data from a 16S rRNA gene analysis are only semi-quantitative. Part of the problem is that endpoint PCR (see Figure 5-1B) is not strictly quantitative. This is due to the fact that some sequences seem to be amplified more efficiently than others (often referred to as PCR bias). Experience has shown that even some major members of the population may be misrepresented due to the absence of a strict quantitative representation of the microbiota. Nonetheless, the analysis gives an idea of what the leading members of the population are and provides a general assessment of their relative abundance.
Quantitative real-time PCR (qPCR). Once the members of a microbial community have been identified, the relative representation or abundance of those bacterial species can be determined by another method, called quantitative real-time PCR (qPCR). In one common variation of qPCR (Figure 5-5), genomic DNA from the microbial community is prepared and used directly as the template in PCR amplification reactions containing primer pairs that anneal specifically to the 16S rRNA genes of the bacterial species of interest in the population. The course of the PCR reaction is followed by an increase in fluorescence caused by intercalation of a dye, such as SYBR green, into the double-stranded PCR products. Alternatively, sequence-specific DNA probes labeled with the fluorescent dye at one end and a fluorescence quencher moiety at the other end can be used to detect the DNA of interest after hybridization of the labeled probe with its complementary sequence. The quencher is removed by the 5′ to 3′ exonuclease activity of the thermostable DNA polymerase, which allows for detection of fluorescence from the probe bound to its complementary DNA. Newer probe designs allow for enhanced specificity and sensitivity of detection, and when coupled with different fluorescent labels enable simultaneous detection of multiple target DNAs (multiplexing).
Figure 5-5. qPCR used to quantify specific bacteria in complex samples. Unlike conventional PCR, quantitative real-time PCR (qPCR) allows for detection of the target DNA in real time during the amplification process by measuring the fluorescence intensity above a given threshold. (A) Unbound fluorescent dye (typically SYBR green) does not fluoresce in the presence of ssDNA template, but does fluoresce when bound to newly formed dsDNA during the amplification process. (B) To eliminate fluorescence due to nonspecific binding of fluorescent dye, fluorescently labeled probes complementary to the target DNA template have quenchers attached that prevent fluorescence emission until the probes are degraded by the 5′ to 3′ exonuclease activity of the polymerase during the PCR amplification reaction. (C) Newer probe designs include fluorescence quenchers attached to the probe. Shown are two examples of such fluorescent dye-quencher probe designs: one as a single probe with step-loop structures to keep the fluorescent dye and quencher together and the other one as a duplex with one strand having the fluorescent dye and the other complementary strand having the quencher. The quenchers are removed during the second amplification cycle, such that the fluorescently labeled probe sequence can then hybridize to the complementary sequence on the newly synthesized target sequence. The blocker prevents the polymerase from extending the PCR primer. The fluorescence detected is directly proportional to the amount of target DNA present in the sample. (D) For qPCR analysis, the fluorescence is plotted versus the number of PCR thermocycles on a logarithmic scale, where the number of cycles at which the fluorescence is detected above the threshold is referred to as the threshold cycle (Ct), which is proportional to the amount of target DNA template in the sample.
For quantification, the qPCR procedure determines the kinetics of the increase in fluorescence intensity after each round of PCR amplification—this is the “real-time” aspect of the method—and relates these kinetics to a parameter called the threshold cycle number (Ct), which is inversely proportional to the starting concentration of the template DNA. Computer software analysis programs are then used to calculate the relative concentrations of each 16S rRNA gene, which is proportional to the relative number of bacteria of each species in the starting microbial community. Relative quantification of target DNA can be determined based on comparison of the Ct obtained with that of an internal reference bacterium. Absolute quantification of a target bacterium in a sample can be determined by comparison of the Ct obtained with DNA standards using a calibration curve generated from titration of serial tenfold dilutions of a known concentration of the target bacterium.
Ultra-High-Throughput, Massively Parallel DNA Sequencing. A revolution has occurred within the last decade that has had a lasting and profound impact on profiling of microbial communities and on microbial functional genetics in general. Technological advances in ultra-high-throughput, massively parallel sequencing have taken place, which allow the simultaneous determination of multiple millions of base pairs of DNA sequences in single reaction runs (hence the term “massively parallel”) at very reasonable costs. New bioinformatic methods now also allow the rapid and accurate assembly of these sequences into large regions of overlapping sequences (called contigs) that can be mapped into a complete genome.
At this writing, Illumina sequencing technology is the prevailing platform for massively parallel sequencing (Figure 5-6A). Despite the limitation of generating rather short read lengths of 50–300 contiguous bases, this method is extremely powerful and inexpensive in terms of capacity and time per sequencing run. At the time of this writing, the Illumina chemistry yields robust sequence determinations, even of long stretches of repeated bases, in as little as 6 hours for data outputs of 0.5–15 gigabases (1–25 million reads) to a few days for outputs of 7.5–35 GB (100–200 million reads). Meanwhile, improvements in DNA sequencing reagents and read detection technologies (Figure 5-6B) are appearing at an astounding pace. Modification to the original Illumina process for library preparation now enables paired-end sequencing (Figure 5-6C), which significantly improves alignment of reads to produce longer high-quality contigs that can be used for genome assembly.
Figure 5-6. Illumina method of massively parallel DNA sequence determination. (A) Shown are the steps in the Illumina platform. (B) Alternative sequence readout can be performed using the less expensive Ion Torrent technology, which detects protons (H+) released during the nucleotide incorporation reaction step as a change in pH for each H+ released during the cycle. (C) Paired-end sequencing entails sequencing both ends of a DNA fragment of defined length (which can be longer than the sequence read), allowing improved sequence alignment of reads during genome assembly. With a small modification of the library preparation process, it is possible to read both the forward and reverse template strands of each cluster (spot), which provides positional information and improved alignment of the paired-end reads with a reference sequence during assembly. Adapted from copyrighted publications from Illumina, with permission.
An alternative sequencing platform that generates much longer sequence reads and is ideal for de novo genome sequencing is the single-molecule, real-time (SMRT) sequencing platform developed by Pacific BioSciences (PacBio). The PacBio SMRT system takes advantage of the intrinsic speed, fidelity (3′ to 5′ exonuclease activity), and importantly the high processivity of a single molecule of a special strand-displacing DNA polymerase, originally obtained from Bacillus subtilis phage phi29, to generate very long reads from a single molecule of DNA template (Figure 5-7). At this writing, each SMRT cell reaction run yields up to 5 GB of sequencing data in about 12 hours. Current chemistry and library preparation advances have pushed the limits of sequence read lengths up to 60 kB (with average read lengths of 10–20 kB), which can yield large overlapping contigs of up to 15 MB in length, enabling the complete assembly of most microbial genomes, including plasmids, without gaps.
