Pool and Denature Libraries

Sequencing System: NovaSeq X Plus, TSO 500 HT

Protocol I

Use this protocol to denature and dilute libraries that have been normalized using standard library quantification and quality control procedures recommended in the library prep documentation.

This protocol requires the following consumables and equipment.

Consumables

Consumables

Supplier

Purpose

1 N NaOH

General lab supplier

Diluting to 0.2 N for denaturing libraries.

Microcentrifuge tube, 1.5 ml

VWR, catalog # 20170-038, or equivalent

Combining volumes when diluting NaOH and library.

Pipette tips, 20 μl

General lab supplier

Pipetting reagents and libraries.

Pipette tips, 200 μl

General lab supplier

Pipetting reagents and libraries.

Pipette tips, 1000 μl

General lab supplier

Pipetting reagents and libraries.

Pre-load buffer

Illumina, provided in the NovaSeq X Plus Kit

Neutralizing denatured libraries.

Resuspension Buffer (RSB)

Illumina, provided in the library prep kit contents

Diluting PhiX Control.

Water, laboratory-grade

General lab supplier

Diluting NaOH for denaturing libraries.

[Optional] PhiX Control v3

Illumina, catalog # FC-110-3001

Spiking in PhiX control.

Prepare NaOH

Prepare a fresh dilution of 0.2 N NaOH to denature libraries for sequencing. Extra volume is prepared to prevent small pipetting errors from affecting the final NaOH concentration.

1. [25B] Combine the following volumes in a microcentrifuge tube.

Reagent

Volume for One 25B Flow Cell (µl)

Volume for Two 25B Flow Cells (µl)

Laboratory-grade water

120

240

Stock 1 N NaOH

30

60

These volumes result in 150 µl 0.2 N NaOH for one flow cell or 300 µl 0.2 N NaOH for two flow cells.

2. [10B] Combine the following volumes in a microcentrifuge tube.

Reagent

Volume for One 10B Flow Cell (µl)

Volume for Two 10B Flow Cells (µl)

Laboratory-grade water

80

160

Stock 1 N NaOH

20

40

These volumes result in 100 µl 0.2 N NaOH for one flow cell or 200 µl 0.2 N NaOH for two flow cells.

3. [1.5B] Combine the following volumes in a microcentrifuge tube.

Reagent

Volume for One 1.5B Flow Cell (µl)

Volume for Two 1.5B Flow Cells (µl)

Laboratory-grade water

20

40

Stock 1 N NaOH

5

10

These volumes result in 25 µl 0.2 N NaOH for one flow cell or 50 µl 0.2 N NaOH for two flow cells.

4. Vortex, and then centrifuge the tube to mix.

Pool Normalized Libraries

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

Preparation

1. If the Normalized Library (NL) plate was stored, bring to room temperature.
2. Centrifuge the plate at 280 × g for 1 minute.

Procedure

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.

Pool RNA and DNA

1. Label a 1.5 ml screw cap microcentrifuge tube PRL (Pooled RNA Libraries) with the flow cell lane number. Repeat for any additional lanes as follows.

Flow Cell Type

Lane 1

Lane 2

Lane 3

Lane 4

Lane 5

Lane 6

Lane 7

Lane 8

25B

PRL_L1

PRL_L2

PRL_L3

PRL_L4

PRL_L5

PRL_L6

PRL_L7

PRL_L8

10B

PRL_L1

PRL_L2

PRL_L3

PRL_L4

PRL_L5

PRL_L6

PRL_L7

PRL_L8

1.5B

PRL_L1

PRL_L2

N/A

N/A

N/A

N/A

N/A

N/A
2. Label a 1.5 ml screw cap microcentrifuge tube PDL (Pooled DNA Libraries) with the flow cell lane number. Repeat for any additional lanes as follows.

Flow Cell Type

Lane 1

Lane 2

Lane 3

Lane 4

Lane 5

Lane 6

Lane 7

Lane 8

25B

PDL_L1

PDL_L2

PDL_L3

PDL_L4

PDL_L5

PDL_L6

PDL_L7

PDL_L8

10B

PDL_L1

PDL_L2

PDL_L3

PDL_L4

PDL_L5

PDL_L6

PDL_L7

PDL_L8

1.5B

PDL_L1

PDL_L2

N/A

N/A

N/A

N/A

N/A

N/A
3. Label a 1.5 ml screw cap microcentrifuge tube PRL+PDL with the flow cell lane number. Repeat for any additional lanes as follows.

Flow Cell Type

Lane 1

Lane 2

Lane 3

Lane 4

Lane 5

Lane 6

Lane 7

Lane 8

25B

PRL+PDL_L1

PRL+PDL_L2

PRL+PDL_L3

PRL+PDL_L4

PRL+PDL_L5

PRL+PDL_L6

PRL+PDL_L7

PRL+PDL_L8

10B

PRL+PDL_L1

PRL+PDL_L2

PRL+PDL_L3

PRL+PDL_L4

PRL+PDL_L5

PRL+PDL_L6

PRL+PDL_L7

PRL+PDL_L8

1.5B

PRL+PDL_L1

PRL+PDL_L2

N/A

N/A

N/A

N/A

N/A

N/A
4. Set a pipette to 30 μl, and then gently pipette five times to mix the libraries in the NL plate.
5. Transfer 5 µl of each normalized RNA library from the NL plate to the PRL tube. Repeat for any additional lanes.
6. Transfer 5 µl of each normalized DNA library from the NL plate to the PDL tube. Repeat for any additional lanes.
7. Vortex each tube to mix.
8. Centrifuge each tube briefly.
9. Transfer the appropriate volume of PRL and PDL to each of the corresponding PRL+PDL tubes.

Flow Cell Type

PRL (µl)

PDL (µl)

Resulting Volume (µl)

25B

11.3

44.7

56

10B or 1.5B

6.75

27

34

Pool DNA Only

1. Label a 1.5 ml screw cap microcentrifuge tube PDL (Pooled DNA Libraries) with the flow cell lane number. Repeat for any additional lanes as follows.

Flow Cell Type

Lane 1

Lane 2

Lane 3

Lane 4

Lane 5

Lane 6

Lane 7

Lane 8

25B

PDL_L1

PDL_L2

PDL_L3

PDL_L4

PDL_L5

PDL_L6

PDL_L7

PDL_L8

10B

PDL_L1

PDL_L2

PDL_L3

PDL_L4

PDL_L5

PDL_L6

PDL_L7

PDL_L8

1.5B

PDL_L1

PDL_L2

N/A

N/A

N/A

N/A

N/A

N/A
2. Label a 1.5 ml screw cap microcentrifuge tube TPDL (Transferred Pooled DNA Libraries) with the flow cell lane number. Repeat for any additional lanes as follows.

Flow Cell Type

Lane 1

Lane 2

Lane 3

Lane 4

Lane 5

Lane 6

Lane 7

Lane 8

25B

TPDL_L1

TPDL_L2

TPDL_L3

TPDL_L4

TPDL_L5

TPDL_L6

TPDL_L7

TPDL_L8

10B

TPDL_L1

TPDL_L2

TPDL_L3

TPDL_L4

TPDL_L5

TPDL_L6

TPDL_L7

TPDL_L8

1.5B

TPDL_L1

TPDL_L2

N/A

N/A

N/A

N/A

N/A

N/A
3. Set a pipette to 30 μl, and then gently pipette five times to mix the libraries in the NL plate.
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. Transfer the appropriate volume of PDL to each of the corresponding TPDL tubes.

Flow Cell Type

Resulting Volume (µl)

25B

56

10B or 1.5B

34

Add PhiX Control (Optional)

1. Spike in 1–2% nondenatured PhiX as follows.

When spiking in PhiX, 1% is the recommended amount for balanced libraries. Low-diversity libraries can require more. To use a PhiX control with low-diversity libraries, contact Illumina Technical Support for guidance.

a. Quantify PhiX.
b. Dilute 10 nM PhiX to 300 pM using RSB.
c. Add the appropriate volume of diluted PhiX to the pooled libraries.

Flow Cell Type

PhiX (µl)

Pooled Libraries (µl)

25B

1.6

56

10B or 1.5B

1

34

Denature Library Pool

1. Add the appropriate volume of 0.2 N NaOH to the pooled libraries and optional PhiX.

Flow Cell Type

0.2 N NaOH (µl)

25B

14

10B or 1.5B

8.5
2. Cap and then vortex briefly.
3. Incubate at room temperature for 5 minutes to denature.
4. Add the appropriate volume of Pre-load buffer to neutralize.

Flow Cell Type

Pre-load Buffer (µl)

25B

210

10B or 1.5B

127.5
5. Cap and then vortex briefly.
6. Centrifuge at 280 × g for up to 1 minute.
7. Store libraries on ice until transferred to the library tube strip.

Revision History - Protocol for TruSight Oncology 500 HT

Document

Date

Description of Change

Document # 200055913 v00

May 2024

Initial release.