NGS Library QC and Quantitation

For next-generation sequencing (NGS), the library is a carefully prepared collection of DNA fragments from which a sequencing sample is taken. The purpose of preparing a library is to make sure the target DNA is of the appropriate size and concentration, free of contaminants, with any required adapters properly ligated. Quantification and quality control (QC) of NGS libraries help researchers proceed to sequencing with confidence. For example, they yield the information required to calculate the final library concentration for careful loading onto the sequencer—hopefully avoiding the sequencing pitfalls that can ensue from over- or underloading. This article discusses the main methods used for quantitation and QC of NGS libraries.

Quantitation

Accurate library quantitation is vital and has a direct positive impact on the quality of your NGS data. There are three main methods of quantifying NGS libraries—qPCR, fluorometry, and electrophoresis—but there is no one best method. Indeed, they are complementary and often used to greatest effect in different situations. For example, Joshua McCauley, Research Associate at Lawrence Berkeley National Laboratory, uses three means of NGS library quantitation: fluorometry using Thermo’s Qubit, electrophoresis using Agilent’s BioAnalyzer, in addition to qPCR. “Each method serves its own purpose, and we use them all to complement the entire story of NGS library prep,” says McCauley, who uses the Illumina Nextera XT Kit to prepare NGS libraries for Illumina-based NGS. “It’s important to understand what each method tells you about the library.”

qPCR. Of course, as in other fields, the main purpose of PCR is to amplify DNA. But the advantage of qPCR is that its quantitative nature eliminates time-consuming gels that would otherwise be required following conventional PCR, in order to analyze the PCR products. “We use qPCR to amplify libraries from a small template input and add adapters to the samples, while also viewing the results of the amplification in real time,” says McCauley. “This is important, because our current workflow of 384+ samples per NGS prep makes running an electrophoresis gel unsustainable at our scale.”

Unlike other methods, qPCR only measures amplifiable DNA fragments (i.e., fragments ligated to adapters); as such, it is a very sensitive method of quantifying an NGS library and may be suitable for detecting more rare sequences, or libraries of lower concentration. Conversely, qPCR is more hands-on, and the reagent costs of many runs can start to add up.

Fluorometry. The fluorometric method involves using an intercalating fluorescent dye that specifically binds with high affinity to DNA or RNA. This method is accurate because only target-bound dye emits a fluorescent signal, as in the popular Qubit quantitation assays. “We use fluorometry to quantify pooled library, after we pool and purify all successfully amplified samples,” says McCauley. “It’s accurate for quantifying a wide variety of DNA fragment sizes with low input volume—we take full advantage of this.”

Recently, a group from Lawrence Berkeley National Laboratory developed a method called fluorescent amplification for next-generation sequencing (FA-NGS), a NGS workflow modification consisting of qPCR using the intercalating fluorophore SYBR Green I. FA-NGS confirms amplification and quantifies pooled libraries, and the authors note that pooling calculations based on the FA-NGS method led to sequencing reads being evenly represented: “We expect that the application of FA-NGS will greatly benefit the production of any NGS library type that is amplified by PCR.” Their open-source FA-NGS software tool calculates pooling volumes of individual libraries based on measured fluorescence values, and is freely available to help others use the workflow modification. The FA-NGS software tool can also be used to evaluate the quality of individual NGS libraries by melting curve analysis.

Electrophoresis. Automated electrophoresis can yield quantitative information for NGS libraries, while providing further library information such as the size of DNA fragments. “qPCR and the Qubit help in showing amplification success and the resulting DNA quantity, but miss the size of the amplicons,” says McCauley. “For this, we use the [automated gel electrophoresis system] Agilent BioAnalyzer since it provides highly accurate size output within our library size range.” Generally, electrophoresis is suitable for quantifying libraries with a narrow size distribution, but is less accurate as the size distribution broadens (when another method is better). The instrument can be a pricey initial investment, but may be available at shared core facilities of research institutions.

Library QC

Besides quantification, assessing other library parameters is important for successful NGS results. Such QC analysis can find whether impurities are present and in what amounts, the lengths of DNA fragments, and the library size distribution. Library QC is also helpful in verifying the insert size, and checking for adapter dimer contaminants prior to sequencing.

Electrophoresis. Depending upon the system used, automated electrophoresis systems can tell you a lot about the fragment length and size distribution of your library (examples include Agilent’s Bioanalyzer and TapeStation). Some instruments only measure size, while others quantify and measure purity. Researchers can use automated capillary electrophoresis to find the desired stoichiometry of adapters onto DNA fragment ends.

MiSeq. Illumina’s MiSeq uses the same sequencing-by-synthesis chemistry as the HiSeq NGS sequencer, so optimizing on the former can streamline your success using the latter. MiSeq returns a plethora of QC parameters about your library, including cluster density, library complexity, degree of duplication, GC bias, and index representation.

UV-Vis Spectroscopy. While UV-Vis spectroscopy is a less accurate QC method than those above, it is quick, inexpensive, and can be used to approximate library concentration. However, it may give a falsely high reading because it cannot differentiate between DNA library fragments versus adapters, excess nucleotides, or other contaminants. As such, it is not a good choice for quantitative measurements, but can be convenient for rough measurements.

When it comes to choosing a method, it’s a good idea to consider the average fragment size of your NGS library, as it might influence your ability to use fluorometric or electrophoretic methods. “Some library prep kits are designed to work within a specified range of fragment sizes, so it’s wise to make sure that your expected range of DNA fragments is compatible with the instrument or system that you plan to use,” says McCauley. Considering the wide range of kits available, finding high-quality preparation tools for your DNA of interest shouldn’t be difficult. Just make sure to head off any future sequencing problems by following the kit’s advice, including the necessary library QC and quantitation steps.

References

1. Importance of QC for the reliable preparation of NGS libraries. BioTechniques. March 29, 2018. https://doi.org/10.2144/btn-2017-0106

2. Chiniquy, J., Garber, M.E., Mukhopadhyay, A. et al. Fluorescent amplification for next generation sequencing (FA-NGS) library preparation. BMC Genomics 21, 85 (2020).