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.

Use this procedure when the Normalize Libraries protocol is not followed.

For sequencing, Illumina recommends the read lengths indicated on the Nextera XT DNA Library Prep compatible products support page. If you would like additional overlapped reads, raw coverage, or adjusted IPB recommendations for ≥ 2 x 250 cycles, you can sequence up to 2 x 250 or 2 x 300, but it is not required.

IDT for Illumina DNA/RNA UD Indexes uses 10 base pair index codes that differ from the Nextera XT and Nextera XT v2 indexes, which use eight base pair index codes. This change in base pair index codes can require adjustments to your sequencing run set up.

1. Calculate the molarity value of the library or pooled libraries using the following formula.
• For libraries qualified on a Bioanalyzer or other trace instrument, use the average size obtained for the library.
• If a trace instrument is not available, use 600 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.
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 of 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.
• For the iSeq 100 System, refer to the system guide for dilution instructions (libraries are automatically denatured).
• For the NovaSeq 6000 System, refer to the system guide for pool and denature instructions.
• For the HiSeq 4000 and HiSeq 3000 Systems, refer to the cBot 2 or cBot system guide for reagent preparation instructions.
• For all other systems, refer to the denature and dilute libraries guide.

Optimize concentrations for your workflow and quantification method over subsequent sequencing runs or by flow cell titration.