Next-generation Sequencing and its Applications in Molecular Diagnostics

Zhenqiang Su; Baitang Ning; Hong Fang; Huixiao Hong; Roger Perkins; Weida Tong; Leming Shi


Expert Rev Mol Diagn. 2011;11(3):333-343. 

In This Article

Abstract and Introduction


DNA sequencing is a powerful approach for decoding a number of human diseases, including cancers. The advent of next-generation sequencing (NGS) technologies has reduced sequencing cost by orders of magnitude and significantly increased the throughput, making whole-genome sequencing a possible way for obtaining global genomic information about patients on whom clinical actions may be taken. However, the benefits offered by NGS technologies come with a number of challenges that must be adequately addressed before they can be transformed from research tools to routine clinical practices. This article provides an overview of four commonly used NGS technologies from Roche Applied Science//454 Life Sciences, Illumina, Life Technologies and Helicos Biosciences. The challenges in the analysis of NGS data and their potential applications in clinical diagnosis are also discussed.


Over the past few years, there have been remarkable advances in DNA sequencing technologies with the emergence and rapid evolution of next-generation sequencing (NGS), also known as massively parallel sequencing.[1] Examples are platforms developed by companies such as Roche Applied Science (454 Genome Sequencer FLX [GS FLX] System; CT, USA), Illumina (Genome Analyzer [GA] II; CA, USA), Life Technologies (Sequencing by Oligonucleotide Ligation and Detection [SOLiD™]; CA, USA) and Helicos BioSciences (HeliScope™ Single Molecule Sequencer; MA, USA). By sequencing DNA in a massively parallel fashion, NGS technologies have dramatically reduced both cost-per-base and time required to decode an entire human genome, making DNA sequencing a cost-effective option for many experimental approaches and allowing investigators to carry out experiments that previously were not technically feasible or affordable (e.g., sequencing thousands of cancer genomes).

Although differing in sequencing chemistries and technical details, all commercialized NGS platforms utilize a similar technical strategy – miniaturization of individual sequencing chemical reactions[1] to overcome the limited scalability of traditional Sanger sequencing,[2] which has been extensively used in somatic and germline genetic studies over the past 30 years and currently remains the gold standard for decoding DNA sequences. The miniaturization of individual sequencing reactions, coupled with other technical breakthroughs, including overcoming the bottlenecks of library preparation and template preparation,[3] allows millions of individual sequencing reactions to occur in parallel. Clonal clusters of an original DNA fragment are sequenced in each miniaturized chemical reaction, and millions of them are spatially arranged so that individual reactions are isolated from one another and can be distinctly detected by digital imaging or other approaches. The results are prodigious volumes of short-read sequence data, unprecedented detail and single-nucleotide resolution of sequence complexity, with consequential challenges in storing, managing, analyzing and interpreting such a wealth of data.

In a relatively short time span since 2005, NGS technologies have fundamentally changed high-throughput genomic research and have opened up many new research areas and novel applications.[4] With the exponential growth of the numbers of NGS-related research articles indexed on Medline (Figure 1), NGS technologies have demonstrated their enormous potential for researchers working in medicine, biology and life sciences. Along with the development of robust informatics tools for nucleotide variant detection,[5] the ongoing evolution of NGS technologies will continually reduce the cost, simplify the workflow for sample preparation and improve the technical robustness,[6,7] paving the path for translating NGS technologies into clinical diagnostics and personalized medicine.

Figure 1.

The number of publications related to next-generation sequencing and indexed in PubMed has been increasing exponentially. The numbers reflect the PubMed search results by using the following query on 11 October, 2010: ('next generation sequencing' OR 'next-generation sequencing' OR 'next generation DNA sequencing' OR 'next-generation DNA sequencing' OR 'RNA-Seq' OR 'Chip-Seq' OR 'mRNA-Seq' OR 'PeakSeq' OR '454 sequencing' OR 'direct RNA sequencing' OR 'massively parallel sequencing' OR 'ultrafast DNA sequencing' OR "deep sequencing" OR '454 Life Sciences') AND (2004[Publication Date]:2010[Publication Date]).

In this article, we first provide an overview of the principles of four commercialized NGS technologies. We then discuss the general challenges in the analyses of NGS short reads and, finally, we discuss the possible impacts and applications of NGS technologies in clinical diagnostics.


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