Analytical Methods
·
2024
·
IF 2.7
Chu Cheng,Qingzhou Cheng,Wei Zhou,Yulong Chen,Wenbin Liu,Zhiling Zhang,Jingsi Ye,Pengfeng Xiao
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.
研究领域:
分子生物学
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基因组学
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医学遗传学
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产品关联
DOI
PubMed
高影响因子
Science Advances
·
2021
·Vol. 7
·期 46
·
IF 14.143
Chengtao Xu,Biao Ma,Zhongli Gao,Xing Dong,Chao Zhao,Hong Liu
DNA has been considered as a compelling candidate for digital data storage due to advantages such as high coding density, long retention time, and low energy consumption. Despite many works reported, the development of a DNA-based database of full integration, high efficiency, and practical applicability is still challenging. In this work, we report the synthesis and sequencing of DNA on a single electrode with scalability for an integrated DNA-based data storage system. The synthesis of DNA is based on phosphoramidite chemistry and electrochemical deprotection. The sequencing relies on charge redistribution originated from polymerase-catalyzed primer extension, leading to a measurable current spike. By regeneration of the electrode after sequencing, repeated sequencing can be achieved to improve the accuracy. A SlipChip device is developed to simplify the liquid introduction involved in DNA synthesis and sequencing. As the proof-of-concept experiment, text information is stored in the system and then accurately retrieved.
研究领域:
生物技术
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分子生物学
·
数据科学
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DOI
高影响因子
Nanoscience and Nanotechnology Letters
·
2019
·Vol. 11
·期 8
·1104-1115
·
IF 0
Bhatti Huma,Jawed Rohil,Liu Hang,Ali Irshad,Zhu Libo,Liu Quanjun
Biological nanopores have been proved as impressive nano-devices capable of holding the detection sensitivity from a nanoparticle to a single macromolecule. Protein pores are efficiently reproducible with assured channel geometry and their propensity to be engineered easily make them
convenient in nanopore sensing techniques. Among various porin-proteins, MspA porin of Mycobacterium smegmatis is quite ideal for nanopore DNA sequencing. However, the traversing speed of singlestranded DNA (ssDNA) through both the mutants of MspA (M1MspA and M2MspA) is too fast to
be analyzed. Therefore, we utilized a new mutant of MspA, termed as M3MspA which was previously purified in our laboratory. We, for the first time, demonstrated that translocation of ssDNA is achieved at a minimum voltage-threshold of 40 mV at pH 8.0. This new mutant of MspA exhibits a crystal-clear
discrimination between two homopolymers, poly dA50 and poly dC50 with duration time up to few milliseconds and well-resolved signature current-amplitudes were obtained for both sides (3′ or 5′) of the polymer entered the pore, without any modification to ssDNA.
Significantly, M3MspA determines a precise length of a hetero-homopolymer, poly(dA50dC50) by producing a dwell time congruent to the sum of the time spent by their component-homopolymers, separately. On account of their specificity and sensitivity towards translocating
polynucleotides, we suggest that this new mutant of MspA would be a sophisticated bio-nanopore in further enlightening the translocation dynamics caused by diverse conformations of a same biomolecule. On top of that, it would also be helpful in nanopore DNA sequencing.
研究领域:
生物物理学
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纳米技术
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分子生物学
·
基因组学
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DOI
ACS Applied Materials & Interfaces
·
2018
·
IF 8.097
Yuqin Wang,Shuanghong Yan,Panke Zhang,Zidao Zeng,Di Zhao,Jiaxin Wang,Hongyuan Chen,Shuo Huang
Recent developments in nanopore sequencing have inspired new concepts in precision medicine but limited in throughput. By optically encoding calcium flux from an array of nanopores, parallel measurements from hundreds of nanopores were reported, while lateral drifts of biological nanopores set obstacles for signal processing. In this paper, optical single-channel recording (oSCR) serves to track nanopores with high precision and a general principle of nanopore motion kinetics is quantitatively investigated. By finely adjusting the osmosis-oriented interactions between the lipid/substrate interfaces, motions of nanopores could be controllably restricted. Improved signal-to-noise ratio is observed from motion-restricted nanopores, which is experimentally demonstrated. To systematically evaluate oSCR with asymmetric salt concentrations, a finite element method simulation is established. oSCR with an array of immobilized nanopores suggests new strategies for sequencing DNA by microscopic imaging in high throughput and is widely applicable to the investigation of other transmembrane proteins.
研究领域:
生物物理学
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纳米技术
·
分子生物学
·
光学
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生物医学工程
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2021
·
IF 6.986
Min Bai,Xiaowen Cao,Feng Chen,Jing Xue,Yue Zhao,Yongxi Zhao
Cellular oxidative thymines, 5-hydroxymethyluracil (5hmU) and 5-formyluracil (5fU), are found in the genomes of a diverse range of organisms, the distribution of which profoundly influence biological processes and living systems. However, the distribution of cellular oxidative thymines has not been explored because of lacking both specific bioorthogonal labeling and sensitivity methods for single-cell analysis. Herein, we report a bioorthogonal chemical signature enabling amplified visualization of cellular oxidative thymines in single cells. The synthesized ATP-γ-alkyne, an ATP analogue with bioorthogonal tag modified on γ-phosphate can be specifically linked to cellular 5hmU by chemoenzymatic labeling. DNA with 5-alkynephosphomethyluracil were then clicked with azide (N3)-modified 5hmU-primer. Identification of 5fU is based on selective reduction from 5fU to 5hmU, subsequent chemoenzymatic labeling of the newly generated 5hmU, and cross-linking with N3-modified 5fU-primer via click chemistry. Then, all of the 5hmU and 5fU sites are encoded with respective circularized barcodes. These barcodes are simultaneously amplified for multiplexed single-molecule imaging. The above two kinds of barcodes can be simultaneously amplified for differentiated visualization of 5hmU and 5fU in single cells. We find these two kinds of cellular oxidative thymines are spatially organized in a cell-type-dependent style with cell-to-cell heterogeneity. We also investigate their multilevel subcellular information and explore their dynamic changes during cell cycles. Further, using DNA sequencing instead of fluorescence imaging, our proposed bioorthogonal chemical signature holds great potential to offer the sequence information of these oxidative thymines in cells and may provide a reliable chemical biology approach for studying the whole-genome oxidative thymines profiles and insights into their functional role and dynamics in biology.
研究领域:
化学生物学
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分子生物学
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细胞生物学
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基因组学
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产品关联
DOI
PubMed
高影响因子
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