Next generation sequencing (NGS) platforms are commercially introduced high throughput devices that allow DNA sequences to be recovered directly from marine environmental samples (amongst others!). NGS are also without need of vector based cloning procedure normally used for amplifying DNA templates. This eliminates cloning bias to sample evenness, but incurs other limitations for each NGS platform.
Despite diverse chemistry and base incorporation/detection techniques, all NGS platforms follow 2 steps; library fragmentation/amplicon library preparation and detection of the incorporated nucleotides
Shokralla et al. (2012) describes and examines NGS techniques for their pros and cons in regard to environmental DNA research.
All PCR-based NGS systems have bias’ introduced during amplification. Bias is initially introduced during amplicon library preparation. PCR bias is also strongly affected by the number of replicating cycles and by annealing temperature. These however can be reduced by using high template concentrations, wise primer selection, low cycle number, low annealing temperature and mixed replicate reaction preparations. Bias’ from amplicon preparation can by exaggerated in the library amplification step. Any bias from library amplification alone is likely eradicated by use of universal probes.
454 pyrosequencing is the most common platform for NGS. Some big advantages are its long read length, short run time, and that it doesn’t necessitate an extra chemical debunking step for DNA extension by DNA polymerase, which is unique among NGS platforms. This reduces the chance of dephasing by lowering the likelihood of premature chain termination, and non-simultaneous extension. Long sequences generated in 454 pyrosequencing will mean higher accuracy when identifying nonmodel organisms in ecological applications. Drawbacks are its high cost per megabase sequencing output, and its lack of terminating moiety to stop the extension run, making it a challenge to read homopolymer regions. The most common error type however is insertion-deletion instead of substitution, causing errors in analysis of environmental DNA by mimicking haplotypes of rare biota. This howeveris significantly reduced with computational tools to highlight and extract such sequences.
Illumia an d SOLiD systems perform individual necleuotide detection, meaning homopolymer regions are accurately sequenced. These systems also have a high output per run compared to 454 pyrosequencing However optical signal decay and dephasing in these platforms leads to short reading lengths. This infers situations were no reference sequence is available to align assaign and annotate generated short sequences. As well as this most NGS workflows are time consuming, tedious and require highly skilled personnel.
Another PCR-based NGS platform not assessed by Shokralla et al. is Life Technologies Ion Torrent, a post light sequencing technology. This relies on real-time detection of hydrogen ion concentration, released as a by-product when a nucleotide is incorporated into a strand of DNA by polymerase action.
Other NGS platforms mentioned by Shokralla et al. include Single-molecule DNA-sequencing technologies, which don’t require a PCR-amplification step and so remove such formerly stated bias’. These are Helicos biosciences HeliScope, which works by sequencing-by-synthesis on a single DNA molecule, and Pacific Biosciences SMRT DNA sequencer, which uses less steps and so a faster process than Heliscope, using the natural capacity of DNA polymerase to incorporate ten or more nucleotides per second in several thousand parallel nano-structures.
An enhancement for NGS technologies is target selection (Sequence Capture), which eliminates initial amplification steps and allows selective analysis of large numbers of target sequences. Sequence capture involves hybridization-based methods using oligonucleotide probes. These are either immobilized to a solid array ‘Capture arrays’ or in solution ‘Baits’ to capture the sequencing targets. The latter is preferable due to higher specificity and uniformity of sequences, and cheaper hardware costs. Target enrichment sequence capture eliminates the initial PCR Step, however it is necessary to start library preparation with a relatively large amount of DNA.
Examples of sequence capture systems are: Roche’s NimbleGen, Agilent’s SureSelect, Rain-Dance Technologies’ RainStorm and Illumina’s TruSeq Exome Enrichment system. Though currently used in studies of human and other model organisms, they show great promise for environmental DNA research.
Modification of sample preparation protocals, like library construction, could also take shape as further enhancements to NGS platforms.
Multiplexing of different target gene markers of a single bulk sample, or multiplexing of a single marker from multiple samples, by tagging or bar coding mixtures of DNA templates could have great ecological application. This process can also be carried out at a reasonable price, however potential biases caused by the addition of multiplexing identifier tags to primer oligos should be considered.
The authors highlight many instances of marine application of such NGS technologies in ecological research of bacterial and viral natures, from surface waters to coral reefs. Following Colin’s last lecture, such applications could also help identify indicator species of disease in the ocean; helping beach goers (locals and tourists alike) make informed decisions before taking a dip!
- Shokralla, S., Spall, J. L., Gibson, J. F., & Hajibabaei, M. (2012). Next‐generation sequencing technologies for environmental DNA research. Molecular Ecology, 21(8), 1794-1805.