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.

For sequencing, Illumina recommends a minimum 1 x 100 bp Sequencing and 10 cycles per Index Read (2). If you would like optional and additional overlapped reads/raw coverage, you can sequence up to 1 x 150, 2 x 100, or 2 x 150. For single-end reads analyze 1 million total reads per sample; for paired-end reads analyze 2 million total reads per sample.

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 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.

3.

Dilute libraries using RSB:

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Libraries quantified as a multiplexed library pool—Dilute the pool 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.