the flow cell lane number. Repeat for any additional lanes.
2. Transfer 5 µl of each normalized DNA library from the NL plate to the PDL tube. Repeat for any additional lanes.
3. Vortex each tube to mix.
4. Centrifuge each tube briefly.
5. If the PDL tube contains more than 40 DNA libraries, transfer 200 µl from the PDL tube to the TPDL tube, and then discard the PDL tube. Repeat for any additional lanes.
1. Vortex and centrifuge each of the following briefly:
• PRL (≤ 40 RNA libraries) or TPRL (> 40 RNA libraries)
• PDL (≤ 40 DNA libraries) or TPDL (> 40 DNA libraries)
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, use one of the following options:
• To sequence libraries derived from RNA samples and DNA samples simultaneously, see Dilute RNA and DNA Libraries.
• To sequence libraries derived from DNA samples only, see Dilute DNA Libraries Only.
1. Label a new 1.5 ml screw cap microcentrifuge tube to combine PRL and PDL libraries. Repeat for any additional lanes. Use the following table as a guide.
Naming Convention for combined PRL and PDL Tubes

Flow Cell

Lane 1

Lane 2

Lane 3

Lane 4

SP/S1

PRL+PDL_L1

PRL+PDL_L2

N/A

N/A

S2

PRL+PDL_L1

PRL+PDL_L2

N/A

N/A

S4

PRL+PDL_L1

PRL+PDL_L2

PRL+PDL_L3

PRL+PDL_L4
2. Vortex and centrifuge each of the following types of tubes briefly:
• PRL (≤ 40 RNA libraries) or TPRL (> 40 RNA libraries)
• PDL (≤ 40 DNA libraries) or TPDL (> 40 DNA libraries)
3. Transfer 5 µl of each PRL or TPRL tube into the corresponding PRL+PDL tube.
4. Transfer 20 µl of each PDL or TPDL tube into the corresponding PRL+PDL tube.
5. Vortex the PRL+PDL tubes to mix.
6. Centrifuge the PRL+PDL tubes briefly.
7. Label a new 1.5 ml screw cap microcentrifuge tube to dilute the combined PRL+PDL libraries. Repeat for any additional lanes. Use the following table as a guide.
Naming Convention for DIL 1 Tubes

Flow Cell

Lane 1

Lane 2

Lane 3

Lane 4

SP/S1

DIL1_L1

DIL1_L2

N/A

N/A

S2

DIL1_L1

DIL1_L2

N/A

N/A

S4

DIL1_L1

DIL1_L2

DIL1_L3

DIL1_L4
8. Transfer the appropriate volume of combined PRL and PDL to each of the corresponding DIL1 tubes.

Flow Cell

PRL+PDL (µl)

SP/S1

4

S2

5

S4

6.8
9. Add the appropriate volume of RSB to each of the corresponding DIL1 tubes.

Flow Cell

RSB (µl)

Resulting Volume (µl)

SP/S1

23

27

S2

28

33

S4

38.2

45
10. [Optional] Add the appropriate volume of denatured 0.25 nM PhiX to each of the corresponding DIL1 tubes.

Flow Cell

0.25 nM PhiX (µl)

Resulting Volume (µl)

SP/S1

0.7

27.7

S2

0.8

33.8

S4

1.1

46.1
11. Vortex the DIL1 tubes to mix.
12. Centrifuge the DIL1 tubes briefly.
13. 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).

Use 10 index cycles when sequencing TSO 500 HT libraries.

1. Label a new 1.5 ml screw cap microcentrifuge tube to dilute the PDL libraries. Repeat for any additional lanes. Use the following table as a guide.
Naming convention for tubes

Flow Cell

Lane 1

Lane 2

Lane 3

Lane 4

SP/S1

DIL1_L1

DIL1_L2

N/A

N/A

S2

DIL1_L1

DIL1_L2

N/A

N/A

S4

DIL1_L1

DIL1_L2

DIL1_L3

DIL1_L4
2. Vortex and centrifuge the PDL or TPDL tubes briefly.
3. Transfer the appropriate volume of PDL or TPDL to each of the corresponding DIL1 tubes.

Flow Cell

PDL or TPDL (µl)

SP/S1

4

S2

5

S4

6.8
4. Add the appropriate volume of RSB to each of the corresponding DIL1 tubes.

Flow Cell

RSB (µl)

Resulting Volume (µl)

SP/S1

23

27

S2

28

33

S4

38.2

45
5. [Optional] Add the appropriate volume of denatured 0.25 nM PhiX to each of the corresponding DIL1 tubes.

Flow Cell

0.25 nM PhiX (µl)

Resulting Volume (µl)

SP/S1

0.7

27.7

S2

0.8

33.8

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).

Use 10 index cycles when sequencing TSO 500 HT libraries.