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Chapter 19 : Next-Generation DNA Sequencing and Microbiology

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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. This chapter reviews on the technology behind these “next-generation” platforms and compares the three that are currently the most popular and also reviews applications to date of these new technologies to microbiology research. In the chapter, steps that are similar and shared among the next-generation sequencing platforms are first described generically. The following sections provide a few examples of microbiological applications of deep-sequencing technologies. The study of vaginal flora has been the subject of many culture-based investigations over the years, but recently it has been recognized that many of the organisms actually present are not represented among those that can be recovered by in vitro cultivation. It may be able to simultaneously determine drug resistance, either at the mutational level within specific viral or bacterial genes or by determining the presence of expressed drug resistance genes such as those encoding beta-lactamases or other markers. In all likelihood, deep-sequencing technologies will make their way into clinical laboratories in the next several years, probably starting out with genetic and oncology testing but ultimately moving into molecular microbiology laboratories as well.

Citation: Higuchi R, Gyllensten U, Persing D. 2011. Next-Generation DNA Sequencing and Microbiology, p 301-312. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch19

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Next-Generation Sequencing Methods
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Microbial Ecology
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Non-Nucleoside Reverse Transcriptase Inhibitors
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Sequencing Methods
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Figures

Image of FIGURE 1
FIGURE 1

Single-molecule amplification. Shown is a representation of single-molecule PCR amplification in a polyacrylamide gel. Isolated DNA molecules that have had adapter sequences ligated to them are shown in the gel. The adapter sequences include at each end different PCR primer annealing sites. Also in the gel from before its casting are PCR components including primers, DNA polymerase, and dNTPs. After casting onto a microscope slide, the gel is subjected to thermocycling. The PCR components, including primers, are able to diffuse within the gel, whereas the higher-molecular-weight template molecules are not. This results in the localized accumulation of amplicon within the gel as depicted. Millions of these nanometer-scale polonies can be formed on a single slide.

Citation: Higuchi R, Gyllensten U, Persing D. 2011. Next-Generation DNA Sequencing and Microbiology, p 301-312. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch19
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Image of FIGURE 2
FIGURE 2

Single-molecule amplification. Shown is a representation of emulsion PCR, a two-phase (aqueous and oil) system in which PCR components are included in the aqueous phase. Also included are capture beads upon which one of the two PCR primers is attached. 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 occurring 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 and a droplet with only either a bead or a template (shown). In these cases no amplification can occur. The possibility of multiple templates and/or beads per droplet, which will result in amplification, is minimized as much as possible by concentration. Multiple templates per droplet will usually result in a mixed sequence read from the capture bead, and these are ultimately filtered out by the software.

Citation: Higuchi R, Gyllensten U, Persing D. 2011. Next-Generation DNA Sequencing and Microbiology, p 301-312. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch19
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Image of FIGURE 3
FIGURE 3

Single-molecule amplification. A representation of single-molecule bridge PCR. On a microscope slide are immobilized high densities 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 the 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: Higuchi R, Gyllensten U, Persing D. 2011. Next-Generation DNA Sequencing and Microbiology, p 301-312. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch19
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Image of FIGURE 4
FIGURE 4

The capture of beads carrying amplicon. (A) In the 454 system, capture beads are mixed in a slurry with other beads containing immobilized enzymes for pyrosequencing. By centrifugal force, the beads in the slurry are packed into nanometer-scale wells of an “optical plate” (represented is one of millions of such wells). The density of capture beads is adjusted such that there are more wells than capture beads and that most wells with capture beads have only one bead. The other beads are in amounts such that they fill up and pack all the wells. Sequencing reagents are flowed over the filled plate, and light emitted as a result from wells containing amplicon is detected through the bottom of the well, as depicted. (B) In the ABI SOLiD system, the 3′ ends of primer extensions on the bead are modified to allow chemical linkage to the modified surface of a glass slide. Beads are captured at a low enough density such that most beads and their DNA do not overlap. Millions of such beads are captured. Sequencing reagents are flowed over the slide (see Fig. 5 ), and fluorescence is excited and emitted as shown.

Citation: Higuchi R, Gyllensten U, Persing D. 2011. Next-Generation DNA Sequencing and Microbiology, p 301-312. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch19
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Image of FIGURE 5
FIGURE 5

Sequencing chemistries. Shown is Illumina Solexa chemistry, in which 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: Higuchi R, Gyllensten U, Persing D. 2011. Next-Generation DNA Sequencing and Microbiology, p 301-312. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch19
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Image of FIGURE 6
FIGURE 6

Sequencing chemistries. Shown is ABI SOLiD chemistry in which labeled octonucleotides with blocked 3′ termini are ligated specifically to the end of a primer one at a time. The specificity is imparted by the central dinucleotide, which is flanked by universal and degenerate nucleotides. Four fluorophore colors are each assigned to 4 of 16 possible dinucleotidecontaining octonucleotides. After each ligation the color is recorded, the block is removed, and the next ligation is made. After five such ligations the extended primer strand is removed, a new primer, offset from the original by –1 nt, is annealed, and a new set of specific incorporations is recorded. This process is repeated with primers that progressively regress to –5 nt, allowing 35 nt of sequence to be read. The sequence is decoded from the series of colors recorded for each ligation.

Citation: Higuchi R, Gyllensten U, Persing D. 2011. Next-Generation DNA Sequencing and Microbiology, p 301-312. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch19
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Image of FIGURE 7
FIGURE 7

Sequencing chemistries. Shown is Roche 454 chemistry, in which nucleotides are flowed one at a time over the nanowell plate. A specific primer is used as shown. If the complementary nucleotide is present in the template, the nucleotide is incorporated. Shown are three dTs in the template strand such that three dATPs are incorporated. For each molecule of dATP incorporated, a pyrophosphate (PP) is released, starting the luminescence process by which light is generated. For three dATPs incorporated, three times as much light is produced than if only one were incorporated. For the next nucleotide flowed, dTTP, no complementary nucleotide is present in the template, so no light is generated. For the next nucleotide flowed, dCTP, a single dG is present in the template, resulting in the incorporation of as much light as the previous incorporation. Then, dGTP is flowed, and the process is begun again with dATP.

Citation: Higuchi R, Gyllensten U, Persing D. 2011. Next-Generation DNA Sequencing and Microbiology, p 301-312. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch19
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