The Diverse Ways in Which the Development of Second Generation Sequencing Technologies - Essay Example

?Second Generation Sequencing extending DNA Barcoding beyond Sanger Sequencing Literature Review Early sequencing technique came into existence in the 70s, and involved the use of bases that are chemically altered known as, dideoxy. Dideoxy terminated DNA fragments that were synthesized in different bases, for example, A, C, T or G. The fragments then, underwent size-separation for purposes of reading the sequence of DNA. On the other hand, DNA barcoding involve characterizing or categorizing species by applying short DNA sequence using an agreed position concerning the genome. The sequence related to DNA barcoding is considered short compared to an entire genome and are beneficial in terms of being obtained fast and cheaply. On the same note, the four components related to barcoding include specimens, laboratory analysis, database and data analysis (CBOL Plant Working Group 2009, p.12794). Since early 90s, DNA sequencing has involved the use of capillary-based and semi-automated techniques related to Sanger biochemistry. The process of DNA sequencing then involved two approaches that include shotgun sequencing and PCR amplification. Shotgun sequencing involves a process of cloning DNA that through a random fragmentation and transformed into high-copy-number plasmid that is used for changing Escherichia coli. PCR amplification, on the other hand involves a process of targeted resequencing where primers are used to flank the target. Following three decades of improvements, the Sanger biochemistry, is now applied to obtain read lengths that average 1000 bp and accuracies in regard to per base raw that average 99.999%(Hutchison 2007, pp.6227-6237). However, the introduction of second generation sequencing techniques continues to expand the field of DNA barcoding beyond the Sanger sequencing technique. The second-generation technologies have contributed to alternative DNA barcoding strategies and can be grouped in a number of categories. This includes sequencing using hybridization, cyclic-array sequencing, microelectrophoretic techniques and observation of single molecules in real-time (Healy 2007; Shendure 2005; Soni & Meller 2007). Second generation technologies as used in the field of barcoding implies to the different types of sequencing that have been introduced recently, in a commercial product and includes 454 sequencing, Solexa technology, Heliscope technology of single molecule sequencer, the Polonator and the SoLiD platform. These products have improved the diversity of sequencing, and have helped in the application of alternative protocols for purposes of generating jumping libraries related to mate-paired tags that contain controlled distance distributions. Further, these new technologies through various approaches, permits the production of amplicons that are clonally clustered, and acts as sequencing features. A common feature among the second-generation technologies in DNA barcoding is that, PCR amplicons emanating from various single library molecules can be spatially clustered on a single site within a planar substrate or on micron-scale bead’s surface. The sequencing process has further improved because of the introduction of alternating cycles related to enzymes-based biochemistry and data acquisition that is based on imaging (Mitra et al. 2003, pp. 55-62). In essence, the benefits of the second-generation technologies in comparison to the Sanger technique in diversifying DNA barcoding includes, the introduction of in vitro construction related to sequencing library. This is followed by cloning amplifications that produce sequencing features and circumvent numerous bottlenecks considered affecting parallelism related to sequencing considered as conventional. Second generation technologies compared to Sanger sequencing, have an advantage in terms of introducing array-based sequencing. Because of the existence of an array-based sequencing, the process of DNA barcoding is able to realize a considerable degree of parallelism compared to capillary-based sequencing. Further, the selection features can now be restrained on the surface of a planar and deployed enzymatically using a volume of single reagent. Mitra et al. (2003, p.65) reiterate that, in considering the benefits of second generation technologies in diversifying DNA barcoding, there are a number of shortfalls that still affects the improvements in sequencing techniques. The most notable in this regard involve the read of length (read lengths in terms of new platforms are presently shorter compared to the conventional sequencing) and raw accuracy (the base-calls resulting from the new platforms are less accurate compared to those produced by Sanger sequencing). Despite the shortfalls resulting in algorithmic challenges, it is necessary to note that, these technologies continue to improve with time and in a similar manner to the gradual improvement of conventional sequencing (Mitra et al. 2003, p. 65). The manner in which diversity of DNA continue to improve beyond what is impossible with Sanger sequencing involve the emergent of second-generation technologies that include, 454 pyrosequencing. The 454 pyrosequencing was the first to come into the existence among the second-generation techniques that are available as a commercial product. 454 pyrosequencing has revolutionized DNA barcoding in terms of improving the construction of libraries using any method that can provide a combination of short and adaptor-flanked fragments. As explained by Dressman et al. (2003, p. 8820), 454 pyrosequencing operates by producing sequencing features through emulsion PCR and after the emulsion has been broken, the beads are processed using denaturant to eliminate untethered strands, followed by hybridization of amplicon-bearing beads. These beads are further preincubated using polymerase known as, Bacillus stearothermophilus in combination with proteins that are single stranded. This is then deposited on a picolitre-scale wells containing micro fabricated array, such a process is considered impossible by relying on the Sanger technique. In essence, the 454 pyrosequencing has improved read length and can generate up to 400,000 reads in every instrument that runs between the lengths of 200 or 300bp (Dressman et al. 2003, p. 8822). The Illumina Genome Analyzer or the Solexa has improved the construction of libraries using various methods that can permit the combination of adaptor-flanked fragments consisting of several base pairs. The analyser produces sequencing features using bridge PCR where, the PCR primers undergo a tethering process to produce a solid substrate using a flexible linker. This process allows amplicons emanating from single template molecule to remain immobilized to allow the clustering in a single site within an array. With the Illumina platform, millions of clusters can be amplified into distinguishable sites or locations and within the independent lanes existing on a single flow-cell. At the present, modifications related to the Illumina platform have improved the mate-paired reads (Fedurco et al. 2006). On the other hand, advancement in DNA barcoding by relying on AB SOLiD platform, has introduced the construction of libraries using a combination of short and adaptor-flanked fragments. This platform focuses more on protocols related to libraries that are mate-paired and involves controlled and distance distributions considered being highly flexible. Further, this platform also produce sequencing features through emulsion PCR; however, the sequencing process involves the use of DNA ligase and not polymerase. Relying on AB SOLiD platform compared to the Sanger sequence, presents an advantage in the sense that, utilizing a high-density array, provides the opportunity of obtaining high data densities (Fedurco et al 2006). Concerning the Heliscope, the process involved also improves DNA sequencing by relying on a cyclic interrogation involving sequencing features with a dense array. This platform is unique compared to other improvements in the sense that, the process of sequencing does not require amplification. In the research by Braslavsyky, Hebert & Kartalov (2003, p. 3962), the Helicos sequencer applies a system of fluorescence detection that is considered to be highly sensitive for interrogating the single DNA molecules. In as much as the second-generation technologies have improved DNA barcoding beyond Sanger sequencing, this does not mean that Sanger sequencing cannot not be used at present. In the explanation by Hert, Fredlake &Barron (2008, pp. 4618-4626), the technique still applies in small-scale experiments and complementing the regions which cannot be sequenced easily, by relying on the second generation platforms such as highly repetitive DNA. However, improvements concerning the second-generation techniques are still regarded as significant in genomics now and in the future. In conclusion, the second-generation technologies are significant at the biological level and assist in opening a new area of research. In essence, these new technologies have diversified DNA barcoding beyond Sanger sequencing in a number of ways. This involves the construction process and amplification concerning the clonal related to the library of in vitro, that solves a number of bottlenecks considered restricting the functions of Sanger sequencing. Further, the array oriented sequencing help to improve parallelism compared to the use of capillary sequencing. The new technologies also contribute to a minimized use of reagent as a resulting on enabling the immobilization related to template DNA within the planar surface. However, the second-generation technologies are faced with a number of limitations that includes a shorter read length compared to the one produced by the Sanger sequence. Raw accuracy is also lower compared to the one obtained by the conventional sequencing. As much as the second-generation technologies have brought considerable changes in the field of DNA barcoding, they are expensive and beyond the reach of many people. As a result, there is a need for more research on how to come up with a cheaper means of improving genomics. References Braslavsyky, I., Hebert, B., Kartalov, E. & Quake, S.R 2003, ‘Sequence information can be obtained from single DNA molecules’, Proc. Natl. Acad. Sci, Vol. 100, pp.3960–3964. CBOL Plant Working Group 2009, ‘A DNA barcode for land plants’, Proc. Natl. Acad. Sci, Vol.106, p.12794. Dressman, D., Yan, H., Traverso, G., Kinzler, K.W. & vogelstein, B 2003, ‘Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations’, Proc. Natl. Acad. Sci, Vol.100, pp. 8817–8822. Fedurco, M., Romieu, A., Williams, S., Lawrence, I. & Turcatti, G 2006, ‘BTA, a novel reagent for DNA attachment on glass and efficient generation of solid-phase amplified DNA colonies’, Nucleic Acids Res, Vol. 34, no.22. Healy, K 2007, ‘Nanopore-based single-molecule DNA analysis’, Nanomed, Vol. 2, pp. 459– 481. Hert, Daniel G., Fredlake, Christopher P., & Barron, Annelise E 2008, ‘Advantages and limitations of next-generation sequencing technologies: A comparison of electrophoresis and non-electrophoresis methods’, Electrophoresis, Vol. 29 no.23, pp. 4618-4626. Hutchison, C.A., III 2007, ‘DNA sequencing: bench to bedside and beyond’, Nucleic Acids Res, Vol. 35, pp. 6227–6237. Mitra, R.D., Shendure, J., Olejnik, J., Edyta Krzymanska, O. & Church, G.M 2003, ‘Fluorescent in situ sequencing on polymerase colonies’. Anal. Biochem, Vol.320, pp.55–65. Shendure, J 2005, ‘Accurate multiplex polony sequencing of an evolved bacterial Genome’, Science, Vol. 309, pp.1728–1732. Soni, G.V. & Meller, A 2007, ‘Progress toward ultrafast DNA sequencing using solid-state nanopores’, Clin. Chem, Vol. 53, p. 1432. Read More