User manual

64 | Droplet Digital
™
PCR Applications Guide
7 Next-Generation
Sequencing Library Analysis
Overview
Next-generation sequencing (NGS) systems are extremely sensitive to the quantity of
library loaded in the sequencing run. Overloading frequently produces unusable data and
underloading wastes reagents and time. Droplet Digital
™
PCR (ddPCR
™
) complements NGS
by offering accurate library concentration measurements and unique quality analyses that
are not available with other methods.
Standard methods for quantifying NGS libraries have disadvantages. Electrophoresis and
spectrophotometry measure total nucleic acid concentrations, whereas optimal cluster
density or template-to-bead ratio depends on the appropriate concentration of PCR-
amplifiable DNA molecules. Incorrectly adapted products or adapter-adapter dimers cannot
be distinguished easily from bona fide library fragments. These undesirable species can
compete for binding sites on the flow cell, inhibit cluster formation, increase the likelihood of
suboptimal loading, and reduce the total number of high-quality reads. These methods also
have low sensitivity, consuming nanograms of precious samples, and are not suitable for
high-throughput workflows. A more accurate method to quantify libraries before sequencing
is required to maximize usage of sequencing platforms.
ddPCR provides an absolute, standard-free method to measure library concentration and
quality, while optimizing overall NGS performance. ddPCR makes very precise and accurate
measurements of the library stock concentrations for flow-cell loading. The digital nature
of droplet partitioning is conducive to accurate quantification by reducing competing
PCR reactions.