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Figure 5-7. The PacBio SMRT sequencing platform. The Pacific Biosciences (PacBio) single-molecule, real-time (SMRT) sequencing technology uses a single highly processive, strand-displacing DNA polymerase that is immobilized to the base of a matrix in a fluorescence-detection well. (A) Each SMRT reaction cell contains 150,000 zero-mode waveguides (ZMWs), which are nanophotonic confinement wells that are illuminated from below and have a detection volume of 20 zeptoliters (10-21 liters). The technology uses ZMWs to observe the base incorporations of an anchored polymerase molecule, which allows light to illuminate only the bottom of the well, in which a single DNA polymerase plus DNA template complex is immobilized. (B) The current version of the SMRT technology uses SMRTbell template preparation to generate a circularized, double-stranded DNA template from appropriately sized fragments of the genomic DNA, generated either by random shearing or by amplification of target regions of interest. Universal hairpin adaptors are then ligated onto the ends of the DNA fragment to generate the SMRTbell library. These hairpin dimer sequences are removed at the end of the library preparation protocol. The sequencing primer is then annealed to the SMRTbell template, followed by binding of the DNA polymerase, to form a complex, which is then immobilized to the bottom of the ZMW well. (C) In each ZMW well, a single molecule of the DNA polymerase sequences a single nucleotide at a time of a single molecule of DNA template. Phospholinked nucleotides, each labeled with a different fluorescently linked dye, are introduced into the ZMW well. As a nucleotide binds the polymerase-DNA complex in the ZMW well, the fluorescently labeled phospholinked nucleotide emits a light pulse that is detected. When the phosphodiester bond is cleaved during the DNA elongation reaction, the fluorescent dye is released and diffuses away from the ZMW detection zone and the light emitted by that nucleotide is no longer detected. The fluorescent read output from incorporation of the four different fluorescent dyes is captured for the entire SMRT cell over time and processed by a computer into sequence reads (one read per ZMW well). (D) With the latest chemical reagent kits, SMRT technology can provide real-time sequence reads of 5–60 kB in length. Adapted from copyrighted publications from PacBio, with permission.
Currently, the Illumina and PacBio sequencing platforms are often used in combination to determine the complete genome sequence of a new bacterial isolate. If a complete genome is already available for a closely related bacterium such that that genome can be used as a template, then sequence assembly of overlapping contigs from a new bacterial isolate can readily be completed without any or only a few gaps remaining to be filled. For this, the shorter reads obtained by the Illumina platform are sufficient. For de novo genome sequencing (where no existing complete genome is available for a related bacterium), the longer reads of the PacBio platform provide a scaffold and high-quality draft sequence that can be rapidly polished by the high accuracy and coverage (i.e., the large number of overlapping sequence reads used to accurately call a sequence) provided by the Illumina sequencing. As these DNA sequencing methods develop, it will become increasingly quick and cost-effective to not only take the census of bacterial species in complex microbial communities, but also to sequence parts or entire genomes directly from the mixture of genomic DNA isolated from these populations.
To understand the power of current whole-genome sequencing technologies, let us consider a common, recurring problem in bacterial genetics. Interesting mutations often arise spontaneously in bacteria whose complete genomes have already been determined. Researchers are often interested in knowing what these mutations are, particularly if the bacterium is a pathogen. Classical bacterial genetics provides exceedingly clever ways to map mutations so that they can be located by conventional sequencing of a limited region of the chromosome of the mutant strain. However, these classical methods are often time-consuming and are far from foolproof, especially in bacterial species lacking powerful genetic systems, such as many bacterial pathogens.
For example, the Illumina sequencing technology is routinely used to locate mutations in bacteria, such as Streptococcus pneumoniae, whose genome contains about 2.2 million base pairs. In this case, chromosomal DNA isolated from mutant strains is sheared into random fragments of about 500 base pairs. Adaptors required for hybridization during the sequencing method are ligated to the ends of the DNA fragments, and the resulting products are amplified by PCR to give random libraries of genomic fragments from each mutant. The adaptors used for each mutant genome have slightly different sequences (“barcodes”), so that DNA sequences from more than one mutant can be sequenced simultaneously and later distinguished. The resulting barcoded libraries are mixed and subjected to Illumina sequencing. Currently, with the highest-capacity instruments, such a sequencing run yields about 120 billion bases of sequence comprised of approximately 400 million reads with lengths of up to 300 bases! Thus, the base-pair coverage of a single S. pneumoniae genome in a reaction lane would be about 27,000-fold in this example. This extraordinarily high level of coverage means that barcoded, fragmented genomic libraries from 27 different mutants could be sequenced simultaneously at 1,000-fold coverage in a single sequencing run in less than a week. Subsequent bioinformatic sequence comparisons with the known genome sequence of S. pneumoniae could then reveal the locations of single-nucleotide point mutations, small and large deletions, or even regions of chromosomal duplication that contribute to the mutant phenotypes. And what is the cost? At this writing, the cost of the sequence determination and bioinformatic analyses per mutant genome is far less than the cost of classical genetic approaches, which previously required weeks to months to obtain and, if they worked, would yield far less information compared to less than one week for the large-scale sequencing analysis.
16S rRNA Gene-Based Profiling of Complex Microbial Communities. Scientists are often interested in understanding the effect of certain conditions or factors over time on composition of the microbial community. For instance, a researcher might be interested in the effect of diet, hormones, or age on the composition of the microbiota, or how antibiotic therapy impacts the microbiota. To accomplish this, it is necessary to collect at any given time a sample of the microbes that is representative of the whole community (a profile) such that changes in abundance or diversity can be monitored.
One profiling method involves DNA microarray chips, called phylochips, which are comprised of tens of thousands of oligonucleotide-containing spots, each corresponding to a set of taxa-defining sequences, including rRNA gene regions and other unique gene sequences that can distinguish among the various microbial species (taxa or “phylotypes”) present in the sample of interest (Figure 5-8). To design appropriate phylochips, the microbial species present in environmental niches, including different areas of the human body, must first be identified by rRNA gene sequencing methods from a representative sample. Oligonucleotide probes that will hybridize to the rRNA genes from each species are then printed onto the microarray. These phylochips can be used to monitor shifts in microbial community compositions in environmental and clinical samples. The greatest advantage of this phylochip technology is that all microorganisms of interest in an entire community, not just bacteria, can be detected in a single assay by multiple probes to give reliable taxonomic information.
Figure 5-8. Phylochips for microbial-community profiling. Current phylochip platforms are a microarray-based method that taxonomically identifies microbes in a given sample through hybridization of the rRNA gene in that sample to all nine of the variable regions of the 16S rRNA gene. (A) General procedure for making a phylochip. Complete genomes are retrieved from the National Center for Biotechnology Information microbial database and a phylogenetic relationship tree is generated. Genome comparison of bacteria within each clade (group of related bacteria) is used to identify core gene sequences that are shared by most members of the clade. After sequence alignment with all other bacterial and archaeal genomes, core gene sequences that are unique to each target clade (species) are identified, and DNA probes are designed against up to 10 of the unique genes for each target species. (B) Example of how a phylochip could be used to distinguish microbes at the genus and species levels in a sample. Positive and negative controls are included to ensure that the hybridization steps worked and that there is no background detection, respectively.
