Protocol E: TruSight Oncology 500 HT Library Denaturation and Dilution Method (Standard Loading)

The NovaSeq Standard workflow for TruSight Oncology 500 HT 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 HT workflow are normalized to a starting concentration that is ready for sample pooling.

Use Protocol E if sequencing TSO 500 HT libraries in SP, S1, S2, or S4 mode with Standard loading. You can sequence up to 16 samples per SP flow cell, 32 samples per S1 flow cell, 72 samples per S2 flow cell, and up to 192 samples per 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 Xp loading, proceed to Protocol F: TruSight Oncology 500 HT 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 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, refer to Pool RNA and DNA.
• To sequence libraries derived from DNA samples only, refer to Pool DNA Only.
1. Label a 1.5 ml screw cap microcentrifuge tube PRL (Pooled RNA Libraries).
• If pooling more than 40 RNA (cDNA) libraries, label an additional 1.5 ml screw cap microcentrifuge tube TPRL (Transferred Pooled RNA Libraries).
2. Label a 1.5 ml screw cap microcentrifuge tube PDL (Pooled DNA Libraries).
• If pooling more than 40 DNA libraries, label an additional 1.5 ml screw cap microcentrifuge tube TPDL (Transferred Pooled DNA Libraries).
3. Transfer 5 µl of each normalized RNA library from the NL plate to the PRL tube.
4. Transfer 5 µl of each normalized DNA library from the NL plate to the PDL tube.
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.
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.
9. Proceed to Denature Library Pool.
1. Label a 1.5 ml screw cap microcentrifuge tube PDL (Pooled DNA Libraries).
• If pooling more than 40 DNA libraries, label an additional 1.5 ml screw cap microcentrifuge tube TPDL (Transferred Pooled DNA Libraries).
2. Transfer 5 µl of each normalized DNA library from the NL plate to the PDL tube.
3. Vortex the PDL tube to mix.
4. Centrifuge the PDL 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.
1. Vortex and centrifuge each of the following tubes 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 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. To dilute the libraries, use 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. 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)
2. Transfer the appropriate volume of denatured PRL or TPRL to the DIL1 tube.

Mode

PRL or TPRL (µl)

SP/S1

10.4

S2

15.6

S4

32.2
3. Transfer the appropriate volume of denatured PDL or TPDL to the DIL1 tube.

Mode

PDL or TPDL (µl)

Resulting Volume (µl)

SP/S1

41.6

52

S2

62.4

78

S4

128.8

161
4. Add the appropriate volume of RSB to the DIL1 tube.

Mode

RSB (µl)

Resulting Volume (µl)

SP/S1

98

150

S2

147

225

S4

304

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

Mode

2.5 nM PhiX (µl)

Resulting Volume (µl)

SP/S1

0.6

150.6

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 full 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. 

Use 10 index cycles when sequencing TSO 500 HT libraries.

1. Vortex the tube and centrifuge briefly.
• PDL (≤ 40 DNA libraries)
• TPDL (> 40 DNA libraries)
2. Add the appropriate volume of PDL or TPDL to the DIL1 tube.

Mode

PDL or TPDL (µl)

SP/S1

52

S2

78

S4

161
3. Add the appropriate volume of RSB to the DIL1 tube.

Mode

RSB (µl)

Resulting Volume (µl)

SP/S1

98

150

S2

147

225

S4

304

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

Mode

2.5 nM PhiX (µl)

Resulting Volume (µl)

SP/S1

0.6

150.6

S2

0.9

225.9

S4

1.9

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

Use 10 index cycles when sequencing TSO 500 HT libraries.