Library quantification and quality control quick reference guide

Accurate quantification and proper quality check of next-generation sequencing libraries is key to a successful sequencing run. There are different methods for quantification and quality control depending on the sequencing library kit being used.

Illumina recommends consulting the library preparation kit specific reference guide/product documentation for the validated library quantification and QC methods to ensure that a compatible method of the appropriate sensitivity is used. Using quantification or QC methods that are not supported, or are of an incorrect sensitivity can lead to inaccurate results, which can impact sequencing performance.

• Note: Certain library types, including those that use Illumina Bead Based Normalization, may not require quantification and/or QC.

Below are high-level overviews of quantification and QC methods commonly used for Illumina sequencing libraries, for reference. These are not interchangeable, and certain library types may only be compatible with one or a subset of methods.

Quantification

Fluorometric-based methods: these methods use fluorometric dye to quantify nucleic acid in a sample. These are often specific to double stranded DNA (dsDNA), single stranded DNA (ssDNA), or RNA, and are offered with various sensitivities to accurately quantify nucleic acid in differing concentration ranges.

qPCR: qPCR for Illumina libraries selectively quantifies DNA with Illumina sequencing adapters added.

Trace/electropherogram instruments: trace instruments are generally not recommended for quantification, though are validated for certain Illumina library types for quantification.

UV Spectrophotometry: Illumina does not recommend UV spectrophotometric quantification.

Quality Control (QC)

Trace/electropherogram instruments: this platforms assess library average fragment size and distributions, and can be used to compare relative abundance of different sized fragments and assess for the presence of adapter dimers, bubble products, and other secondary peak types.

Agarose gels: agarose gels can be used for size verification, though are generally not recommended for most Illumina library types.

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