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.
[Show abstract][Hide abstract] ABSTRACT: Two years ago we described the first droplet digital PCR (ddPCR) system aimed at empowering all researchers with a tool that removes the substantial uncertainties associated with using the analogue standard, quantitative real-time PCR (qPCR). This system enabled TaqMan hydrolysis probe-based assays for the absolute quantification of nucleic acids. Due to significant advancements in droplet chemistry and buoyed by the multiple benefits associated with dye-based target detection, we have created a "second generation" ddPCR system compatible with both TaqMan-probe and DNA-binding dye detection chemistries. Herein, we describe the operating characteristics of DNA-binding dye based ddPCR and offer a side-by-side comparison to TaqMan probe detection. By partitioning each sample prior to thermal cycling, we demonstrate that it is now possible to use a DNA-binding dye for the quantification of multiple target species from a single reaction. The increased resolution associated with partitioning also made it possible to visualize and account for signals arising from non-specific amplification products. We expect that the ability to combine the precision of ddPCR with both DNA-binding dye and TaqMan probe detection chemistries will further enable the research community to answer complex and diverse genetic questions.
[Show abstract][Hide abstract] ABSTRACT: In this study we present a highly customizable method for quantifying copy number and point mutations utilizing a single-color, droplet digital PCR platform. Droplet digital PCR (ddPCR) is rapidly replacing real-time quantitative PCR (qRT- PCR) as an efficient method of independent DNA quantification. Compared to its highly variable counterpart, ddPCR eliminates standards and measures target and reference DNA within the same well. The applications for ddPCR are widespread including targeted quantitation of genetic aberrations, which is commonly achieved with a two-color fluorescent oligonucleotide probe (TaqMan) design. The cost and optimization can be greatly reduced, however, with an alternative method of distinguishing between target and reference products using the non-specific DNA binding properties of EvaGreen (EG) dye. By manipulating the length of the target and reference amplicons we can distinguish between their fluorescent signals and quantify each independently. We demonstrate the effectiveness of this method by examining copy number in the proto-oncogene FLT3 and the common V600E point mutation in BRAF. Using a series of well-characterized control samples and cancer cell lines, we confirmed the accuracy of our method in quantifying mutation percentage and integer value copy number changes. Additionally, our assay was able to detect a mutation comprising less than 1% of an otherwise wild-type sample, as well as a copy number change in a sample of 20% amplified tumor. This flexible and cost-effective method of independent DNA quantification proves to be a robust alternative to the commercialized TaqMan assay.
[Show abstract][Hide abstract] ABSTRACT: Cancer is malignant disease that causes many deaths worldwide every year, with most deaths occurring in the middle and advanced stages of cancer. Numerous deaths can be avoided by detecting cancer at an early stage, making early diagnosis and timely therapy critical for cancer treatment. Analyses at the level of nucleic acids rather than phenotypes can eliminate various false-positive and -negative results, and diagnoses can occur at an earlier stage. Many techniques have been developed for this purpose, including capillary electrophoresis (CE), which has the advantages of high-efficiency, high-speed, high-throughput, automation, cleanliness, and versatility, and CE can be conducted on a microscale or coupled with other separation techniques. These advantages afford this technique the ability to meet the future medical requirements that will undoubtedly call for amassing large numbers of samples for analysis, suggesting that CE may become an important tool for providing data in clinical cancer diagnosis and therapy. This review focuses on CE-based nucleic acid detection as it is applied to cancer diagnosis and therapy, and provides an introduction to the drawbacks and future developments of analysis with CE.
The Analyst 05/2014; 139(14). DOI:10.1039/c4an00400k · 4.11 Impact Factor
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