Protocol A: Pool and Denature Libraries for Sequencing (Standard Loading)

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

For Xp loading, proceed to Protocol B: Pool and Denature Libraries for Sequencing (Xp Loading).

Use the following instructions to normalize libraries to the appropriate concentration and then pool. Libraries sequenced on the same flow cell must be combined into a single normalized pool.

1. Refer to the following table for the typical number of reads and recommended plexity by application and flow cell type.
Recommended Library Pool Plexity

Application

Flow cell Type

Paired-End Reads Passing Filter per flow cell (B)

Libraries per Lane

Human Genomes

SP

1.3–1.6

~2

S1

2.6–3.2

~4

S2

6.6–8.2

~10

S4

16–20

~24

Exomes

SP

1.3–1.6

~20

S1

2.6–3.2

~40

S2

6.6–8.2

~100

S4

16–20

~250

Transcriptomes

SP

1.3–1.6

~16

S1

2.6–3.2

~32

S2

6.6–8.2

~82

S4

16–20

~200
1. Determine the required pooled library concentration based on the desired final loading concentration.

Refer to Recommended Loading Concentrations.

Final Loading Concentration (pM)

Pooled Library Concentration (nM)

100

0.50

150

0.75

200

1

250

1.25

300

1.50

350

1.75

400

2

450

2.25

500

2.50
2. Normalize libraries to the desired pooled library concentration using 10 mM Tris-HCl, pH 8.5.

For assistance diluting libraries to the appropriate concentration, refer to the Pooling Calculator on the Illumina website.

The optimal DNA loading concentration depends on the library type and insert size. The following table provides DNA loading concentrations that are recommended based on Illumina libraries with insert sizes that are ≤ 450 bp. Load libraries with smaller insert sizes at the lower end of the recommended range. For libraries > 450 bp, higher loading concentrations might be necessary.

For libraries generated from non-Illumina library prep methods, you may need to perform a titration of your specific library type initially to obtain optimal seeding concentration to yield best %PF. When optimal loading concentration is determined, it should be applicable for identical library types later.

Recommended Loading Concentrations for Standard Workflow (Software Version 1.1, or later)

Library Type

Final Loading Concentration (pM)

Pooled Loading Concentration (nM)

PhiX¹

250

1.25

Illumina DNA PCR-free library pool

400–600²

2–3²

TruSeq DNA PCR-free library pool

175–350

0.875–1.75

DNA PCR-amplified library pool

300–600

1.5–3.0

Single cell³

250–500

1.25–2.5

¹ For a PhiX-only run.

² Calculated based on 450 bp as the median insert size, 660 g/mol as the DNA mass, and ssQubit concentration values.

³ Single Cell has been verified for the Xp workflow only.

If you have optimized a final loading concentration for HiSeq™ X, HiSeq® 4000, or HiSeq® 3000, use 1.5× that concentration for NovaSeq 6000. For example, if the final loading concentration for HiSeq X is 200 pM, use 300 pM for NovaSeq 6000.

1. Combine the appropriate volume of each normalized library in a new microcentrifuge tube to result in one of the following final volumes:

Mode

Final Volume (µl)

SP/S1

100

S2

150

S4

310

For example, for a six-plex library pool and S2 mode, combine 25 µl of each library that has been normalized to the same concentration. For a four-plex library pool and S1 mode, combine 25 µl of each normalized nondenatured library.

2. [Optional] Store remaining unpooled libraries at ‑25°C to ‑15°C.
3. [Optional] Spike-in 1% nondenatured PhiX as follows.
a. Dilute 10 nM PhiX to 2.5 nM using 10 mM Tris-HCl, pH 8.5.
b. Add the appropriate volume of nondenatured 2.5 nM PhiX to the tube of nondenatured library pool.

Mode

Nondenatured 2.5 nM PhiX (µl)

Nondenatured Library Pool (µl)

SP/S1

0.6

100

S2

0.9

150

S4

1.9

310

When spiking in 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.

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

Freshly diluted 0.2 N NaOH is essential to the denaturation process. Improper denaturation can reduce yield.

1. Combine the following volumes in a microcentrifuge tube to dilute 1 N NaOH to 0.2 N:
SP/S1/S2 Mode

Reagent

Volume for One Flow Cell (µl)

Volume for Two Flow Cells (µl)

Laboratory-grade water

40

80

Stock 1 N NaOH

10

20

These volumes result in 50 µl 0.2 N NaOH for one flow cell or 100 µl 0.2 N NaOH for two flow cells.

S4 Mode

Reagent

Volume for One Flow Cell (µl)

Volume for Two 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.

2. Invert several times to mix, or vortex thoroughly. Keep the tube capped and use within 12 hours.
1. Add 0.2 N NaOH to the tube of nondenatured library pool and optional PhiX as follows.

Flow Cell

0.2 N NaOH (μl)

Nondenatured Library Pool (µl)

Resulting Volume

SP/S1

25

100

125 µl, or 125.6 µl with PhiX

S2

37

150

187 µl, or 187.9 µl with PhiX

S4

77

310

387 µl, or 388.9 µl with PhiX
2. Cap and then vortex briefly.
3. Centrifuge at 280 × g for up to 1 minute.
4. Incubate at room temperature for 8 minutes to denature.
5. Add 400 mM Tris-HCl, pH 8.0 to neutralize as follows.

Mode

400 mM Tris-HCl, pH 8.0 (µl)

Resulting Volume

SP/S1

25

150 µl, or 150.6 µl with PhiX

S2

38

225 µl, or 225.9 µl with PhiX

S4

78

465 µl, or 466.9 µl with PhiX
6. Cap and then vortex briefly.
7. Centrifuge at 280 × g for up to 1 minute.
8. Transfer the entire volume of denatured library or denatured library and PhiX to the library tube provided with the NovaSeq 6000 Reagent Kit.
9. Immediately proceed to loading the library tube into the cluster cartridge and setting up the run.

The reagent cartridges, including the library tube, must be loaded onto the instrument within 30 minutes.

10. [Optional] If you cannot immediately proceed, cap the library tube and store at -25°C to -15°C for up to 3 weeks. Do not refreeze after thawing.

Store the library tube only if necessary. Long term storage at -25°C to -15°C can increase duplicates, which decrease yield.

After denaturing and diluting the libraries and preparing the optional PhiX control, proceed to Prepare SBS and Cluster Cartridges in the Standard Workflow section of the NovaSeq 6000 System Guide (document # 1000000019358).