Application and Prospect of Nanopore Sequencing Technology in Etiological Diagnosis of Blood Stream Infection / 中国医学科学院学报

Blood stream infection (BSI),a blood-borne disease caused by microorganisms such as bacteria,fungi,and viruses,can lead to bacteremia,sepsis,and infectious shock,posing a serious threat to human life and health.Identifying the pathogen is central to the precise treatment of BSI.Traditional blood culture is the gold standard for pathogen identification,while it has limitations in clinical practice due to the long time consumption,production of false negative results,etc.Nanopore sequencing,as a new generation of sequencing technology,can rapidly detect pathogens,drug resistance genes,and virulence genes for the optimization of clinical treatment.This paper reviews the current status of nanopore sequencing technology in the diagnosis of BSI.

Monkeypox virus genome sequence from an imported human case in Colombia / Secuencia genómica del virus de la viruela símica de un caso importado en Colombia

Introduction: Monkeypox virus (MPXV) is an enveloped double-stranded DNA virus with a genome of approximately 197.209 bp. The current classification divides MPXV into three clades: Clade I (Central African or Congo Basin clade) and clades IIa and IIb (West African clades). Objective: To report the complete genome and phylogenetic analysis of a human monkeypox case detected in Colombia. Materials and methods: Exudate from vesicular lesions was obtained from a male patient with recent travel history to Spain. A direct genomic approach was implemented in which total DNA from the sample was purified through a column-based method, followed by sequencing on the Nanopore GridION. Reads were aligned against the MPXV reference genome using minimap2 v.2.24 and phylogenetic inference was performed using maximum likelihood estimation. Results: A total of 11.951 reads mapped directly to a reference genome with 96.8% of coverage (190.898 bp). Conclusion: Phylogenetic analysis of the MPXV circulating in Colombia demonstrated its close relationship to clade IIb responsible for the multi-country outbreak in 2022.

Introducción. El virus de la viruela del mono (MPXV) está compuesto por un genoma de ADN bicatenario, aproximadamente, de 197.209 pb. La clasificación actual agrupa el MPXV en tres clados: clado I (de la cuenca del Congo en África central), y clados IIa y IIb (de África occidental). Objetivo. Reportar el genoma completo y el análisis filogenético de un caso humano de viruela símica detectado en Colombia. Materiales y métodos. Se obtuvo exudado de lesiones vesiculares de un paciente varón con el antecedente de un viaje reciente a España. Se implementó un enfoque directo, en el cual se purificó el ADN total de la muestra mediante un método basado en columnas, seguido de la secuenciación directa en la plataforma Nanopore GridION. Las lecturas se alinearon con el genoma de referencia del MPXV, utilizando minimap2, v.2.24, y la inferencia filogenética fue realizada mediante la estimación por máxima verosimilitud. Resultados. Un total de 11.951 lecturas se alinearon directamente con el genoma de referencia con una cobertura del 96,8 % (190.898 pb). Conclusión. El análisis filogenético del MPXV circulante en Colombia demostró su estrecha relación con el clado de África occidental (clado IIb) responsable del brote en múltiples países en el 2022.

Unsupervised deep learning for identifying the O 6-carboxymethyl guanine by nanopore sequencing / 生物医学工程学杂志

O 6-carboxymethyl guanine(O 6-CMG) is a highly mutagenic alkylation product of DNA that causes gastrointestinal cancer in organisms. Existing studies used mutant Mycobacterium smegmatis porin A (MspA) nanopore assisted by Phi29 DNA polymerase to localize it. Recently, machine learning technology has been widely used in the analysis of nanopore sequencing data. But the machine learning always need a large number of data labels that have brought extra work burden to researchers, which greatly affects its practicability. Accordingly, this paper proposes a nano-Unsupervised-Deep-Learning method (nano-UDL) based on an unsupervised clustering algorithm to identify methylation events in nanopore data automatically. Specially, nano-UDL first uses the deep AutoEncoder to extract features from the nanopore dataset and then applies the MeanShift clustering algorithm to classify data. Besides, nano-UDL can extract the optimal features for clustering by joint optimizing the clustering loss and reconstruction loss. Experimental results demonstrate that nano-UDL has relatively accurate recognition accuracy on the O 6-CMG dataset and can accurately identify all sequence segments containing O 6-CMG. In order to further verify the robustness of nano-UDL, hyperparameter sensitivity verification and ablation experiments were carried out in this paper. Using machine learning to analyze nanopore data can effectively reduce the additional cost of manual data analysis, which is significant for many biological studies, including genome sequencing.

Application of nanopore sequencing in environmental microbiology research / 生物工程学报

The development of high-throughput sequencing techniques enabled a deeper and more comprehensive understanding of environmental microbiology. Specifically, the third-generation sequencing techniques represented by nanopore sequencing have greatly promoted the development of environmental microbiology research due to its advantages such as long sequencing reads, fast sequencing speed, real-time monitoring of sequencing data, and convenient machine carrying, as well as no GC bias and no PCR amplification requirement. This review briefly summarized the technical principle and characteristics of nanopore sequencing, followed by discussing the application of nanopore sequencing techniques in the amplicon sequencing, metagenome sequencing and whole genome sequencing of environmental microorganisms. The advantages and challenges of nanopore sequencing in the application of environmental microbiology research were also analyzed.

Application of Nanopore Sequencing Technology in the Clinical Diagnosis of Infectious Diseases / 生物医学与环境科学（英文）

Infectious diseases are an enormous public health burden and a growing threat to human health worldwide. Emerging or classic recurrent pathogens, or pathogens with resistant traits, challenge our ability to diagnose and control infectious diseases. Nanopore sequencing technology has the potential to enhance our ability to diagnose, interrogate, and track infectious diseases due to the unrestricted read length and system portability. This 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 communities. Finally, we discuss the current challenges, potential opportunities, and future outlook for applying nanopore sequencing technology in the diagnosis of infectious diseases.

Analysis of gene inversion in Hemophilia A by Nanopore sequencing / 中华医学遗传学杂志

OBJECTIVE@#To detect gene inversion in two pedigrees affected with Hemophilia A by using Nanopore sequencing technology.@*METHODS@#Peripheral blood samples were taken from members of the two pedigrees. Following extraction of genome DNA, genetic variants of the carriers were detected by Nanopore sequencing and subjected to bioinformatic analysis.@*RESULTS@#Nanopore sequencing has identified the niece of the proband of the pedigree 1 as carrier of Hemophilia A Inv22, and the mother of the proband of the pedigree 2 as carrier of Hemophilia A Inv1, which was consistent with clinical findings. Breakpoint sites in both pedigrees were accurately mapped. Statistical analysis of the sequencing results revealed a large number of variations in the carriers' genomes including deletions, duplications, insertions, inversions and translocations.@*CONCLUSION@#Nanopore sequencing can be used to analyze gene inversions associated with Hemophilia A, which also provided a powerful tool for the diagnosis of diseases caused by gene inversions.

Research progress and application of nanopore sequencing technology / 生物工程学报

Sequencing technology has been greatly improved in terms of throughput and cost. The single-molecule nanopore DNA sequencing, one of the major branches of the third-generation sequencing technology, has made great contributions in the fields of medicine and life sciences due to its advantages of ultra-long reading length, real-time detection and direct detection of base methylation modification, etc. This article briefly describes the principle of nanopore sequencing technology, and discusses its application in clinical, animal, plant, bacterial and virus fields and its future development direction.