# 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](https://support-docs.illumina.com/IN/NovaSeqX_DnD-A/Content/NovaSeqX/protocol-A-NVX.htm) 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)**
   * Volume needed by flow cell type = **34 µl** for 10B or 1.5B, and **56 µl** for 25B flow cells
   2. Volume library needed = Z
   3. 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](https://support-docs.illumina.com/IN/NovaSeqX_DnD-A/Content/NovaSeqX/protocol-A-NVX.htm) 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**

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #9000), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000009000%20-%20Library%20Preparation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |


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