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Generating barcodes for nanopore sequencing data with PRO

Fundamental Research 2024年第4期4卷 785-794页

作者： Ting Yu Zitong Ren Xin Gao Guojun Li Renmin Han Research Center for Mathematics and Interdisciplinary Sciences Frontiers Science Center for Nonlinear Expectations(Ministry of Education)Shandong UniversityShandong 266000China Computer Electrical and Mathematical Sciences and Engineering Division&Computational Bioscience Research CenterKing Abdullah University of Science and TechnologyThuwal 23955Saudi Arabia

DNA barcodes,short and unique DNA sequences,play a crucial role in sample identification when processing many samples simultaneously,which helps reduce experimental ***,the low quality of long-read sequencing makes it difficult to identify barcodes accurately,which poses significant challenges for the design of barcodes for large numbers of samples in a single sequencing ***,we present a comprehensive study of the generation of barcodes and develop a tool,PRO,that can be used for selecting optimal barcode sets and *** formulate the barcode design problem as a combinatorial problem and prove that finding the optimal largest barcode set in a given DNA sequence space in which all sequences have the same length is theoretically *** practical applications,we developed the novel method PRO by introducing the probability divergence between two DNA sequences to expand the capacity of barcode kits while ensuring demultiplexing ***,the maximum size of the barcode kits designed by PRO is 2,292,which keeps the length of barcodes the same as that of the official ones used by Oxford nanopore Technologies(ONT).We validated the performance of PRO on a simulated nanopore dataset with high error *** demultiplexing accuracy of PRO reached 98.29%for a barcode kit of size 2,922,4.31%higher than that of Guppy,the official demultiplexing *** the size of the barcode kit generated by PRO is the same as the official size provided by ONT,both tools show superior and comparable demultiplexing accuracy.

关键词： Third-generation sequencing nanopore sequencing DNA barcode Farthest point sampling algorithm High throughput

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Cas9-targeted nanopore sequencing rapidly elucidates the transposition preferences and DNA methylation profiles of mobile elements in plants

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Journal of Integrative Plant Biology 2023年第10期65卷 2242-2261页

作者： Pavel Merkulov Sofya Gvaramiya Maxim Dudnikov Roman Komakhin Murad Omarov Alina Kocheshkova Zakhar Konstantinov Alexander Soloviev Gennady Karlov Mikhail Divashuk Ilya Kirov All‐Russia Research Institute of Agricultural Biotechnology Moscow 127550Russia Moscow Institute of Physics and Technology Dolgoprudny 141701Russia All‐Russia Center for Plant Quarantine Ramenski 140150Russia N.V.Tsitsin Main Botanical Garden of the Russian Academy of Sciences Moscow 127276Russia

Transposable element insertions(TEIs) are an important source of genomic innovation by contributing to plant adaptation, speciation, and the production of new varieties. The often large,complex plant genomes make identifying TEIs from short reads difficult and expensive. Moreover, rare somatic insertions that refect mobilome dynamics are difficult to track using short *** address these challenges, we combined Cas9-targeted nanopore sequencing(CANS) with the novel pipeline Nano Cas TE to trace both genetically inherited and somatic TEIs in plants. We performed CANS of the EVADé(EVD) retrotransposon in wild-type Arabidopsis thaliana and rapidly obtained up to 40× sequence *** of hemizygous T-DNA insertion sites and genetically inherited insertions of the EVD transposon in the ddm1(decrease in DNA methylation1) genome uncovered the crucial role of DNA methylation in shaping EVD insertion *** also investigated somatic transposition events of the ONSEN transposon family, finding that genes that are downregulated during heat stress are preferentially targeted by ONSENs. Finally, we detected hypomethylation of novel somatic insertions for two ONSENs. CANS and Nano Cas TE are effective tools for detecting TEIs and exploring mobilome organization in plants in response to stress and in different genetic backgrounds, as well as screening T-DNA insertion mutants and transgenic plants.

关键词： Arabidopsis DNA methylation nanopore sequencing transposon insertions

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Application of nanopore sequencing Technology in the Clinical Diagnosis of Infectious Diseases

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Biomedical and Environmental Sciences 2022年第5期35卷 381-392页

作者： ZHANG Lu Lu ZHANG Chi PENG Jun Ping NHC Key Laboratory of Systems Biology of Pathogens Institute of Pathogen BiologyChinese Academy of Medical Sciences&Peking Union Medical CollegeBeijing 100176China Key Laboratory of Respiratory Disease Pathogenomics Chinese Academy of Medical SciencesBeijing 100176China

Infectious diseases are an enormous public health burden and a growing threat to human health *** or classic recurrent pathogens,or pathogens with resistant traits,challenge our ability to diagnose and control infectious *** sequencing technology has the potential to enhance our ability to diagnose,interrogate,and track infectious diseases due to the unrestricted read length and system *** review focuses on the application of nanopore sequencing technology in the clinical diagnosis of infectious diseases and includes the following:(i)a brief introduction to nanopore sequencing technology and Oxford nanopore Technologies(ONT)sequencing platforms;(ii)strategies for nanopore-based sequencing technologies;and(iii)applications of nanopore sequencing technology in monitoring emerging pathogenic microorganisms,molecular detection of clinically relevant drug-resistance genes,and characterization of disease-related microbial ***,we discuss the current challenges,potential opportunities,and future outlook for applying nanopore sequencing technology in the diagnosis of infectious diseases.

关键词： nanopore sequencing Infectious diseases Pathogen Oxford nanopore Technologies

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GREPore-seq: A Robust Workflow to Detect Changes After Gene Editing Through Long-range PCR and nanopore sequencing

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Genomics, Proteomics & Bioinformatics 2023年第6期21卷 1221-1236页

作者： Zi-Jun Quan Si-Ang Li Zhi-Xue Yang Juan-Juan Zhao Guo-Hua Li Feng Zhang Wei Wen Tao Cheng Xiao-Bing Zhang State Key Laboratory of Experimental Hematology National Clinical Research Center for Blood DiseasesHaihe Laboratory of Cell EcosystemInstitute of Hematology&Blood Diseases HospitalChinese Academy of Medical Sciences&Peking Union Medical CollegeTianjin 300020China Center for Stem Cell Medicine Chinese Academy of Medical SciencesTianjin 300020China Department of Stem Cell&Regenerative Medicine Peking Union Medical CollegeTianjin 300020China

To achieve the enormous potential of gene-editing technology in clinical therapies,one needs to evaluate both the on-target efficiency and unintended editing consequences ***,there is a lack of a pipelined,large-scale,and economical workflow for detecting genome editing outcomes,in particular insertion or deletion of a large ***,we describe an approach for efficient and accurate detection of multiple genetic changes after CRISPR/Cas9 editing by pooled nanopore sequencing of barcoded long-range PCR *** the high error rates of Oxford nanopore sequencing,we developed a novel pipeline to capture the barcoded sequences by grepping reads of nanopore amplicon sequencing(GREPore-seq).GREPore-seq can assess nonhomologous end-joining(NHEJ)-mediated double-stranded oligodeoxynucleotide(dsODN)insertions with comparable accuracy to Illumina next-generation sequencing(NGS).GREPore-seq also reveals a full spectrum of homology-directed repair(HDR)-mediated large gene knock-in,correlating well with the fluorescence-activated cell sorting(FACS)analysis *** note,we discovered low-level fragmented and full-length plasmid backbone insertion at the CRISPR cutting ***,we have established a practical workflow to evaluate various genetic changes,including quantifying insertions of short dsODNs,knock-ins of long pieces,plasmid insertions,and large fragment deletions after CRISPR/Cas9-mediated ***-seq is freely available at GitHub(https://***/lisiang/GREPore-seq)and the National Genomics Data Center(NGDC)BioCode(https://***/biocode/tools/BT007293).

关键词： CRISPR/Cas9 Genetic change Long-range PCR nanopore sequencing GREPore-seq

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Rapid Acquisition of High-Quality SARS-CoV-2 Genome via Amplicon-Oxford nanopore sequencing

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Virologica Sinica 2021年第6期36卷 901-912页

作者： Yi Yan Ke Wu Jun Chen Haizhou Liu Yi Huang Yong Zhang Jin Xiong Weipeng Quan Xin Wu Yu Liang Kunlun He Zhilong Jia Depeng Wang Di Liu Hongping Wei Jianjun Chen CAS Key Laboratory of Special Pathogens and Biosafety Wuhan Institute of VirologyCenter for Biosafety MegaScienceChinese Academy of SciencesWuhan 430071China National Virus Resource Center Wuhan Institute of VirologyChinese Academy of SciencesWuhan 430071China Computational Virology Group Center for Bacteria and Viruses Resources and BioinformationWuhan Institute of VirologyChinese Academy of SciencesWuhan 430071China University of Chinese Academy of Sciences Beijing 101409China Wuhan Pulmonary Hospital Wuhan Tuberculosis Prevention and Treatment InstituteWuhan 430030China National Biosafety Laboratory Chinese Academy of SciencesWuhan 430071China GrandOmics Biosciences Wuhan 430000China GrandOmics Biosciences Beijing 102200China GrandOmics Diagnostics Wuhan 430000China Key Laboratory of Biomedical Engineering and Translational Medicine Ministry of Industry and Information TechnologyChinese PLA General HospitalBeijing 100039China Beijing Key Laboratory for Precision Medicine of Chronic Heart Failure Chinese PLA General HospitalBeijing 100039China First Affiliated Hospital of Xinjiang Medical University Urumqi 830054China

Genome sequencing has shown strong capabilities in the initial stages of the COVID-19 pandemic such as pathogen identification and virus preliminary *** the rapid acquisition of SARS-Co V-2 genome from clinical specimens is limited by their low nucleic acid load and the complexity of the nucleic acid *** address this issue,we modified and evaluated an approach by utilizing SARS-Co V-2-specific amplicon amplification and Oxford nanopore Prometh ION *** workflow started with the throat swab of the COVID-19 patient,combined reverse transcript PCR,and multi-amplification in one-step to shorten the experiment time,then can quickly and steadily obtain high-quality SARS-Co V-2 genome within 24 h.A comprehensive evaluation of the method was conducted in 42 samples:the sequencing quality of the method was correlated well with the viral load of the samples;high-quality SARS-Co V-2 genome could be obtained stably in the samples with Ct value up to 39.14;data yielding for different Ct values were assessed and the recommended sequencing time was 8 h for samples with Ct value of less than 20;variation analysis indicated that the method can detect the existing and emerging genomic mutations as well;Illumina sequencing verified that ultra-deep sequencing can greatly improve the single read error rate of nanopore sequencing,making it as low as 0.4/10,000 *** summary,high-quality SARS-Co V-2 genome can be acquired by utilizing the amplicon amplification and it is an effective method in accelerating the acquisition of genetic resources and tracking the genome diversity of SARSCo V-2.

关键词： SARS-CoV-2 Genome Amplicon nanopore sequencing

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Applications and potentials of nanopore sequencing in the(epi)genome and(epi)transcriptome era

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The Innovation 2021年第4期2卷 11-26页

作者： Shangqian Xie Amy Wing-Sze Leung Zhenxian Zheng Dake Zhang Chuanle Xiao Ruibang Luo Ming Luo Shoudong Zhang Key Laboratory of Ministry of Education for Genetics and Germplasm Innovation of Tropical Special Trees and Ornamental Plants College of ForestryHainan UniversityHaikou 570228China Department of Computer Science The University of Hong KongHong Kong 999077China Beijing Advanced Innovation Centre for Biomedical Engineering Key Laboratory for Biomechanics and Mechanobiology of Ministry of EducationSchool of Biological Science and Medical EngineeringBeihang UniversityBeijing 100083China State Key Laboratory of Ophthalmology Zhongshan Ophthalmic CentreSun Yat-sen UniversityGuangzhou 510060China Agriculture and Biotechnology Research Center Guangdong Provincial Key Laboratory of Applied BotanyCenter of Economic BotanyCore Botanical GardensSouth China Botanical GardenChinese Academy of SciencesGuangzhou 510650China School of Life Sciences The Chinese University of Hong KongShatinHong Kong 999077China Center for Soybean Research of the State Key Laboratory of Agrobiotechnology The Chinese University of Hong KongShatinHong Kong 999077China

The Human Genome Project opened an era of(epi)genomic research,and also provided a platform for the development of new sequencing *** and after the project,several sequencing technologies continue to dominate nucleic acid sequencing ***,Illumina(short-read),PacBio(long-read),and Oxford nanopore(longread)are the most popular sequencing *** PacBio or the popular short-read sequencers before it,which,as examples of the second or so-called Next-Generation sequencing platforms,need to synthesize when sequencing,nanopore technology directly sequences native DNA and RNA *** sequencing,therefore,avoids converting mRNA into cDNA molecules,which not only allows for the sequencing of extremely long native DNA and full-length RNA molecules but also document modifications that have been made to those native DNA or RNA *** this review on direct DNA sequencing and direct RNA sequencing using Oxford nanopore technology,we focus on their development and application achievements,discussing their challenges and future *** also address the problems researchers may encounter applying these approaches in their research topics,and how to resolve them.

关键词： nanopore sequencing direct DNA sequencing direct RNA sequencing base modification base-calling long-read sequencing tools and algorithms

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Cancer biomarkers discovery of methylation modification with direct high-throughput nanopore sequencing

Cancer biomarkers discovery of methylation modification with...

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2021年中国肿瘤标志物学术大会暨第十五届肿瘤标志物青年科学家论坛

作者： Junjie Zhang Shuilian Xie Shaogui Wan Gannan Medical University

Cancer is a complex disease, driven by a combination of genetic and epigenetic alterations. DNA and RNA methylation modifications are the most common epigenetic events that play critical roles in cancer development and progression. Bisulfite converted sequencing is a widely used technique to detect base modifications in DNA methylation, but its main drawbacks lie in DNA degradation, lack of specificity,or short reads with low sequence diversity. The nanopore sequencing technology can directly detect base modifications in native DNA as well as RNA without harsh chemical treatment, compared to bisulfite sequencing. Furthermore, CRISPR/Cas9-targeted enrichment nanopore sequencing techniques are straightforward and cost-effective when targeting genomic regions are of interest. In this review, we mainly focus on DNA and RNA methylation modifications detection in cancer with the current nanopore sequencing approaches. We also present the respective strengths, weaknesses of nanopore sequencing techniques and their future translational applications in identification of epigenetic biomarkers for cancer detection and prognosis.

关键词： nanopore sequencing Cancer biomarker Cas9 enrichment DNA methylation RNA methylation

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Determining antimicrobial resistance profiles and identifying novel mutations of Neisseria gonorrhoeae genomes obtained by multiplexed MinION sequencing

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Science China(Life Sciences) 2020年第7期63卷 1063-1070页

作者： Chi Zhang Feng Wang Cansheng Zhu Leshan Xiu Yamei Li Li Li Bo Liu Yizhun Li Yaling Zeng Boyang Guo Junping Peng NHC Key Laboratory'of Systems Biology of Pathogens Institute of Pathogen BiologyChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing 100176China Shenzhen Center for Chronic Disease Control Shenzhen 518020China Department of STD Laboratory Shaanxi Provincial Institute for Skin Disease and STD ControlXi'an 710003China

Gonorrhea is one of the most common sexually transmitted diseases worldwide. To cure infection and prevent transmission,timely and appropriate antimicrobial therapy is necessary. Unfortunately, Neisseria gonorrhoeae, the etiological agent of gonorrhea, has acquired nearly all known mechanisms of antimicrobial resistance(AMR), thereby compromising the efficacy of antimicrobial therapy. Treatment failure resulting from AMR has become a global public health concern. Whole-genome sequencing is an effective method to determine the AMR characteristics of N. gonorrhoeae. Compared with next-generation sequencing, the MinION sequencer(Oxford nanopore Technologies(ONT)) has the advantages of long read length and portability. Based on a pilot study using MinION to sequence the genome of N. gonorrhoeae, we optimized the workflow of sequencing and data analysis in the current study. Here we sequenced nine isolates within one flow cell using a multiplexed sequencing strategy. After hybrid assembly with Illumina reads, nine integral circular chromosomes were obtained. By using the online tool Pathogenwatch and a BLAST-based workflow, we acquired complete AMR profiles related to seven classes of antibiotics. We also evaluated the performance of ONT-only assemblies. Most AMR determinants identified by ONT-only assemblies were the same as those identified by hybrid assemblies. Moreover, one of the nine assemblies indicated a potentially novel antimicrobial-related mutation located in mtrR which results in a frame-shift, premature stop codon, and truncated *** addition, this is the first study using the MinION sequencer to obtain complete genome sequences of N. gonorrhoeae strains which are epidemic in China. This study shows that complete genome sequences and antimicrobial characteristics of *** can be obtained using the MinION sequencer in a simple and cost-effective manner, with hardly any knowledge of bioinformatics required. More importantly, this strategy provid

关键词： nanopore sequencing MinION sequencer Neisseria gonorrhoeae whole-genome sequencing antimicrobial resistance

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DNA sequencing using nanopores and kinetic proofreading

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Quantitative Biology 2020年第3期8卷 187-194页

作者： Xinsheng Sean Ling Department of Physics Brown UniversityProvidenceRI 02912USA

We discuss the feasibility of using a nanopore sandwich device to implement the principle of kinetic proofreading to discriminate incorrect hybridizing oligonucleotides on a target DNA or *** propose a method of sequencing DNA or RNA using this *** design parameters for such a DNA sequencer are estimated from the Hopfield・Ninio theory of kinetic proofreading and Schrodinger's first-passage-time distribution function.

关键词： DNA sequencing nanopore sequencing biosensing using kinetics

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WaveNano:a signal-level nanopore base-caller via simultaneous prediction of nucleotide labels and move labels through bi-directional WaveNets

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Frontiers of Electrical and Electronic Engineering in China 2018年第4期6卷 359-368页

作者： Sheng Wang Zhen Li Yizhou YU Xin Gao Computational Bioscience Research Center (CBRC) King Abdullah University of Science and Technology (KAUST)Thuwa23955-6900Kingdom of Saudi Arabia Department of Computer Science University of Hong KongHong Kong SAR 999077China School of Science and Engineering Shenzhen Research Institute of Big DataThe Chinese University of Hong Kong Shenzhen 518172China

The Oxford MinION nanopore sequencer is the recently appealing third-generation genome sequencing device that is portable and no larger than a cellphone. Despite the benefits of MinION to sequence ultra-long reads in real-time, the high error rate of the existing base-calling methods, especially indels (insertions and deletions), prevents its use in a variety of applications. In this paper, we show that such indel errors are largely due to the segmentation process on the input electrical current signal from MinION. All existing methods conduct segmentation and nucleotide label prediction in a sequential manner, in which the errors accumulated in the first step will irreversibly influence the final base-calling. We further show that the indel issue can be significantly reduced via accurate labeling of nucleotide and move labels directly from the raw signal, which can then be efficiently learned by a bi-directionalWaveNet model simultaneously through feature sharing. Our bi-directional WaveNet model with residual blocks and skip connections is able to capture the extremely long dependency in the raw signal. Taking the predicted move as the segmentation guidance, we employ the Viterbi decoding to obtain the final base-calling results from the smoothed nucleotide probability matrix. Our proposed base-caller, WaveNano, achieves good performance on real MinION sequencing data from Lambda phage. The signal-level nanopore base-callerWaveNano can obtain higher base-calling accuracy, and generate fewer insertions/deletions in the base-called sequences.

关键词： nanopore sequencing bi-directional WaveNets base-calling third generation sequencing deep learning

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