Quantify Enriched Libraries and Manual Normalization

In this step, the enriched libraries are quantified in preparation for manual library normalization. Library normalization is the process of diluting libraries of variable concentrations to the same concentration to ensure uniform library representation in the sequencing pool.

Quantify Enriched Libraries

Assess the quantity of enriched libraries before normalization using a fluorometric quantification method (user-supplied). The Qubit dsDNA BR Assay Kit has been demonstrated to be effective for quantifying libraries in this protocol.

Recommended Guidelines

1.

Prepare Qubit dsDNA BR reagents according to manufacturer recommendations.

2.

Remove the PL plate from storage and bring to room temperature.

3.

Centrifuge at 280 × g for 1 minute.

4.

Pipette to mix.

5.

Calibrate the Qubit reader by running standards prepared with the same Qubit working solution used for library quantification.

6.

Run 2 µl each enriched library following the instructions of the manufacturer. It is recommended to quantify all libraries that are sequenced together on the same quantification event.

Expect post-enrichment library yields of ≥ 3 ng/µl. Library yields can vary based on FFPE DNA quality.

Manual Normalization

Refer to the system guide for your sequencing system to prepare consumables for the sequencing run. Library loading concentrations vary depending on the sequencing system used. For instructions on how to normalize, pool, denature, and dilute libraries to the loading concentration, follow the denature and dilute instructions for your system.