Dilute Libraries to the Starting Concentration

This step dilutes libraries to the starting concentration for your sequencing system and is the first step in a serial dilution. After diluting to the starting concentration, libraries are ready to be denatured and diluted to the final loading concentration.

Illumina recommends setting up a paired-end run with 151 cycles per read (2 x 151) and 0 cycles per Index Read with a single sample per lane for the NovaSeq 6000 S4 flow cell using the NovaSeq Xp workflow.

1. Calculate the molarity value of the libraries using the following formula:

2. Using the molarity value, calculate the volumes of RSB and library needed to dilute libraries to the starting concentration for your system.

Sequencing System

Starting Concentration (nM)

Final Loading Concentration (pM)

NovaSeq 6000

0.875

175

NovaSeq X Series

2

150
3. Dilute each library to the starting concentration for your system.
4. Follow the denature and dilute instructions for your system.

For NovaSeq 6000, refer to the NovaSeq 6000 Xp loading denature and dilute instructions in the NovaSeq 6000 System Denature and Dilute Guide (document # 1000000106351). Proceed to the 1% PhiX spike-in instructions without pooling.

The final loading concentrations are a starting point and general guideline. Optimize concentrations for your workflow and quantification method over subsequent sequencing runs or by flow cell titration.