A Real-Time Decoding Sequencing With Dual Mononucleotide Addition And Its Applications

DNA sequencing technologies are the basis for further research and improvement of the genes of interest. They are such most important tools in the life sciences and other fields that they have a wide range of applications for modern biological research. In this dissertation, we propose a real-time decoding sequencing technology. This technology is based on the principle that different types of nucleotides involved in the polymerization generate the same detection molecules. It will increase read length and provide a new method for high-throughput DNA sequencing. Meanwhile, it will also have the potential in the real-time analysis of PCR products. The contents of this doctoral thesis contain,1. Principle and characteristic of the decoding sequencing technology based on dual mononucleotide additionWe propose a real-time decoding sequencing technology in which a template is determined without directly measuring base sequence but by decoding two sets of encodings obtained from two parallel sequencing runs. These encodings contains the type of incorporated nucleotides and the number of incorporated nucleotides. In this part, the following items are investigated:(i) Theoretically investigate the feasibility of the sequencing technology. Generally, any two of nucleotides A, G, C and T, are able to form six combinations of two different nucleotide pairs. These include AC, AG, AT, CG, CT, and GT. The combinations can form three sets of dual mononucleotide addition (AG/CT, AC/GT, and AT/GC). This strategy can be performed by the three sets of dual mononucleotide addition, and three sets of encodings can be obtained. However, given any two sets of encodings, the nucleic acid sequence is allowed to be sequentially reconstructed. For any template sequence, two sets of encodings can be obtained. By decoding the two sets of encodings, the queried templates can be recovered.(ii) Apply pyrosequencing instrument (Pyrosequencing PSQ 96MATM system, Biotage) to investigate the feasibility of this technology. Synthetic template is interrogated by two parallel sequencing runs, and two sets of encodings are obtained. These two sets of encodings allow for a queried template sequence to be sequentially decoded, moving from first to last, in a deterministic manner. Thus, it enbles to interrogate unknown DNA sequence,(iii) Optimize the reaction conditions for this technology. Here, the amount of DNA template for decoding sequencing, the optimal amount of dNTPs and the relationship between the identical nucleotides in the homopolymeric regions and the released molecules in the polymerization are investigated. First, we investigate how many DNA templates are needed to successfully perform this technique. The results demonstrate that the technique can be successfully performed with 0.3-5 pmol of DNA template; Second, The relationship between the released molecules and the incorporated nucleotides in the homopolymeric region are investigated. We find when the homopolymeric region is≤7 bp in length, the released pyrophosphates are proportional to the number of incorporated nucleotides; Third, The optimal amount of DNA templates for homopolymeric regions are studied, the results show that pyrosequencing on homopolymeric regions with 7 identical nucleotides using dual mononucleotide addition can be successfully performed with optimal amount of DNA (0.3-1 pmol). Finally, the optimal amount of dNTPs are investigated and the results display that the amount of dNTPs should be less than 250 pmol in the technology.(iv) Theoretically discuss the potentials of this technology in high-throughput DNA sequencing platforms. We first investigate the advantages of this technology for high-throughput DNA sequencing technology and then provide the strategies for homopolymeric regions and chain decoding errors to provide a new sequencing strategy for increasing read length in high-throughput DNA sequencing.2. Qualitative analysis of PCR products using pyrosequencing with dual mononucleotide additionWe investigate the feasibility of pyrosequencing with dual mononucleotide addition for qualitative analysis of PCR products only in a single sequencing run. First, based on the principle that encodings obtained in a single run is useful to qualitative analysis of PCR products as each PCR product with different size and base composition will give a specific pattern, partial rnpB genes from S. infantis, S. peroris, S. anginosus and S.constellatus are interrogated via pyrosequencing with dual mononucleotide addition. The results indicated that Streptococcal strains could be successfully differentiated by comparing signal intensity in each cycle and encoding size of each template. This strategy is likely to be applied to differentiate nucleic acid sequence as encoding size and signal intensity in each cycle vary with the base size and composition. Furthermore, the high similarity between S. infantis and S. peroris in the P3 region makes the differentiation of the two species by traditional pyrosequencing impossible. However, there is 16% encoding difference between these strains in a single run when these species are analyzed by pyrosequencing with dual mononucleotide addition. This implied that few nucleotide differences in the P3 region were species specific and this strategy was possible to amplify sequence differences among the species. This enables the application of this technology to differentiate microbial strains.In addition, we apply this technology to simultaneously genotype several closely located SNPs (single nucleotide polymorphisms) in a single sequencing run. Pyrosequencing with dual mononucleotide addition was developed and only a single sequencing run was required to genotype several SNPs located in the uridine diphosphoglucuronosyl transferase 1A1 (UGT1A1) gene at different positions. Templates with two SNPs covering 37 bp and with three SNPs covering 58 bp as well as 82 bp are interrogated with specific dual mononucleotide chose from six kinds of different dual mononucleotides (AG, AC, AT, GT, CT and CG). These SNPs were successfully genotyped by using only a sequencing primer in a single PCR/sequencing run. Compared with traditional pyrosequencing, the read length of per flow is increased, making simultaneous identification of multiple SNPs in a single sequencing run possible. Furthermore, higher signals are produced and thus relatively lower sample amount will be required. By using this technology, as low as 50 copies of DNA templates were accurately sequenced. Our results demonstrated that the technology can be potentially developed into a powerful methodology to accurately determine SNPs so as to diagnose clinical settings.3. Quantitative analysis of PCR products using pyrosequencing with dual mononucleotide additionIn this technology, the released pyrophosphates during polymerization are proportional to the incorporated nucleotides, and the incorporated nucleotides are proportional to the amount of DNA template. Thus, it makes the quantitative analysis of PCR products possible. First, 2-fold and 1.5-fold serial dilutions of wild-type PCR products including variants rs6717546 (or rs4148324) are used to generate concentration-response curves. The essentially linear region in the concentration-response curves is determined, and the data from the linear portion are plotted separately on a decimal scale to establish a function of template concentration:Y=aX+b. To assess the precision and accuracy of quantification of the genotypes by the this technology, we test a series of standard mixtures with different ratios (100%,90%...10%,0%) of wild-type and mutant templates. The corresponding formulas and the normalized signal intensities from pyrosequencing with dual mononucleotide addition are used to provide ratio of each genotype in the mixed sample and are compared to the original spike-in ratios. The results show excellent correlation between the spike-in ratios and the detected ratios (R2>0.95). Second,24 samples are analyzed with pyrosequencing with dual mononucleotide addition. The normalized signal intensities from the SNP positions and the corresponding formulas for rs6717546 and rs4148324 are used to quantify the allelic frequency. The results show that there is no significant difference (P>0.05) when compared to conventional pyrosequencing. Finally, different ratios (1%,3%,5% and 7%) of wild-type and mutant templates are used to investigate the detect limit of this quantitative method. The results show that the detect limit is 3% which is better than that of conventional pyrosequencing.