How to prepare < 2 nM libraries for sequencing on NovaSeq X/X Plus

The tables in the NovaSeq X Plus Product Documentation provide the µl of 2 nM libraries + µl of RSB to combine to obtain the specified Final Loading Concentration (pM) after the addition of NaOH and Pre-load Buffer. The following math accounts for the later additional dilutions with NaOH and Pre-Load Buffer. Note that sequencing of libraries less than 2 nM has not been validated by Illumina, and is not supported.

1. Measure the library pool concentration in pM (Y)

• eg, 1000 pM

2. Determine the final target loading concentration in pM (X)

• eg, 180 pM

3. Calculate the volume of library and RSB required using the following formula:

   1. (Y pM) (Z µl) = (5*X) (volume needed by flow cell type)

   2. Volume needed by flow cell type = 34 µl for 10B or 1.5B, and 56 µl for 25B flow cells

   3. Volume library needed = Z

   4. Volume RSB needed = (Volume needed by flow cell type) - Z

4. Move to step 4 "[Optional] Spike in 1-2% nondenatured PhiX as follows" of the protocol if adding PhiX, or the "Denature Libraries" section if not adding PhiX.

Example calculation for 1.5 B or 10B flow cell: (1000 pM) (Z µl) = (5 * 180 pM) (34 µl)

Volume library needed (Z) = 30.6 µl Volume RSB needed = 34 - 30.6 = 3.4 µl

Example calculation for 25B flow cell: (1000 pM) (Z µl) = (5 * 180 pM) (56 µl)

Volume library needed (Z) = 50.4 µl Volume RSB needed = 56 - 50.4 = 5.6 µl

For any feedback or questions regarding this article (Illumina Knowledge Article #9000), contact Illumina Technical Support [e-mail].