A high-quality library is the key to successful NGS. Library construction includes complex steps, such as fragmenting the sample, repairing ends, adenylation of ends, ligation of adapters, and amplifying the library. These steps may vary depending on different platforms and library types. Monitoring of each step is highly recommended, including checking sizes after sample fragmentation, and a size and concentration check after ligation of adapters. Library validation serves as the final library quality control step, which analyzes the library size and quantity.
Assessment of library size
Agarose and PAGE gel electrophoresis are traditional methods of assessing size and can be time-consuming.
In recent years, microfluidics-based electrophoresis or capillary electrophoresis have become more popular for ascertaining size and concentration. Ready-to-use chip or gel cartridges are user friendly, omitting the need for gel pouring. They have much higher throughput and require far less hands-on time. In addition, they are more sensitive (low detection limit) and are fully automated for data acquisition and digital data output. These machines detect size and concentration simultaneously.
As mentioned above, microfluidics-based electrophoresis or capillary electrophoresis provides quantification data in addition to size information. However, one limitation to electrophoresis, spectrophotometry, and fluorometry is that all 3 methods measure the total nucleic acid concentrations, not just molecules with adapters added.
The presence of adapter sequences at both ends of the library molecules enables the amplification of millions of individual DNA molecules in parallel PCR amplification step (emulsion PCR or bridge PCR). On some instruments, emulsion PCR is performed to amplify a single DNA molecule to millions of copies of the same sequence all attached to a single bead. With another platform, bridge PCR amplification converts a single DNA molecule into a cluster with many copies of the same sequence. Therefore, the amplifiable molecules, which are appended with adapter sequences at both ends, are the ones that determine the template-to-beads ratio in emulsion PCR or optimal cluster generated by bridge PCR.
Accurate quantification of amplifiable library molecules is essential for ensuring quality reads and efficient data generation. Underestimation of amplifiable library molecules leads to mixed signals and non-resolvable data; conversely, overestimation results in poor yield of template-carrying beads or clusters and reduced usage of sequencing capacity.
Real-time PCR can specifically quantify DNA molecules with adapters at both ends, and therefore provides highly accurate quantification of amplifiable library molecules. The high sensitivity of real-time PCR allows quantification of libraries with very low concentration, even below the detection threshold of conventional methods. Thus, further amplification of the library is minimized and reduces potential bias.
Digital PCR for absolute quantification
Digital PCR provides absolute quantification of NGS libraries with no need for a standard curve. A limiting dilution of library is made across a large number of separate PCR reactions; therefore, most of the reactions have no templates and yield negative results. A single, positive PCR reaction at the endpoint is counted as one individual template molecule. In counting all the positive PCR reactions, the total absolute number of library molecules can be derived. The major advantages of digital PCR include:
Single molecule sensitivity
Independence from variations in PCR amplification efficiency, since successful amplification is counted as one molecule, independent of the endpoint amount of product.
However, very specific equipment is required and is relatively expensive, so this technique is not widely used for library quantification yet.