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Chapter 5 : Massively Parallel DNA Sequencing and Microbiology

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Massively Parallel DNA Sequencing and Microbiology, Page 1 of 2

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Abstract:

Recent, dramatic increases in the throughput of DNA sequencing instruments provide opportunities for microbiology research that previously were limited to large genome centers. In sequencing the human genome, what once took years and hundreds of instruments and workers to complete can now be done in days on just one instrument. Moreover, such increases in throughput are likely to continue over the next few years. Here we review the different platforms for massively parallel sequencing (also called next-generation sequencing, or NGS) and compare those that are currently most popular. As evidenced by other chapters of this book, some of the exciting applications of NGS for microbiology research are the abilities to accurately deduce microbial population structure (also known as metagenomics), discover new pathogens, detect rare pathogens within a complex background, and elucidate the dynamics of drug resistance—even when culture of the microorganisms is impossible. These new technologies are directly applicable to and have significant ramifications for both research and clinical microbiology.

Citation: Gyllensten U, Higuchi R, Persing D. 2016. Massively Parallel DNA Sequencing and Microbiology, p 58-67. 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.ch5
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Figures

Image of FIGURE 1
FIGURE 1

Emulsion PCR for single-molecule amplification. A two-phase (aqueous and oil) system in which PCR components are included in the aqueus phase. Also included are capture beads upon which one of the two PCR primers is attached. In the Ion Torrent system these beads are magnetic to facilitate their subsequent capture and washing. With vigorous agitation, emulsification takes place such that millions of tiny water droplets form in the oil. The concentration of DNA templates, beads, and water droplets is such that the frequency of there being one bead and one DNA template in one droplet is optimized. In this case, an amplicon attached to the bead as shown will form. Other possibilities include a droplet with neither bead nor template or a droplet with only a bead or a template (shown). In these cases no amplification can occur. The possibility of multiple templates and/or beads per droplet, which results in amplification, is minimized as much as possible by concentration. Multiple templates per droplet usually result in a mixed sequence read from the capture bead and are ultimately filtered out in software.

Citation: Gyllensten U, Higuchi R, Persing D. 2016. Massively Parallel DNA Sequencing and Microbiology, p 58-67. 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.ch5
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Image of FIGURE 2
FIGURE 2

A representation of single-molecule, bridge PCR. Immobilized on a microscope slide are a high density of both forward and reverse primers with the 3′ ends free. Single DNA template molecules are flowed over the slide and captured by the PCR primers as shown. Rounds of replication and denaturation through thermocycling are allowed such that 3′ ends of primer extensions can anneal and reanneal to reverse primers, each time being copied again. The original template strands get released to solution in this process but are washed away. All the copies of the original template strand stay localized as shown.

Citation: Gyllensten U, Higuchi R, Persing D. 2016. Massively Parallel DNA Sequencing and Microbiology, p 58-67. 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.ch5
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Image of FIGURE 3
FIGURE 3

The capture of beads carrying amplicon and sequencing chemistry: Ion Torrent system. (A) A single bead in a single well of the array of wells on the sequencing chip. The beads—enriched for those that are carrying amplified template—are deposited in the chip wells by a short centrifugation step. The density of capture beads is adjusted such that there are more wells than capture beads and most wells with capture beads contain only one bead. Shown attached to the beads are single-stranded amplicon templates extended from a primer attached to the bead. Annealed to each template is a sequencing primer. (B) A closeup of the bead shows the sequencing chemistry employed. Sequencing reagents are flowed over the filled plate, including one of the four dNTPs at a time. For each incorporated nucleotide molecule, a hydrogen ion (H) is released. The sum of all such incorporations over all template molecules is recorded by the ion-sensitive field-effect transistor fabricated into the bottom of the well. The nucleotides are not modified or terminated such that if the template contains, for example, three sequential deoxyribosyladenines, three dTTP molecules are incorporated and three hydrogen ions are released. This is recorded as three times the signal as a single incorporation. If there is no base in the template complementary to the currently flowed dNTP, no signal is generated.

Citation: Gyllensten U, Higuchi R, Persing D. 2016. Massively Parallel DNA Sequencing and Microbiology, p 58-67. 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.ch5
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Image of FIGURE 4
FIGURE 4

Illumina sequencing chemistry. Different fluorophore-labeled nucleotides (f1 through f4) with blocked 3′ termini are added specifically to the end of a primer one at a time. After each addition the fluorophore color is recorded by the imaging system, the block and fluorophore are removed, and the next addition is made.

Citation: Gyllensten U, Higuchi R, Persing D. 2016. Massively Parallel DNA Sequencing and Microbiology, p 58-67. 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.ch5
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Image of FIGURE 5
FIGURE 5

Single-molecule sequencing: the Pacific Biosciences system. Shown is a single, micro-fabricated nanowell with a light-transmitting bottom and with dimensions smaller than the wavelengths of light used to illuminate the well. This results in a “zero mode waveguide” with a narrow zone of illumination as shown. The nanowell has a single DNA polymerase molecule attached to its bottom within this zone. When overlaid with a solution of prepared DNA templates (also known as a library) at a suitable concentration, the polymerase is able to capture and bind a single primer-template molecule, as shown. Present in the solution are the four dNTPs, each labeled with a different color of fluorescent dye as shown and mostly outside the zone of illumination. Also shown is one of the four dNTPs bound to the polymerase-template-primer complex. This is the next base to be incorporated opposite its complementary base. The fluorescent tag is bound long enough for its presence and color to be detected. Subsequently, completion of the incorporation reaction results in cleavage of the fluorescence tag and its diffusion away from the polymerase such that it will not interfere with the detection of the next, incoming dNTP.

Citation: Gyllensten U, Higuchi R, Persing D. 2016. Massively Parallel DNA Sequencing and Microbiology, p 58-67. 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.ch5
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