During library creation, adaptor sequences are appended to both ends of the DNA molecules in a sample. The workflow for these new sequencing technologies proceeds as follows: library creation, library quantification, massively parallel clonal PCR amplification of library molecules, and sequencing. A key advance facilitating higher throughput and lower costs for several of these platforms was migration from the clone-based sample preparation used in Sanger sequencing to the massively parallel clonal PCR amplification of sample molecules on beads (Roche 454 and ABI Solid) or on a surface (Solexa). The digital PCR assay allows absolute quantification of sequencing libraries, eliminates uncertainties associated with the construction and application of standard curves to PCR-based quantification, and with a coefficient of variation close to 10%, is sufficiently precise to enable direct sequencing without titration runs.Ī new generation of sequencing technologies based on sequencing by synthesis and sequencing by ligation are revolutionizing biology, biotechnology, and medicine. This study is the first to definitively demonstrate the successful sequencing of picogram quantities of input DNA on the 454 platform, reducing the sample requirement more than 1000-fold without pre-amplification and the associated bias and reduction in library depth. We successfully sequenced low-nanogram scale bacterial and mammalian DNA samples on the 454 FLX and Solexa DNA sequencing platforms. We demonstrate the use of digital PCR to accurately quantify 454 and Solexa sequencing libraries, enabling the preparation of sequencing libraries from nanogram quantities of input material while eliminating costly and time-consuming titration runs of the sequencer. Second, each library requires a titration sequencing run, thereby increasing the cost and lowering the throughput of sequencing. For many applications, including metagenomics and the sequencing of ancient, forensic, and clinical samples, the quantity of input DNA can be critically limiting. First, large amounts of sample-typically micrograms-are needed for library preparation, thereby limiting the scope of samples which can be sequenced. This requirement has two unfavorable consequences. Yu B (2014) Setting up next-generation sequencing in the medical laboratory.Next-generation DNA sequencing on the 454, Solexa, and SOLiD platforms requires absolute calibration of the number of molecules to be sequenced. Xuan J, Yu Y, Qing T et al (2013) Next-generation sequencing in the clinic: promises and challenges. Williams R, Peisajovich SG, Miller OJ et al (2006) Amplification of complex gene libraries by emulsion PCR. Meyerhans A, Vartanian JP, Wain-Hobson S (1990) DNA recombination during PCR. J Biotechnol 102:117–124ĭressman D, Yan H, Traverso G et al (2003) Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations. Nakano M, Komatsu J, Matsuura S et al (2003) Single-molecule PCR using water-in-oil emulsion. Nat Methods 7:111–118īuermans HP, den Dunnen JT (2014) Next generation sequencing technology: advances and applications. Mamanova L, Coffey AJ, Scott CE et al (2010) Target-enrichment strategies for next-generation sequencing. Gullapalli RR, Desai KV, Santana-Santos L et al (2012) Next generation sequencing in clinical medicine: challenges and lessons for pathology and biomedical informatics. Metzker ML (2010) Sequencing technologies – the next generation. For routine clinical testing, following library generation, we employ the automated Ion OneTouch™ System that includes the Ion OneTouch™ 2 and the Ion OneTouch™ ES instruments for template generation and enrichment of template-positive ISPs, respectively. We describe the methods of preparation and enrichment of template-positive Ion PGM™ Template OT2 200 Ion Sphere™ Particles (ISPs) on the Ion Personal Genome Machine ® (PGM™) System. Here, we discuss the basic principles, advantages, and challenges of applications of emPCR in clinical testing. Ideally, the dilution is to a degree where each droplet contains a single template molecule and functions as a micro-PCR reactor. The basic principle of emPCR is dilution and compartmentalization of template molecules in water droplets in a water-in-oil emulsion. Emulsion PCR (EmPCR) is a commonly employed method for template amplification in multiple NGS-based sequencing platforms.
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