Protocol B: Pool and Denature Libraries for Sequencing (Xp Loading)

Use Protocol B to denature and dilute libraries that have been normalized using standard library quantification and quality control procedures recommended in the library prep documentation. For addressable lane loading, refer to the NovaSeq Xp Workflow chapter in the NovaSeq 6000 System Guide (document # 1000000019358).

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

Use the following instructions to normalize libraries to the appropriate concentration and then pool. Libraries sequenced on the same lane must be combined into a single pool. The total volume per lane of each normalized pool is shown in the following table. If the same pool is sequenced across more than one lane, multiply the value from by the number of lanes.

Total Volume of Pooled Library

Mode

Total Volume of Pool Per Lane (µl)

SP/S1

18

S2

22

S4

30

For the Xp workflow, the data output is obtained for each lane, as opposed to all the lanes in aggregate for the Standard workflow. As a result, library pools for the Xp workflow contain fewer libraries compared to the Standard workflow.

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 Lane (B)

Libraries per Lane

Human Genomes

SP

.65–.8

1

S1

1.3–1.6

~2

S2

3.3–4.1

~5

S4

4.0–5.0

~6

Exomes

SP

.65–.8

~10

S1

1.3–1.6

~20

S2

3.3–4.1

~50

S4

4.0–5.0

~62

Transcriptomes

SP

.65–.8

~8

S1

1.3–1.6

~16

S2

3.3–4.1

~41

S4

4.0–5.0

~50
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

0.5

150

0.75

200

1.0

250

1.25

300

1.5

350

1.75

400

2.0

450

2.25

500

2.5
2. Normalize libraries to the desired pooled library loading 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.

Recommended Loading Concentrations

Library Type

Final Loading Concentration (pM)

Pooled Loading Concentration (nM)

PhiX¹

100

0.5

Illumina DNA PCR-free library pool

300–400²

1.5–2.0²

TruSeq DNA PCR-free library pool

115–235

0.575–1.175

DNA PCR-amplified library pool

200–400

1.0–2.0

Single Cell

175–275

.875–1.375

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.

If you have optimized the loading concentration for HiSeq X®, HiSeq® 4000, or HiSeq® 3000, use approximatelythe same concentration for the NovaSeq Xp workflow. If you have optimized the loading concentration for the NovaSeq Standard workflow, use approximately 1/3 less for the NovaSeq Xp workflow.

Libraries might need to be titrated to obtain optimal seeding concentration. When the optimal loading concentration is determined, it is applicable for identical library types.

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

Mode

Total Volume of Pool Per Lane (µl)

SP/S1

18

S2

22

S4

30

For example, for a six-plex library pool and S4 mode, combine 5 µl of each library that has been normalized to the same concentration.

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 0.25 nM using 10 mM Tris-HCl, pH 8.5.
b. Add the appropriate volume of PhiX to the tube of nondenatured library pool.

Mode

Nondenatured 0.25 nM PhiX (µl)

Nondenatured Library Pool (µl)

SP/S1

0.7

18

S2

0.8

22

S4

1.1

30

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 minimize pipetting errors that could affect the final NaOH concentration, prepare at least 30 µl diluted NaOH per flow cell. For a dual flow cell run, prepare 60 µl diluted NaOH.

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

1. Prepare 0.2 N NaOH by diluting stock NaOH with laboratory-grade water.
2. For one flow cell, combine the following volumes in a microcentrifuge tube to dilute 1 N NaOH to 0.2 N.
• Laboratory-grade water (24 µl)
• Stock 1 N NaOH (6 µl)

These volumes result in 30 µl 0.2 N NaOH. For two flow cells, double the volumes.

3. 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.

Mode

0.2 N NaOH (µl)

Nondenatured Library Pool (µl)

Resulting Volume

SP/S1

4.0

18.0

22.0 µl, or 22.7 µl with PhiX

S2

5.0

22.0

27.0 µl, or 27.8 µl with PhiX

S4

7.0

30.0

37.0 µl, or 38.1 µl with PhiX
2. Cap and then vortex briefly.
3. Centrifuge at a maximum of 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

5.0

27.0 µl, or 27.7 µl with PhiX

S2

6.0

33.0 µl, or 33.8 µl with PhiX

S4

8.0

45.0 µl, or 46.1 µl with PhiX
6. Cap and then vortex briefly.
7. Centrifuge at a maximum of 280 × g for up to 1 minute.
8. Place on ice until use.
9. Keep denatured libraries on ice until ready to add the ExAmp master mix.
10. [Optional] If you cannot proceed immediately, cap the tube and store at -25°C to -15°C for up to 3 weeks. Do not refreeze after thawing.

Store denatured library pools only if necessary. Long term storage can increase duplicates, which decrease yield.

After denaturing and diluting thelibraries 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).