Pool and Denature Libraries

Sequencing System: NovaSeq X, standard

Protocol A

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. The NovaSeq X Series 10B Flow Cell, NovaSeq X Series 5B Flow Cell, and the NovaSeq X Series 1.5B Flow Cell have the same recommendations per lane. However, the NovaSeq X Series 10B Flow Cell and NovaSeq X Series 5B Flow Cell have eight lanes, and the NovaSeq X Series 1.5B Flow Cell has two lanes.

To obtain the optimal seeding concentration to yield the best %PF, you might need to perform a titration of your library type.

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 Series reagent kit

Neutralizing denatured libraries.

Resuspension Buffer (RSB)

Illumina, provided in the library prep kit contents

Diluting libraries to the required loading concentration.

Water, laboratory-grade

General lab supplier

Diluting NaOH for denaturing libraries.

[Optional] PhiX Control v3

Illumina, catalog # FC-110-3001

Spiking in PhiX control.

Equipment

Equipment

Supplier

Microcentrifuge

General lab supplier

Vortexer

General lab supplier

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. Combine the volumes appropriate for your flow cell type in a microcentrifuge tube. Multiply each volume by the number of flow cells.

Flow Cell Type

Laboratory-grade water (µl)

Stock 1 N NaOH (µl)

Total (µl)

25B

120

30

150

10B or 5B

80

20

100

1.5B

20

5

25

2. Vortex to mix, and then centrifuge briefly.

Dilute Libraries (without PhiX Control)

1. Dilute libraries to 2 nM using RSB.
2. In a new 1.5 ml microcentrifuge tube, dilute libraries to the desired final loading concentration using RSB. Use the table appropriate for your flow cell type.
• For NovaSeq X Series 25B, the final volume should be 56 µl per sample well.
• For NovaSeq X Series 10B, 5B, or 1.5B, the final volume should be 34 µl per sample well.
NovaSeq X Series 25B

Final Loading Concentration (pM)

2 nM Library (µl)

RSB (µl)

90

12.6

43.4

100

14

42

110

15.4

40.6

120

16.8

39.2

130

18.2

37.8

140

19.6

36.4

150

21

35

160

22.4

33.6

170

23.8

32.2

180

25.2

30.8

190

26.6

29.4

200

28

28
NovaSeq X Series 10B, 5B, 1.5B

Final Loading Concentration (pM)

2 nM Library (µl)

RSB (µl)

90

7.7

26.3

100

8.5

25.5

110

9.4

24.6

120

10.2

23.8

130

11.1

22.9

140

11.9

22.1

150

12.8

21.2

160

13.6

20.4

170

14.5

19.5

180

15.3

18.7

190

16.2

17.8

200

17

17
3. Vortex to mix, and then centrifuge briefly.

Dilute Libraries (with PhiX Control)

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

If using NovaSeq X Series Control Software version v1.3 or later, you can use a lower amount of PhiX spike in for low-diversity libraries.

1. Dilute libraries to 2 nM using RSB.
2. Prepare nondenatured PhiX as follows.
a. Quantify PhiX.
b. Dilute PhiX to 300 pM using RSB.
3. In a new 1.5 ml microcentrifuge tube, dilute libraries to the desired final loading concentration using RSB and spike in nondenatured PhiX.

The following tables show example volumes.

• For NovaSeq X Series 25B, the final volume should be 56 µl per sample well.
• For NovaSeq X Series 10B, 5B, or 1.5B, the final volume should be 34 µl per sample well.
NovaSeq X Series 25B

Final Loading Concentration (pM)

2 nM Library (µl)

RSB (µl)

300 pM PhiX (µl)

90

12.6

42.4

1

100

14

40.9

1.1

110

15.4

39.3

1.3

120

16.8

37.8

1.4

130

18.2

36.3

1.5

140

19.6

34.8

1.6

150

21

33.3

1.7

160

22.4

31.8

1.8

170

23.8

30.3

1.9

180

25.2

28.7

2.1

190

26.6

27.2

2.2

200

28

25.7

2.3
NovaSeq X Series 10B, 5B, 1.5B

Final Loading Concentration (pM)

2 nM Library (µl)

RSB (µl)

300 pM PhiX (µl)

90

7.7

25.7

0.6

100

8.5

24.8

0.7

110

9.4

23.8

0.8

120

10.2

23

0.8

130

11.1

22

0.9

140

11.9

21.1

1

150

12.8

20.1

1.1

160

13.6

19.3

1.1

170

14.5

18.3

1.2

180

15.3

17.4

1.3

190

16.2

16.5

1.3

200

17

15.6

1.4
4. Vortex to mix, and then centrifuge briefly.

Denature Libraries

1. Add the appropriate volume of 0.2 N NaOH to the nondenatured library tube, which can optionally include PhiX.

Flow Cell Type

0.2 N NaOH (µl)

25B

14

10B, 5B, or 1.5B

8.5
2. Vortex to mix, and then centrifuge 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, 5B, or 1.5B

127.5
5. Vortex to mix, and then centrifuge briefly.
6. Store libraries on ice until transferred to the library tube strip.

Revision History - Protocol for Standard Loading

Document

Date

Description of Change

Document # 200047034 v02

May 2026

Added information for NovaSeq X Series 5B Flow Cell.

Added centrifuge step after diluting libraries.

Updated PhiX spike-in instructions and recommendations.

Updated vortex and centrifuge steps for consistency.

Document # 200047034 v01

September 2025

Added guidance for diluting libraries to a final loading concentration of 190pM and 200pM.

Document # 200047034 v00

June 2024

Initial release.