Introduction
Determination of the genome of a bacteria culture is one of the complex biotechnological techniques. In this case, there is the genomic DNA of Bacillus Subtilis given to compare the utility of different types of DNA sequencing technologies. Currently, this technique is based on the determination of nucleic acids in polynucleotide chains based on first, second, or third-generation sequencing (Heather & Chain, 2016). All the strategies have their pros and cons and differences that are detailed and compared further in the text.
First Generation Sequencing Technology
To begin with, first-generation sequencing technology has undergone numerous improvements since it was invented. It is focused on the sequencing of clonal DNA populations, especially those of relatively pure RNA species such as the one proposed in this case. The strategy combines techniques from analytical chemistry intended to measure nucleotide composition with selective ribonuclease treatments to produce degraded RNA fragments possible to be observed (Heather & Chain, 2016). After a number of improvements involving the inclusion of fluorometric-based detection, fully-automated first-generation DNA sequencing machines were invented and became of high prevalence.
With respect to the properties of first-generation sequencing technology, which also are to further compare with the other two strategies, they are the following. Fundamental technology is based on the separation of specifically end-labeled DNA fragments by size (Kchouk et al., 2017). The resolution of the technique is averaged across many DNA molecule copies. Read accuracy is high, and read length is between 800 and 1000 base pairs. Current throughput is low, that considered the weakness of first-generation sequencing. At the same time, the cost of conducting is high per base but low per run. The time required to obtain result is comparatively short and measured in hours. Sample preparation is moderately complex, and data analysis is comparatively simple. Primary results are base calls with quality values, which is also true for two other techniques.
Second Generation Sequencing Technology
This technology is focused on the luminescent method for pyrophosphate synthesis measuring, which also enables to increase throughput by parallelizing reactions. The approach incorporates a two-enzyme process and measures pyrophosphate production. Each nucleotide is washed through the system in turn, over the template DNA affixed to a solid phase (Kchouk et al., 2017, Second-Generation DNA Sequencing section). The pyrosequencing technique is considered more beneficial than the ones incorporated by the first-generation strategy. It is so due to using natural nucleotides instead of modified dNTPs and incorporating real-time observing instead of lengthy electrophoreses. This strategy has also undergone numerous transformations, leading to that the sequencing machines increased amount of DNA that can be produced in one run.
A brief explanation of second-generation sequencing technology’s properties is the following. It is based on wash-and-scan SBS fundamental technology, and equally to the first strategy, its resolution is averaged across DNA molecule copies (Kchouk et al., 2017). Read accuracy is high, read length is short, the cost per base is low, but the cost per run is high. It takes days to obtain a result, while sample preparation is complex with obligatory PCR amplification. Data analysis is complicated with large data volumes and short reads.
Third Generation Sequencing Technology
Third-generation sequencing strategy is of particular interest, as it enables single-molecule sequencing (SMS) while also supporting real-time sequencing. The requirement for DNA amplification is also neglected by this technology. The core of third-generation sequencing is attaching DNA templated to a planar surface, and further fluorescent reversible terminator dNTPs are washed over one base at a time and imaged phase (Kchouk et al., 2017, Third-Generation DNA Sequencing section). Despite the considerable expenses related to running this analysis, no other methodic can avoid amplification of DNA that is known to raise biases and errors. Single-molecule real-time (SMRT) third-generation sequencing is currently prevalent and the most advanced one. DNA polymerization, exploited in the process, occurs in arrays of microfabricated nanostructures, and using the properties of light passing through diameters smaller than wavelengths, visualization of single molecules is possible.
Finally, third-generation sequencing technology is based on single-molecule real-time sequencing and its resolution, respectively, single DNA molecule. While read accuracy is lower than two other techniques can offer, third-generation sequencing’s ‘read length is the highest and equals more than 1000 base pairs (Kchouk et al., 2017). Current throughput is high, the cost per base is low, and the cost per run is high. It usually takes less than one day to obtain results via this technology, and the difficulty of sample preparation is variable, while data analysis is complex.
Conclusion
DNA sequencing techniques have been being improved for more than fifty years, resulting in the invention and constant review of three distinct strategies. With respect to the pros and cons of each sequencing method, it is not possible to state the complete advantage of any over two others. Although the following methods were intended to replace previous ones, all three concepts are still viable and continue to be developed further. The determinants of most appropriate, for a particular case, technique are costs, time of analysis, the complexity of preparation, and read properties required.
References
Heather, J. M., & Chain, B. (2016). The sequence of sequencers: The history of sequencing DNA. Elsevier, 107(1), 1-8. Web.
Kchouk, M., Gibrat, J. F., & Elloumi, M. (2017). Generations of sequencing technologies: From first to next generation. Biology and Medicine, 9(3), Web.