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Simultaneous digital quantification and fluorescence-based size characterization of massively parallel sequencing libraries
Matthew T. Laurie1, Jessica A. Bertout1, Sean D. Taylor1, Joshua N. Burton2, Jay A. Shendure2, and Jason H. Bielas1,3, 4
1Translational Research Program, Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA
2Department of Genome Sciences, University of Washington, Seattle, WA
3Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA
4Department of Pathology, University of Washington, Seattle, WA
BioTechniques, Vol. 55, No. 2, August 2013, pp. 61–67
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Due to the high cost of failed runs and suboptimal data yields, quantification and determination of fragment size range are crucial steps in the library preparation process for massively parallel sequencing (or next-generation sequencing). Current library quality control methods commonly involve quantification using real-time quantitative PCR and size determination using gel or capillary electrophoresis. These methods are laborious and subject to a number of significant limitations that can make library calibration unreliable. Herein, we propose and test an alternative method for quality control of sequencing libraries using droplet digital PCR (ddPCR). By exploiting a correlation we have discovered between droplet fluorescence and amplicon size, we achieve the joint quantification and size determination of target DNA with a single ddPCR assay. We demonstrate the accuracy and precision of applying this method to the preparation of sequencing libraries.

Massively-parallel next-generation sequencing (NGS) technology is rapidly revolutionizing the fields of genomics, molecular diagnostics, and personalized medicine through the increasingly efficient and economical generation of unprecedented volumes of data (1-7). A common characteristic of the various commercially available NGS technologies is the need to load a precise number of viable DNA library molecules onto the instrument to optimize the yield of data from an individual sequencing experiment (8-11). Performing a sequencing run with either too many or too few library molecules results in compromised data yields or completely failed sequencing runs that waste sample, expensive reagents, user time, and instrument time. Similarly, if library molecules are not the appropriate length to fully utilize the capabilities of the sequencing platform, fewer bases can be sequenced in an NGS run and the throughput is wasted. Thus, accurate quantification and size determination of library DNA is essential for achieving optimal data yield and maximizing a laboratory's efficiency and sequencing throughput.

Protocols for the preparation of NGS libraries include quality control steps to validate the size and concentration of amplifiable library molecules (i.e., molecules properly ligated to NGS adapter sequences) before committing to a sequencing run. Manufacturers typically recommend quantification with quantitative real-time PCR (qPCR) and size determination with gel or capillary electrophoresis. It is also possible to enumerate library DNA using UV spectrophotometry, the Quant-iT PicoGreen assay, or the Agilent BioAnalyzer, but these methods are not ideal because they quantify amplifiable and non-amplifiable molecules equally (12-14). These methods are also only capable of measuring mass per volume, which must be converted to copy number using an estimated average size of library molecules which can introduce further error (15). Although qPCR is widely considered the best option for library quantification, there are considerable drawbacks to the method, including amplification biases due to template size and GC-content as well as the need for a standard curve to estimate the absolute quantity of DNA (16). Creating a standard curve for each sample to be analyzed is a difficult and uncertain process that leads to inaccuracies in measurements of absolute target quantity (15, 17). When intercalating dyes are used for quantification, the concentration reading can include non-amplifiable DNA as dyes measure dsDNA indiscriminately. Because of these potential inaccuracies, some NGS platform manufacturers recommend performing titration runs on their instrument to determine the proper loading amount. The high cost of reagents and the length of NGS runs make this an expensive and time-consuming step.

Method summary

QuantiSize allows for the determination of absolute quantity and size distribution of target DNA molecules in a single experiment. This assay exploits a correlation, reported herein, between the length of an amplified DNA molecule and the fluorescence amplitude produced in droplet digital PCR (ddPCR), to allow the user to calculate the size of unknown DNA. As ddPCR simultaneously measures the concentration of target DNA, the user can accurately determine the target population size and quantity in a single step.

We have developed a new assay capable of concurrently measuring the absolute concentration and length of unknown amplifiable DNA templates, making it well suited for quality control of NGS libraries. The assay, which we have termed QuantiSize, is based on the previously validated droplet digital PCR (ddPCR) absolute quantification system (18, 19) and adds the ability to calculate the size of target DNA by exploiting a linear correlation we have discovered between the fluorescence amplitude of ddPCR droplets and the size of amplicons within them. As a quantification method, ddPCR has demonstrated greater precision and sensitivity than real-time PCR (18). We demonstrate that QuantiSize accurately measures the size and concentration of target DNA simultaneously while avoiding the limitations of other quantification systems and, we highlight the utility of this assay for preparation of NGS libraries.

Materials and methods

Purification of DNA size standards

An exACTGene 50 bp DNA Ladder (Fisher, Waltham, MA) and 1 kb Plus DNA Ladder (Fisher) were run on a 1.0% UltraPure Low-Melting Point Agarose (Invitrogen, Carlsbad, CA) electrophoresis gel and the 25, 50, 100, 200, 300, 400, 500, 600, 700, 800, and 1000 bp ladder bands were manually excised. The DNA in these gel slices was purified using the QIAcube automated gel extraction protocol with the QIAquick Gel Extraction Kit (QIAGEN, Hilden, Germany). The size and purity of all DNA fragments were verified by gel electrophoresis.

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