Pool and Dilute Libraries

After diluting to the starting concentration of 4 nM (or the recommended starting concentration for the sequencing system), libraries are ready to be denatured and diluted to the final loading concentration.

Calculate the number of samples per run based on the number of reads per sample required and the flow cell type. For example, 0.5 M clusters and 1M total paired-end reads per sample (0.5M read-pairs) is a standard starting point. Challenging or diluted samples may require more reads, typically >2 M total paired-end reads per sample.

Transfer the designated volume of normalized libraries containing the appropriate index adapter sets to a new microcentrifuge tube for each number of samples. The following table lists an example at 1M total paired-end M reads.

For multiple normalized pools, combine the designated volume of each normalized pool in the tube. This process produces a final pool of samples diluted to a starting concentration of 4 nM. Do not combine pools with the same index adapter set. If additional indexes are needed, use Unique Dual Index Plate A or Plate C.

* Multiplexing 384 samples requires (8) Illumina Microbial Amplicon Prep—Influenza A/B kits and (4) IDT for Illumina DNA/RNA UD Indexes (Set A, B, C and D). These index plates must be used in place of the Illumina Unique Dual Indexes, LT that come with these library prep kits.

1.

Follow the denature and dilute instructions for your sequencing system to dilute to the final loading concentration.

Refer to the Illumina support site for more information on diluting to the final loading concentration.

2.

Use the following loading concentrations for your sequencing system.

Adjustments to final loading concentration should follow the denature and dilute instructions for your sequencing system.