Dilute Libraries to the Starting Concentration

This step dilutes the pooled libraries to the starting concentration for the 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.

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

Illumina does not recommend including a PhiX control in the sequencing run. As there are < 26 cycles per read, no PhiX metrics can be generated.

Procedure

1.

Calculate the molarity value of the libraries. Use the following formula:

Use 164 bp as the average library size.

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, S4	1	200
NovaSeq X Series, 10B	2	65
NovaSeq X Series, 25B	2	85

3.

Dilute each library pool to the starting concentration for your system.

Illumina recommends combining up to two plates by taking 1 nM (NovaSeq 6000) or 2 nM (NovaSeq X Series) diluted libraries and adding 1:1 volume. This process allows for the sequencing of two full plates.

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If you are combining two plates for sequencing, each sample ID must be unique per sample sheet.

Sequencing System	Total Library Pool Required (µl)	Minimum Volume if Combining Two Pools
NovaSeq 6000, S4	310	155 µl at 1 nM (each plate)
NovaSeq X Series, 10B	52	26 µl at 2 nM (each plate)
NovaSeq X Series, 25B	108	27 µl at 2 nM (each plate)