Group-1-Assignment-1-Info.pdf

Name: Abratique, Patrick Hanz Bugayong, Jamaicah Rae Bunzo, Kernsten Paz, Mark Angelo Tarun, Nichols Amy Date submitted: 7/12/2023 Second Generation Sequencing - Sequencing by Ligation/ SOLiD Supported Oligonucleotide Ligation and Detection (SOLiD) introduces a new technique of DNA sequencing whic...

Name: Abratique, Patrick Hanz Bugayong, Jamaicah Rae Bunzo, Kernsten Paz, Mark Angelo Tarun, Nichols Amy Date submitted: 7/12/2023 Second Generation Sequencing - Sequencing by Ligation/ SOLiD Supported Oligonucleotide Ligation and Detection (SOLiD) introduces a new technique of DNA sequencing which is the sequencing-by-ligation and it is a second-generation DNA sequencing technology developed by Applied Biosystems (Applied Biosystems, 2008). Sequencing-by-ligation is dependent on the ligation of DNA fragments through the usage of DNA ligase in order to determine the underlying sequence of the target DNA compared to the technique introduced by its predecessors which focuses on the sequencing-by-synthesis principles where the addition of nucleotides with DNA polymerase (DNAP) is utilized to sequence the target DNA (Nguyen, 2021). Emulsion PCR is used to amplify a ssDNA primer-binding region, which is known as an adapter, which has been conjugated to the target sequence on a bead. The beads mentioned will be deposited onto a glass surface (ATD Bio, n. d.). Once a bead is deposited, a primer of length N is hybridized to the adapter. Then after, the beads are exposed to a number of 8-mer probes which contain different fluorescent dye at the 5' end and a hydroxyl group at the 3' end. The bases 1 and 2 are complementary to the nucleotides to be sequenced. On the other hand, the bases 3-5 are degenerates and the bases 6-8 are inosine bases. The complementary probe will hybridize to the target sequence that is adjacent to the primer. The DNA ligase is then used to join an 8-mer probe with the primer. A phosphorothioate bond between the bases 5 and 6 allows the fluorescent dye to be cleaved from the fragment using silver ions. This cleavage allows fluorescence to be measured which uses four different fluorescent dyes that have different emission spectra. Also, this cleavage generates a 5’-phosphate group which can undergo further ligation. The extension product is melted off after the first round of sequencing is completed. Then, a second round of sequencing is performed with a primer that has a length of N-1, succeeding sequencing then contains primers that are shorter than the previous primers. The SOLiD platform uses four (4) fluorescent dyes for detection and two-base encoding to analyze the sequence, in which there are sixteen (16) possible combinations of two (2) nucleotide bases associated with the fluorophores (Choudhori, 2014). The raw data acquired after a cycle cannot be translated directly into a sequence because a single color can be any of the four (4) nucleotide combinations as seen in Figure 1 (Garrido-Cardenas et al., 2017). With this, successive cycle is required to identify the base at a certain position by ruling out the other possible combinations using the fluorophore emitted in the next cycle. In this way, each nucleotide base of the sequence is interrogated twice by different probes. Therefore, the next base can be deduced if the previous base is known until the whole sequence is read (Menon, 2021). The whole sequence can be deduced if one base is known as seen in Figure 2. (Applied Biosystems, n.d.). Figure 1. Association of nucleotide pairs to the fluorescent dyes used in the SOLiD platform. Retrieved from Garrido-Cardenas et al., 2017. Figure 2. An example on reading a sequence using the 2 base encoding by Applied Biosystems. The SOLiD can generate over six gigabases of mappable data and more than two hundred forty million tags per run that’s why it is advantageous when it comes to ultra high throughput. SOLiD sequencing is considered to be one of the most accurate second-generation sequencing technologies at 99.94% because of its capability for intensive sequencing (Ho et al., 2011). The ability of SOLiD sequencing to reduce the measurement errors and superior SNP detection contributes to its robust accuracy (Castellana et al., 2012). Furthermore, this technology is easy to implement and is readily accessible for the reason that it can be performed with off-the-shelf reagents (Applied Biosystems, 2008). In addition, SOLiD allows users to track run status in real time to help ensure that runs are completed successfully. Also, the independent flow cell configuration enables users to run two completely independent experiments on a single SOLiD Analyzer. The SOLiD System's open slide format and flexible bead densities enable increases in throughput on the current system with modest protocol and chemistry optimizations. On the other hand, one of the disadvantages of SOLiD sequencing is the fact that it is slow when it comes to sequencing due to the fact that it takes up to seven days to complete a single run (ATD Bio, n. d.). Moreover, it has a short read length of 35 bp which is significantly smaller than what is being offered by competing sequencing technologies. Additionally, recent research has demonstrated that palindromic sequences are difficult to sequence effectively using sequencing-by-ligation techniques (Applied Biosystems, 2008). SOLiD sequencing was able to make a lasting impact on a number of applications in the domains of transcriptomics, bacterial genome research, and chromatin immunoprecipitation since the benefits of the technology vastly exceed the disadvantages. REFERENCES Applied Biosystems. (2007). SOLiD Data - 2 Base Encoding. Los Alamos National Laboratory. https://www.lanl.gov/conferences/finishfuture/pdfs/2007%20talks/SOLiD%20Data%20V1. 0.8.pdf. Applied Biosystems. (2008). SOLiD System Brochure. Retrieved on July 10, 2023 from http://www.columbia.edu/cu/biology/courses/w3034/Dan/readings/SOLiD_System_Broch ure.pdf. ATD Bio. (n. d.). Next-generation sequencing. Retrieved on July 10, 2023 from https://atdbio.com/nucleic-acids-book/Next-generation-sequencing. Castellana, S., Romani, M., Valente, E., & Mazza, T. (2012). A solid quality-control analysis of AB SOLiD short-read sequencing data. Brief. Bioinform. 14(6):684-695. Choudhuri, S. (2014). Bioinformatics for Beginners: Genes, Genomes, Molecular Evolution, Databases and Analytical Tools. Elsevier Inc. https://doi.org/10.1016/C2012-0-07153-0. Garrido-Cardenas, J. A., Garcia-Maroto, F., Alvarez-Bermejo, J. A., & Manzano-Agugliaro, F. (2017). DNA sequencing sensors: an overview. Sensors, 17(3), 588. https://doi.org/10.3390/s17030588. Ho, A., Murphy, M., Wilson, S., & Edwards, J. (2011). Sequencing by ligation variation with endonuclease V digestion and deoxyinosine-containing query oligonucleotides. BMC Genom. 12 (1). Menon, S. (2021). Comparison of High-Throughput Next generation sequencing data processing pipelines. International Research Journal of Modernization in Engineering Technology and Science (IRJMETS), 3(8), 125-136. Nguyen, J. (2021). Solid Sequencing. Retrieved https://apollo-institute.org/solid-sequencing/. on July 10, 2023 from Voelkerding, K. V., Dames, S. A., & Durtschi, J. D. (2009). Next-generation sequencing: From basic research to diagnostics. Clinical Chemistry, 55(4), 641–658. https://doi.org/10.1373/clinchem.2008.112789