Dilute Libraries to the Starting Concentration

This step dilutes exome-enriched 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.

For sequencing, regardless of the enrichment probe panel you are using, Illumina recommends setting up a paired-end run with 101 cycles per read (2 × 101) and 10 cycles per Index Read. If you want optional and additional overlapped reads/raw coverage, you can sequence up to 2 × 126 or 2 × 151.

1. Calculate the molarity value of the library or pooled libraries using the following formula.
• For libraries qualified on a Bioanalyzer, use the average size obtained for the library.
• For all other qualification methods, use 350 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)

NextSeq 550 and NextSeq 500

2

1.4–1.5

NextSeq 1000 and NextSeq 2000*

2

1000

NovaSeq 6000

2

175–185

NovaSeq X

2

150

* For the NextSeq 1000 and NextSeq 2000 systems, use the RSB with Tween 20 supplied with the system to dilute below 10 nM.

3. Dilute libraries using RSB:
• Libraries quantified as a multiplexed library pool—Dilute the pool to the starting concentration for your system.
• Libraries quantified individually—Dilute each library to the starting concentration for your system.

Add 10 µl each diluted library to a tube to create a multiplexed library pool.

4. Follow the denature and dilute instructions for your system to dilute to the final loading concentration.
• Refer to the Illumina Denature and Dilute protocol generator and the Illumina support site for pool and denature instructions.
• 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.