期刊：
Journal of Pharmaceutical and Biomedical Analysis,2024年249:116397 ISSN：0731-7085
通讯作者：
Cheng, C
作者机构：
[Cheng, Qingzhou; Cheng, Chu; Zhou, Wei; Chen, Yulong] Wuhan Polytech Univ, Coll Med & Hlth Sci, Wuhan, Peoples R China.;[Xiao, Pengfeng] Southeast Univ, Sch Biol Sci & Med Engn, State Key Lab Digital Med Engn, Nanjing, Peoples R China.
通讯机构：
[Cheng, C ] W;Wuhan Polytech Univ, Coll Med & Hlth Sci, Wuhan, Peoples R China.
关键词：
Decoding sequencing;High accuracy;Long read length;Rare mutations;Single-color fluorogenic DNA
摘要：
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.
作者机构：
[Cheng, Qingzhou; Cheng, Chu; Zhou, Wei; Chen, Yulong; Liu, Wenbin] College of Medicine and Health Science, Wuhan Polytechnic University, Wuhan, China. chengchu@whpu.edu.cn;[Zhang, Zhiling; Ye, Jingsi] Huaren Technology Co., Ltd, Wuhu, China;[Xiao, Pengfeng] State Key Laboratory of Digital Medical Engineering, School of Biological Science and Medical Engineering, Southeast University, Nanjing, China
摘要：
Eliminating errors in next-generation sequencing has proven to be challenging. Here we present a novel strategy for DNA sequencing, called correctable two-color fluorogenic DNA decoding sequencing, which can significantly improve sequencing accuracy and throughput by employing a dual-nucleotide addition combined with fluorogenic sequencing-by-synthesis (SBS) chemistry. This sequencing method involves introducing a mixture of natural nucleotide X, labeled unblocked nucleotide X', 3' blocked nucleotide Y*, and labeled 3' blocked nucleotide Y* into each reaction cycle. By cyclically interrogating a template twice with different nucleotide combinations, two sets of base-encoding are sequentially obtained, enabling accurate deduction of base sequence. We demonstrate the remarkable efficacy of this approach in detecting and correcting sequencing errors, achieving a theoretical error rate of 0.0005%, which is twice as accurate as Sanger sequencing. Furthermore, we show the capability to detect known mutation sites using information from only a single sequencing run. The correctable two-color fluorogenic DNA decoding sequencing approach should enable accurate identification of extremely rare genomic variations in diverse applications in biology and medicine.
关键词：
Genetic diversity;hepatitis C virus infection;people who inject drugs;social network analysis
摘要：
Social networks facilitate the transmission of hepatitis C virus (HCV) in people who inject drugs (PWID). The aim of this study was to assess how certain network structural characteristics are related to HCV infections in PWID and to determine the most susceptible individuals for HCV transmission in a network of PWID. PWID (N= 80) from central China were recruited from a previous follow-up case-control study. Demographic and behavioural information was obtained from a computerized database for each group. HCV RNA was extracted from blood specimens. Sequences were used to construct a phylogenetic tree and to determine genetic distances. Socio-metric social links were established between participants. Network measures were calculated using UCINET. Three HCV genotypes were identified, covering five subtypes. The density of the social networks for the whole sample (N = 80), case group (n = 31) and control group (n = 49) was 0.038, 0.054 and 0.008, respectively. PWID infected with HCV were in frequent contact with others within their group. There were four pairs of nodes with genotypic distances of 0.000 that were identified and clustered in subtypes 6a and 1b; each subject pair was linked and found in one clique. Three of the five most active nodes were infected with HCV. These three nodes served as a bridge, contributing to the connection of other nodes. These findings identify susceptible individuals for HCV transmission in PWID based on their frequent contact with others in the network. These results provide data that could be used for modelling HCV transmission patterns and in public health policies.
