Dilute Libraries to the Starting Concentration

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

Illumina recommends setting up a paired-end run with 101 cycles per read (2 × 101) and 10 cycles per Index Read. If you would like additional overlapped reads or additional raw coverage, you can sequence up to 2 × 126 or 2 × 151, but it is not required.

1.

Calculate the molarity value of the library or pooled libraries using the following formula.

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For libraries qualified on a Bioanalyzer, use the average size obtained for the library.

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For other qualification methods, use 350 bp as the average library size.

2.

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

3.

Dilute libraries using RSB as follows.

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Libraries quantified as a pool—Dilute the pool to the starting concentration for your system.

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Libraries quantified individually—Dilute each library to the starting concentration for your system. Add 10 µl from 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.

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.