Library Quantification

Inaccurate library quantification often leads to suboptimal clustering. Quantification is necessary for protocols that do not include a final bead-based normalization step.

Recommended Methods

The following table describes recommended library quantification methods. See the library prep documentation for product-specific recommendations and instructions.

Quantification Method	Description
qPCR¹	qPCR is the most effective method of library quantification when paired with a standard of similar size range. This method measures only functional library fragments instead of all DNA species in a library (such as primer dimers, free nucleotides, library fragments).²
PicoGreen or Qubit¹	PicoGreen, Qubit, and other fluorometric methods measure dsDNA only and are best for libraries with a broad fragment size range. These methods can overestimate library concentration because they measure all dsDNA in a pool, including partially constructed and adapter-dimer contaminants. However, PicoGreen and Qubit are highly accurate when the Bioanalyzer quality assessment indicates low levels of library contamination.
Bioanalyzer	Although recommended for quality control purposes, use the Bioanalyzer to quantify three types of libraries only: TruSeq Small RNA, TruSight Tumor 26, and TruSeq Targeted RNA Expression. Due to decreasing accuracy with increasing library fragment size range, the Bioanalyzer is not optimal for quantifying other library types.

¹ Quantifying libraries is distinct from checking library quality, which can require use of the Bioanalyzer. See Library Quality.

² For more information, see the Sequencing Library qPCR Quantification Guide (document # 11322363) and Nextera Library Validation and Cluster Density Optimization (Pub. No. 770-2013-003).