Library loading concentrations and considerations for the NovaSeq X/X Plus instruments

Recommended Loading Concentrations

Important Note: The following recommendations are general starting points. The optimal loading concentration depends on the library type and insert size. Titration of each library type is recommended to obtain optimal seeding concentration to yield the best clusters passing filter percentage (%PF). Optimize concentrations over subsequent runs to identify a loading concentration that consistently yields data that meets run specifications.

Note: the above Illumina DNA PCR-Free values are based on quantification with qPCR.

Dilute and Denature Protocol

Prepare Sodium Hydroxide (NaOH)

1. According to the Flow Cell type being used, combine the following volumes in a microcentrifuge tube.

2. Vortex and then centrifuge the tube to mix.

[25B]

** **

[10B]

** **

[1.5B]

** **

Dilute Libraries and Add Optional PhiX Control

1. Dilute libraries to 2 nM using Resuspension Buffer (RSB).

2. According to the Flow Cell type, choose desired final loading concentration and dilute libraries in a new 1.5 ml microcentrifuge tube using RSB according Tables below.

3. [Optional] Spike in 1% to 2% non-denatured PhiX for well balanced libraries.

   1. Quantify PhiX.

   2. Dilute 10 nM PhiX to 300 pM using RSB.

   3. Add the appropriate volume of PhiX according to the Flow Cell configuration:

      1. [25B] Add 1.6 µl of diluted PhiX to 56 µl non-denatured library.

      2. [10B, 1.5B] Add 1 µl of diluted PhiX to 34 µl non-denatured library.

[25B]

[10B, 1.5B]

Denature Libraries

1. Add the appropriate volume of NaOH to the nondenatured libraries according to the Flow Cell type:

   1. [25B] Add 14 µl of 0.2 N NaOH to the nondenatured library tube and optional PhiX.

   2. [10B, 1.5B] Add 8.5 µl of 0.2 N NaOH to the nondenatured library tube and optional PhiX.

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 the denatured libraries according to Flow Cell type:

   1. [25B] Add 210 µl of Pre-load Buffer to neutralize.

   2. [10B, 1.5B] Add 127.5 µl of Pre-load Buffer to neutralize.

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 (refer to Load Lyo Insert and Library Tube Strip).

On the NovaSeq X/X Plus Instruments, see the following for summary of volumes and final volume of denatured, neutralized library solution based on Flow Cell type.

[25B]

Library Pool: 56 µl (~57.6 µL if using optional PhiX) 0.2 N NaOH: 14 µl Pre-load Buffer: 210 µl Total Volume: 280 µL (~281.6 µL if if using optional PhiX)

[10B, 1.5B]

Library Pool: 34 µl (35 µL if using optional PhiX) 0.2 N NaOH: 8.5 µl Pre-load Buffer: 127.5 µl Total Volume: 170 µL (171 µL if if using optional PhiX)

For further information about Dilute and Denaturing, visit the Protocol section in the NovaSeq X Product Documentation.

For any feedback or questions regarding this article (Illumina Knowledge Article #7778), contact Illumina Technical Support [e-mail].