Highly accurate single-color fluorogenic DNA decoding sequencing for mutational genotyping.

Autor: Cheng C; College of Medicine and Health Science, Wuhan Polytechnic University, Wuhan, China. Electronic address: chengchu@whpu.edu.cn., Cheng Q; College of Medicine and Health Science, Wuhan Polytechnic University, Wuhan, China., Zhou W; College of Medicine and Health Science, Wuhan Polytechnic University, Wuhan, China., Chen Y; College of Medicine and Health Science, Wuhan Polytechnic University, Wuhan, China., Xiao P; State Key Laboratory of Digital Medical Engineering, School of Biological Science and Medical Engineering, Southeast University, Nanjing, China.
Jazyk: angličtina
Zdroj: Journal of pharmaceutical and biomedical analysis [J Pharm Biomed Anal] 2024 Oct 15; Vol. 249, pp. 116397. Date of Electronic Publication: 2024 Aug 05.
DOI: 10.1016/j.jpba.2024.116397
Abstrakt: We proposed a single-color fluorogenic DNA decoding sequencing method designed to improve sequencing accuracy, increase read length and throughput, as well as decrease scanning time. This method involves the incorporation of a mixture of four types of 3'-O-modified nucleotide reversible terminators into each reaction. Among them, two nucleotides are labeled with the same fluorophore, while the remaining two are unlabeled. Only one nucleotide can be extended in each reaction, and an encoding that partially defines base composition can be obtained. Through cyclic interrogation of a template twice with different nucleotide combinations, two sets of encodings are sequentially obtained, enabling the determination of the sequence. We demonstrate the feasibility of this method using established sequencing chemistry, achieving a cycle efficiency of approximately 99.5 %. Notably, this strategy exhibits remarkable efficacy in the detection and correction of sequencing errors, achieving a theoretical error rate of 0.00016 % at a sequencing depth of ×2, which is lower than Sanger sequencing. This method is theoretically compatible with the existing sequencing-by-synthesis (SBS) platforms, and the instrument is simpler, which may facilitate further reductions in sequencing costs, thereby broadening its applications in biology and medicine. Moreover, we demonstrate the capability to detect known mutation sites using information from only a single sequencing run. We validate this approach by accurately identifying a mutation site in the human mitochondrial DNA.
Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2024 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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