NGS Library Quantification and Quality Control

Next-generation sequencing (NGS) is a powerful and increasingly common research tool. Numerous library-preparation methods are now available, and library quality control has become an increasingly important step in the NGS workflow.

NGS library preparation requires considerable time and effort. It often involves DNA fragmentation (mechanical or enzymatic), followed by DNA repair and end-polishing and finally, platform-specific adaptor ligation.

Table 1: Methods for Quantification and Evaluation of NGS Libraries

* Absolute concentration of total DNA; ** Absolute concentration of target DNA.

Two determinants of sequencing success are the quality and quantity of the library material (Table 1). Using too little DNA, libraries with inappropriate fragment size ranges or libraries rich in adapter-adapter dimers, tends to underutilize sequencing runs, resulting in reduced coverage and read depth, empty runs and increased costs. Loading too much DNA onto the sequencer will produce mixed signals, unresolved data and fewer single reads.

Accurate library concentrations are even more important if several libraries are pooled for sequencing in parallel. Here, balancing the library is essential to ensure even sequence yield across all samples in the library pool.

Users have several approaches to library quantification and quality control:

1. Digital PCR (dPCR)

dPCR systems separate nucleic-acid molecules into a large number of partitions, each of which contains an individual PCR reaction. Partitioning may occur in droplets, microwell plates or capillaries.

Using standard PCR, fluorescence-based detection and Poisson statistics, the starting concentration of a DNA template in a sample can be quantified with high precision without the need for standard curves. Unlike qPCR, dPCR does not rely on the assumption that all sequences amplify with similar efficiency. This approach also reduces input-sample requirements.

dPCR assays can be designed for detection of library concentration and quality. This enables more accurate DNA loading for sequencing, and end users can choose to remake their library if it has a high concentration of adapter-adapter dimers, thereby saving time and money.

On the other hand, dPCR requires specialized equipment. It is, however, particularly useful when libraries need to be balanced for parallel sequencing. dPCR is also a promising surveillance tool for illnesses such as cancer, and it is a vital front end to determining genomic content, including sequencing the human genome.

2. qPCR

Quantitative real-time PCR, or qPCR, is a fluorescence-based PCR assay that uses either dyes that bind double-stranded DNA (dsDNA) or fluorophore-labeled oligonucleotide probes designed to bind specific target sequences.

As the PCR process generates increasing amounts of dsDNA, fluorescence in the sample increases. This increase is monitored in real time and compared to standards of known concentration to determine library concentration.

qPCR assays can be designed such that only fragments with the required adapter sequences are quantified. This ensures that only DNA that can be sequenced is quantified.

However, it is difficult using qPCR to accurately determine fragment size ranges. This method also relies heavily on the assumption that all DNA is amplified with the same efficiency. In addition, dye-based qPCR protocols cannot distinguish between library fragments and adapter-adapter dimers, potentially causing overestimation of library concentration.

3. Agarose gel electrophoresis

Agarose gel electrophoresis is a simple and inexpensive way to assess library purity and fragment size range. Size range is estimated by comparison to a standard DNA ladder, and RNA contamination and primer dimers are visible as a small-molecular-weight smear at the bottom of the gel.

Concentrations determined by this method are approximations, and libraries with large fragment size ranges are particularly difficult to quantify accurately. In addition, all dsDNA is quantified using this method, regardless of whether it is flanked by the necessary adapter sequences or represents adapter-adapter dimers without the DNA insert.

4. Spectrophotometry

Nucleic-acid concentration also can be determined using a standard UV spectrophotometer by measuring sample absorption at 260 nm. Because UV spectrophotometers are common laboratory equipment, this is a convenient and inexpensive means of library quantification.

Readings need to be within the linear range of your spectrophotometer—as a rule of thumb, between 0.1 and 1.0. Unfortunately, many other molecules absorb light in the 260-nm range, potentially causing overestimation of DNA concentration.

DNA quality can be assessed by measuring absorbance at 280 nm and calculating the A260/A280. “Pure” DNA should have an A260/A280 of approximately 1.8, but DNA quality is considered acceptable as long as its A260/A280 is between 1.7 and 2.0. Low A260/A280 readings can be indicative of protein or phenol contamination or very low DNA concentrations.

RNA contamination is more difficult to assess by spectrophotometry and the go-to method is often still an agarose gel.

With these limitations in mind, a combination of gel electrophoresis and spectrophotometry may yield adequate results for quality control of NGS libraries, in which case there is no need to invest in more laborious and expensive techniques.

Related Products from: Bio-Rad Laboratories

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  QX200 ™ Droplet Digital ™ PCR System