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

The tables in the Denature and Dilute Protocol Generator for NovaSeq X Plus 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 "Dilute Libraries and Add PhiX Control" section 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].