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Sanger Sequencing vs. Illumina Sequencing for Aptamer Selections
Sanger Sequencing vs. Illumina Sequencing for Aptamer Selections
Sanger Sequencing vs. Illumina Sequencing
Sanger Sequencing vs. Illumina Sequencing
Sanger and Illumina sequencing are both methods of sequencing which can be utilized to identify aptamer candidates, however, they differ in their processes, resulting in different sequencing outcomes. Sanger sequencing can only sequence one DNA fragment, one aptamer candidate, at a time.1 In Illumina sequencing, sequencing occurs in each individual round of aptamer selection, and Illumina can sequence millions of DNA fragments at a time, which greatly increases sequencing volume.2 In Sanger Sequencing, we typically only sequence the pool of DNA strands in the final round of selection. Using per-round sequencing, Illumina can measure the enrichment (the prevalence of a DNA sequence after each round of selection) of each sequence at the end of selection.
Using Illumina sequencing in our research project allows for more data to be collected from our aptamer selections which we can then analyze for potential aptamer candidates.
Keywords
Keywords
Reuter J., et al. (2015). High Throughput Sequencing Technologies. Molecular Cell 58(4), 586-597. https://doi.org/10.1016/j.molcel.2015.05.004
High Throughput Sequencing
High Throughput Sequencing
A new method of sequencing involving multiple sequencing rounds. This method allows for running of more samples to get more sequencing data back in return in a fast, cost-effective manner. High throughput sequencing can be applied to other areas of sequencing outside of aptamers as well.
Gauthier, M. (2007). The Sanger Sequencing method in 7 steps. https://www.researchgate.net/figure/The-Sanger-sequencing-method-in-7-steps-1-The-dsDNA-fragment-is-denatured-into-two_fig2_234248746
Sanger Sequencing
Sanger Sequencing
A traditional method of nucleic acid sequencing in which a single DNA strand is sequenced in vitro and detected using dyes and computer technology. Sanger is used in aptamer sequencing in what is known as, "end-point sequencing", in which a single step of sequencing occurs after selection.
Genomic Sequencing Core. About Next-Gen Sequencing. The University of Kansas. https://gsc.ku.edu/about
Illumina sequencing
Illumina sequencing
A method of Next-Generation Sequencing, more recently developed, in which the large pool of DNA strands are sequenced simultaneously using "flow cells". Flow cells are pads of genetic material that attract and bind to the target strands. Illumina is used in aptamer sequencing in what is known as, "Dynamic Sequencing", in which sequencing occurs after each aptamer selection round.
Sanger vs. Illumina Comparison
Sanger vs. Illumina Comparison
Relevant Papers
Relevant Papers
(1) Differential binding cell-SELEX method to identify cell-specific aptamers using high-throughput sequencing
(1) Differential binding cell-SELEX method to identify cell-specific aptamers using high-throughput sequencing
Pleiko, K., Saulite, L., Parfejevs, V. et al. Differential binding cell-SELEX method to identify cell-specific aptamers using high-throughput sequencing. Sci Rep 9, 8142 (2019). https://doi.org/10.1038/s41598-019-44654-w
This study by Pleiko, et al. (2019) discusses benefits of using high throughput methods of aptamer sequencing to identify aptamers that can bind to live cells. To conduct the study, the live cell sample used was of clear cell renal cell carcinoma cell line. Illumina sequencing and in-silico approaches were used after cell-SELEX to form cell-specific aptamer candidates. This paper used software such as edgeR package, GraphPad Prism software, ExpressPro software, and more to analyze different aspects of the sequencing results. This study shows one benefit of high throughput methods of sequencing: the use of software to improve the analyzation process to look for patterns beyond just nucleotide sequences. This paper relates to this topic as it discusses the benefits to using Illumina (high throughput) methods of sequencing over the traditional Sanger Sequencing method.
(2) Selection of DNA aptamers for ovarian cancer biomarker HE4 using CE-SELEX and high-throughput sequencing
(2) Selection of DNA aptamers for ovarian cancer biomarker HE4 using CE-SELEX and high-throughput sequencing
Eaton, R.M., Shallcross, J.A., Mael, L.E. et al. (2015) Selection of DNA aptamers for ovarian cancer biomarker HE4 using CE-SELEX and high-throughput sequencing. Anal Bioanal Chem 407, 6965–6973. https://doi.org/10.1007/s00216-015-8665-7
This study, conducted by Eaton, et al. (2015), develops aptamers targeting the HE4 ovarian cancer biomarker through the use of high throughput Illumina sequencing. The results of this study show one benefit of using Illumina over Sanger sequencing, as Illumina can not only determine sequence abundance, but also enrichment. Enrichment can be determined using Illumina sequencing since the pool of aptamer candidates are sequenced after each round of aptamer selection. The results found four aptamer candidates, based on levels of enrichment, that were then tested for binding affinity. This study relates to our project as it is a direct application of Illumina sequencing to identify aptamer candidates.
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