Chapter 6 : Next-Generation Sequencing

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Next-generation sequencing (NGS), otherwise known as deep or massively parallel sequencing, refers to the technological advances in DNA sequencing instrumentation that enable the generation of hundreds of thousands to millions of sequence reads per run. Sequencing of the human genome, which was once a >10-year endeavor by the NIH at the cost of approximately $3 billion (1), can now be done routinely on a single instrument. Rapid advances in technology led to the first-ever FDA clearance of an NGS instrument, the Illumina MiSeq, in 2014 (2), and the development of rapid, miniaturized sequencing devices such as the Oxford Nanopore are ongoing (3). The applications of NGS are wide-ranging and include (i) whole-genome sequencing, (ii) pathogen discovery, (iii) metagenomic/microbiome analyses, (iv) transcriptome profiling, and (vi) infectious disease diagnosis. Here we will focus on NGS technology and the last three applications, because the first two topics are described in detail elsewhere.

Citation: Chiu C, Miller S. 2016. Next-Generation Sequencing, p 68-79. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch6
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Image of FIGURE 1

Sequencing methods for currently available NGS platforms. (A) The sequencers manufactured by Roche/454 (left), SOLiD (middle), and Ion Torrent (right) all use bead-based emulsion PCR (rectangular inset) in the library generation process, followed by different approaches to fluorescent-based sequencing. (B) Illumina sequencing involves library generation on a flow cell via a sequencing-by-synthesis approach and the imaging of millions of fluorescent flow cell clusters. (C) PacBio sequencing is performed by a DNA polymerase enzyme affixed to a glass substrate in a zero-mode waveguide nanostructure. Each nanostructure generates an individual sequence. (D) Nanopore sequencing, as performed by the Oxford Nanopore MinION instrument, leverages the voltage conductance changes (left) that occur in response to passage of DNA through a nanopore (right), a protein in the lipid-bilayer membrane containing a single hole that allows a single molecule of DNA to pass through.

Citation: Chiu C, Miller S. 2016. Next-Generation Sequencing, p 68-79. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch6
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Image of FIGURE 2

Schematic overview of an NGS pipeline. Sample processing for NGS involves a stepwise process of nucleic acid extraction, library preparation, and sequencing on a dedicated instrument (left). Following generation of raw data, bioinformatics analysis of metagenomic or microbial NGS data includes preprocessing, assembly, host subtraction, pathogen identification, taxonomic classification, and results reporting/visualization (right). The asterisks denote optional steps in the procedure.

Citation: Chiu C, Miller S. 2016. Next-Generation Sequencing, p 68-79. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch6
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Generic image for table

Comparison of NGS platforms

Citation: Chiu C, Miller S. 2016. Next-Generation Sequencing, p 68-79. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch6

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