The rapid advancement of next-generation sequencing (NGS) technology has impacted all branches of life science research, from microbial genomics to the study of human disease. Compared to the previous generation of sequencing, based on Sanger chemistry and capillary electrophoresis (CE), NGS offers massively higher throughput at a fraction of the cost. For example, a typical CE sequencing run generates 50-80kb of sequence information, while the newest Illumina HiSeq® X sequencing systems produce 1.6-1.8 Tb of data in a single run.
Current Sanger CE systems can sequence a 1 megabase (1Mb) genome at a cost of approximately $2,400; by comparison, the same genome would cost just $0.07 on an Illumina HiSeq 2000 system.
Although several NGS platforms are available, the market at present is largely dominated by Illumina sequencing systems, with the Ion Torrent® systems from Life Technologies running a distant second.
For any platform, the success of NGS depends, to a large extent, on using high-quality starting DNA that is quantitated accurately. This article discusses the role of DNA quantitation during typical NGS library preparation workflows.
Current Sanger CE systems can sequence a 1-megabase (1Mb) genome at a cost of approximately $2,400; by comparison, the same genome would cost just $0.07 on an Illumina HiSeq 2000 system.
How Should I Purify Genomic DNA for NGS to Get the Best Results?
Several methods for purifying genomic DNA are commonly used prior to NGS library preparation. Some library preparation methods are more sensitive to DNA quality than others. However, all purification methods should yield DNA that is free from organic contaminants, such as phenol or ethanol. In addition, the DNA solution should not contain EDTA concentrations higher than 1mM.
Resin-based methods are popular for purifying genomic DNA for NGS, because they are rapid, produce high-quality DNA, and can be automated for processing large numbers of samples simultaneously. The Promega Maxwell® DNA Purification Kits are suitable for automated workflows and are available in a variety of formats for purifying genomic DNA from cells, tissues, and blood samples. For manual processing of samples, Promega offers the ReliaPrep® and Wizard® Genomic DNA Purification Kits.
All purification methods should yield DNA that is free from organic contaminants, such as phenol or ethanol.
How Much DNA Do I Need to Prepare a NGS Library?
The amount of input DNA depends on the NGS library preparation method, as well as the specific type of NGS application. For example, Illumina mate-pair genomic DNA sequencing libraries require as much as 10-20µg of DNA, compared to chromatin-immunoprecipitation sequencing (ChIP-Seq), which uses 10–50ng of input DNA.
Regardless of the platform, NGS library preparation methods can be divided into two stages.
In the first stage, genomic DNA is broken into smaller pieces to accommodate the relatively short read lengths of NGS. This fragmentation can be accomplished by mechanical techniques that shear the DNA (such as sonication with Covaris® instruments, or nebulization using compressed air). Mechanical fragmentation typically results in substantial DNA losses; consequently, NGS library preparation workflows based on mechanical fragmentation methods generally require higher amounts of input DNA, approximately 1–5µg. They are best suited for samples where DNA amount is not limiting, e.g., cultured cells, bacteria, or fresh/frozen tissue samples.
In contrast, enzyme-based methods such as the NEB® Fragmentase™ Enzyme can be used for limiting samples—DNA from formalin-fixed paraffin-embedded (FFPE) tissues, stem cells, or laser-capture microdissected (LCM) samples. The NEBNext Ultra™ DNA Library Prep Kits require as little as 5ng of DNA to prepare Illumina sequencing libraries; Ion Torrent® libraries require 100ng to 1µg of DNA.
NGS library preparation can be divided into two stages: fragmentation and fragment-end repair.
In the second stage of NGS library preparation, the ends of the fragmented DNA are repaired (blunted) using a mixture of DNA polymerases. Next, specific adapter sequences are ligated to the ends of the fragmented DNA. The sequence of these adapters will vary, depending on the sequencing system being used. In most workflows, the library is then amplified by PCR to enrich for ditagged fragments (those DNA molecules that have the appropriate adapter sequences on each end) and to produce sufficient DNA for sequencing. For Illumina libraries, PCR-free workflows are also available for situations where it is desirable to minimize amplification bias. PCR-free methods require larger amounts of input DNA than conventional library preparation workflows—typically 1–2µg of DNA for Illumina libraries.
Illumina’s Nextera® library preparation kits adopt a unique approach that combines both these stages into a single enzymatic reaction known as tagmentation. An engineered transposase enzyme fragments and tags the DNA in a single-tube process, eliminating the need for intermediate purification and ligation steps. Nextera® library preparation kits require as little as 50ng of purified genomic DNA.
Why Is Accurate DNA Quantitation Important for Preparing NGS Libraries?
Quality sequencing libraries should provide even coverage with minimal bias. Accurate quantitation of the input genomic DNA ensures consistent and reproducible results from library preparation. It is important to choose fluorometric methods of DNA quantitation that are specific to double-stranded DNA (dsDNA), since single-stranded DNA (ssDNA) is not a suitable substrate for most library preparation technologies. Further, spectrophotometric methods (e.g., NanoDrop® systems) that measure ultraviolet (UV) absorbance should be avoided. These methods measure total nucleic acids in a sample as well as impurities. Thus, the amount of genomic dsDNA actually present can be overestimated, due to the presence of contaminating RNA, ssDNA, or oligonucleotides that contribute to overall UV absorbance.
Accurate quantitation of the input genomic DNA ensures consistent and reproducible results from library preparation.
What Methods Are Commonly Used for Input DNA Quantitation?
All sequencing system manufacturers recommend dsDNA-specific fluorometric quantitation methods. The Qubit® Fluorometer (Life Technologies) and Quantus™ Fluorometer (Promega Corp) are popular methods for quantitating genomic DNA for NGS library preparation. PicoGreen® (Life Technologies) and the QuantiFluor® dsDNA System (Promega Corp) are dsDNA-specific fluorescent dyes that can be used with any fluorometer.
All sequencing system manufacturers recommend dsDNA-specific fluorometric quantitation methods.
Illumina recommends using Promega’s QuantiFluor® dsDNA System for more accurate measurement of DNA when preparing Nextera Rapid Capture DNA libraries.
The QuantiFluor® dye is specific for dsDNA, with minimal binding to ssDNA, RNA, protein, and interfering compounds. The sensitivity of QuantiFluor® dye is comparable to PicoGreen®, down to DNA sample concentrations of 10pg/µL (assay concentrations of 50pg/mL).
The QuantiFluor® dsDNA System was developed using Promega detection systems. The assay can be used in both single-tube and multiwell plate formats. The QuantiFluor® dsDNA System is compatible with any fluorometer that is capable of measuring fluorescence at the appropriate excitation and emission wavelengths (504nm and 531nm, respectively), including the Quantus™ Fluorometer and the GloMax® Discover and Explorer Systems. It can also be used with the GloMax®-Multi Jr Single Tube Multimode Reader. A standard curve obtained using the QuantiFluor® dsDNA System is shown in Figure 1.
Figure 1. Representative dsDNA standard curve in a 96-well plate format. Bottom panel. Expanded view of the low end of the standard curve.
The QuantiFluor® dye offers similar performance specifications to PicoGreen® (Figure 2). Under conditions used to generate the standard curves for the assay, the dynamic range for both dyes is approximately 100pg to 200ng per well (in 200μl total volume). The limit of detection for the QuantiFluor® Dye is approximately 10pg per well, as defined by greater than three standard deviations above background fluorescence.
Figure 2. Comparison of QuantiFluor® dsDNA Dye and PicoGreen.
How Should I Validate Finished Libraries before Sequencing?
Accurate library quantitation is critical to obtaining optimal NGS data. Loading more than the recommended amount of DNA can lead to instrument read problems associated with saturation of the flowcell or beads, while loading less can cause reduced coverage and read depth.
While fluorometric methods can provide accurate and sensitive measurement of the amount of DNA present in a sequencing library, quantitative polymerase chain reaction (qPCR) methods are recommended by sequencing system manufacturers for final library validation before sequencing. Any library preparation method will produce some DNA molecules that cannot be sequenced; this issue is particularly important for PCR-free workflows, since there is no amplification step to enrich the library in DNA molecules that contain both adapter sequences. Library quantitation by qPCR allows the measurement of only DNA molecules that contain the correct adapter sequences, because the PCR primers are designed to match the specific adapters for the sequencing platform being used.
Although library quantity can also be measured using a Bioanalyzer, qPCR and fluorometric dye methods are still preferred for accurate quantitation.
Illumina recommends using KAPA® Library Quantification Kits for its sequencing systems. Standard qPCR library quantitation kits are available for all platforms from several vendors, and many researchers choose to prepare their own qPCR reagents. For Ion Torrent® sequencing libraries, Life Technologies provides qPCR kits based on TaqMan® fluorescent probe technology.
In addition to quantity, library quality must be validated. Typically, this is done using a 2100 Bioanalyzer (Agilent) electrophoresis instrument. The Bioanalyzer provides a visual representation of the range of DNA fragment sizes constituting the library, making it easy to detect potential problems like a high percentage of short DNA fragments or adapter-dimers. Although library quantity can also be measured using a Bioanalyzer, qPCR and fluorometric dye methods are still preferred for accurate quantitation.
Accurate quantitation of both input DNA and the finished library is vital for successful NGS. Since library preparation methods use enzymatic reactions, it is important to use purification systems, including Promega Maxwell® , ReliaPrep™, and Wizard® Systems, that avoid contaminants that can interfere with enzyme activity. Quantitation methods that are specific for dsDNA will give the most accurate results. While qPCR is recommended for final library validation, fluorometric methods are quick and sensitive enough for accurate quantitation of DNA prior to library preparation. The QuantiFluor® dsDNA System is a florescent dye–based assay that can measure DNA sample concentrations as low as 10pg/µL using a standard fluorometer. The QuantiFluor® Assay and Quantus™ Fluorometer have been used successfully to quantitate input DNA for NGS library preparation in a variety of applications.