November 1, 2015 (Vol. 35, No. 19)
Nathan A. Tanner Ph.D. Staff Scientist New England Biolabs
Janine G. Borgaro Ph.D. Application and Product Development Scientist New England Biolabs
Erbay Yigit, Ph.D. Applications and Product Development Scientist New England Biolabs
Don Johnson Ph.D. was Application and Product Development Scientist New England Biolabs
Julie F. Menin Ph.D. Application and Product Development Associate New England Biolabs
Eileen T. Dimalanta Ph.D. Group Leader in Applications and Product Development New England Biolabs
Nicole Nichols Ph.D. Group Leader in Applications and Product Development New England Biolabs
NEBNext Library Quant Kit Improves Accuracy and Consistency for a Broad Range of Library Types
Accurate quantitation of a next-generation sequencing (NGS) library is essential for maximizing data output and quality from each sequencing run. More specifically, in the case of Illumina sequencing, the underlying chemistries require an optimal amount of library to be loaded into the cluster generation step. Thus, the library concentration must be known precisely.
Also, if multiple libraries are sequenced in one run, it is desirable for the sequence coverage to be equal for each library and so an equal amount of each library should be moved into the cluster generation step. Again, this requires knowledge of the concentration of each library.
During library preparation, adaptors containing sequences required downstream in the sequencing workflow are attached to each end of the DNA fragments to be sequenced. With efficient library preparation, the majority of DNA fragments have an adaptor at each end, but DNA fragments with only one adaptor attached, or with no adaptors may also be present. Since these incomplete molecules cannot be sequenced, it is important that they not be included in the quantitation measurement of the library.
Multiple options are available for quantitation of NGS libraries, with advantages and disadvantages for each. Some methods such as spectrophotometry are straightforward, but they quantitate the total nucleic acid in the sample and not only the molecules of interest. Fluorometry-based methods can specifically quantitate different types of nucleic acid (dsDNA, ssDNA, RNA, etc.) but cannot specifically quantitate the molecules that will be successfully sequenced. An important feature of electrophoresis-based methods is the ability to provide library size information, but quantitation values obtained by this method can be variable.
Therefore there is a need for a quantitation method that not only quantitates accurately and consistently, but specifically quantitates only those molecules that can be sequenced (i.e., those molecules with an adaptor at each end). New England Biolabs’ NEBNext® Library Quant Kit for Illumina employs qPCR to achieve this.
Here we examine the utility of the NEBNext Library Quant Kit for a broad range of library types and sizes, while also highlighting the advantages offered by qPCR quantitation for obtaining optimal cluster density and performance consistency.
The NEBNext Library Quant Kit uses four prediluted DNA standards of known concentration and size (399 bp) and primers that target the P5 and P7 Illumina adaptor sequences to quantitate diluted library samples of interest. The use of four standards maximizes the number of libraries that can be quantitated without sacrificing performance.
The NEBNext Library Quantitation Workflow is shown in Figure 1. The full protocol can be found in the product manual, which can be downloaded at www.neb.com/e7630. This kit can be used to quantitate any Illumina library containing the P5 and P7 sequences, and so the kit quantitates only molecules with an adaptor on each end.
To analyze the use of qPCR versus electrophoretic methods for library quantitation, concentrations of four libraries were determined by the NEBNext Library Quant Kit and compared to values measured by the Agilent Bioanalyzer® (Figure 2). Compared to the qPCR-based NEBNext, concentrations obtained by the Bioanalyzer displayed a greater level of variation. This finding demonstrates the benefits of qPCR for library quantitation.
Concentrations of four libraries were determined by the NEBNext Library Quant Kit (orange) and compared to values measured using the Aglient Bioanalyzer (blue).
In addition to the method itself, consistency of the reagents involved and simplicity of a protocol both minimize variability. Replicates of 340–400 bp libraries from E. coli, H. influenzae, and human (IMR-90) genomic DNA were quantitated by four different users with the NEBNext Library Quant Kit. The NEBNext kit was able to successfully quantitate the various sample types with good consistency.
The most relevant measure of accuracy of library quantitation is the density of clusters achieved after loading the recommended amount of library. If the quantitation value is too low, more library than desired will be loaded, and over-clustering will result. In contrast, if the quantitation value is too high, less library will be loaded, producing under-clustered samples. Seven different libraries at a range of concentrations were quantitated using the NEBNext Library Quant Kit, then diluted to 8 pM and loaded into cluster generation. We observed a raw cluster density average of 1160 k/mm2 for libraries prepared with the NEBNext Kit. This falls directly in the optimal range of 900–1300 k/mm2 .
The consistent and reliable performance of a method or kit with a variety of libraries, in terms of GC content and insert size, is critical for practical utility. The ability of the NEBNext Library Quant Kit to accurately quantitate a wide range of library types was tested by using libraries from 10 different sources, including human and microbial DNA, of high GC and high AT content, and a broad range of library sizes (150–963 bp) (Figure 3). Optimal cluster density was achieved using concentrations determined by the NEBNext Library Quant Kit.
Furthermore, the NEBNext Library Quant Kit has been used to quantitate successfully libraries from 20–70% GC, with a broad range of sizes and made with several library prep kits, including NEBNext, Illumina TruSeq® Nano, and Kapa™ Hyper library prep kits.
Libraries of 310–963 bp from the indicated sources were quantitated using the NEBNext Library Quant Kit, then diluted to 8 pM and loaded onto a MiSeq (v2 chemistry; MCS v220.127.116.11). Library concentrations ranged from 7–120 nM, and resulting raw cluster density for all libraries was 965–1300 k/mm2 (ave. =1199). Optimal cluster density was achieved using concentrations determined by the NEBNext Library Quant Kit.
The NEBNext Library Quant Kit provides accurate and reliable qPCR-based library quantitation of Illumina libraries. This is shown by the production of optimal cluster densities. The NEBNext kit demonstrates improved reproducibility and consistency when compared to alternative methods and kits. Furthermore, this kit can successfully quantitate samples from a wide variety of sample types, as well as a broad range of sizes and GC-content.
NEW ENGLAND BIOLABS®, NEB® and NEBNEXT® are registered trademarks of New England Biolabs. ILLUMINA®, MISEQ® and TRUSEQ® are registered trademarks of Illumina. BIOANALYZER® is a registered trademark of Agilent Technologies. KAPA™ is a trademark of Kapa Biosystems.
Nathan A. Tanner, Ph.D. (firstname.lastname@example.org), is a staff scientist, Janine G. Borgaro, Ph.D., and Erbay Yigit, Ph.D., are applications & product development scientists, Don Johnson, Ph.D., was an applications & product development scientist, Julie F. Menin, Ph.D., is an applications & product development associate, Eileen T. Dimalanta, Ph.D., and Nicole Nichols, Ph.D., are group leaders in applications & product development, all at New England Biolabs.