Oxford Nanopore MinION Sequencing and Genome Assembly

The revolution of genome sequencing is continuing after the successful secondgeneration sequencing （SGS） technology. The third-generation sequencing （TGS） technology, led by Pacific Biosciences （PacBio）, is progressing rapidly, moving from a technology once only capable of providing data for small genome analysis, or for performing targeted screening, to one that promises high quality de novo assembly and structural variation detection for human-sized genomes. In 2014, the MinION, the first commercial sequencer using nanopore technology, was released by Oxford Nanopore Technologies （ONT）. MiniON identifies DNA bases by measuring the changes in electrical conductivity generated as DNA strands pass through a biological pore. Its portability, affordability, and speed in data production makes it suitable for real-time applications, the release of the long read sequencer MiniON has thus generated much excitement and interest in the genomics community. While de novo genome assemblies can be cheaply produced from SGS data, assem- bly continuity is often relatively poor, due to the limited ability of short reads to handle long repeats. Assembly quality can be greatly improved by using TGS long reads, since repetitive regions can be easily expanded into using longer sequencing lengths, despite having higher error rates at the base level. The potential of nanopore sequencing has been demonstrated by various studies in genome surveillance at locations where rapid and reliable sequencing is needed, but where resources are limited.

Application of Nanopore Sequencing Technology in the Clinical Diagnosis of Infectious Diseases

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.

Improved genome annotation of Brassica oleracea highlights the importance of alternative splicing

Brassica oleracea has been developed into many important crops,including cabbage,kale,cauliflower,broccoli and so on.The genome and gene annotation of cabbage(cultivar JZS),a representative morphotype of B.oleracea,has been widely used as a common reference in biological research.Although its genome assembly has been updated twice,the current gene annotation still lacks information on untranslated regions(UTRs)and alternative splicing(AS).Here,we constructed a high-quality gene annotation(JZSv3)using a full-length transcriptome acquired by nanopore sequencing,yielding a total of 59452 genes and 75684 transcripts.Additionally,we re-analyzed the previously reported transcriptome data related to the development of different tissues and cold response using JZSv3 as a reference,and found that 3843 out of 11908 differentially expressed genes(DEGs)underwent AS during the development of different tissues and 309 out of 903 cold-related genes underwent AS in response to cold stress.Meanwhile,we also identified many AS genes,including BolLHCB5 and BolHSP70,that displayed distinct expression patterns within variant transcripts of the same gene,highlighting the importance of JZSv3 as a pivotal reference for AS analysis.Overall,JZSv3 provides a valuable resource for exploring gene function,especially for obtaining a deeper understanding of AS regulation mechanisms.

Transcriptomic Analysis and Comparison of the Gene Expression Profiles in Fast- and Slow-Growing Pearl Oysters Pinctada fucata martensii

The pearl oyster Pinctada fucata martensii is an economically valuable shellfish that is cultured for seawater pearl pro-duction,which mainly depends on oyster growth.However,the growth mechanisms of the pearl oyster are still poorly understood.In this study,oysters were grouped with relative growth rate,including fast-growing(FG)group and slow-growing(SG)group.Oxford Nanopore Technologies(ONT)long-read sequencing was applied to investigate the molecular mechanisms involved in the growth of this species.Five alternative splicing(AS)types were analyzed in both FG and SG groups,which include alternative 3’splice site,alternative 5’splice site,exon skipping,intron retention,and mutually exclusive exon.Transcriptome analysis showed that four of five different AS events(excluding mutually exclusive exons)occurred more frequently in FG than in SG oysters,and the five main AS types exhibited different characteristics.The AS events that were detected may be involved in growth,and the difference in ex-pression of AS events between FG and SG oysters may be involved in the mechanism underlying the difference in growth.Fifty dif-ferentially expressed genes(DEGs)were identified between the FG and SG oysters.The results showed that 40 genes were signifi-cantly up-regulated in FG oysters,while 10 genes were significantly down-regulated in SG oyster.Several genes related to nutrient metabolism,shell formation,and immunity were more highly expressed in FG oysters than in SG oysters.In summary,FG oysters exhibited higher metabolic and biomineralization activities and had a more powerful immune system than SG oysters.These results provide insight into the growth of P.f.martensii that can be used to improve breeding programs.

Clinical applications of metagenomics next-generation sequencing in infectious diseases

Infectious diseases are a great threat to human health.Rapid and accurate detection of pathogens is important in the diagnosis and treatment of infectious diseases.Metagenomics next-generation sequencing(mNGS)is an unbiased and comprehensive approach for detecting all RNA and DNA in a sample.With the development of sequencing and bioinformatics technologies,mNGS is moving from research to clinical application,which opens a new avenue for pathogen detection.Numerous studies have revealed good potential for the clinical application of mNGS in infectious diseases,especially in difficult-to-detect,rare,and novel pathogens.However,there are several hurdles in the clinical application of mNGS,such as:(1)lack of universal workflow validation and quality assurance;(2)insensitivity to high-host background and low-biomass samples;and(3)lack of standardized instructions for mass data analysis and report interpretation.Therefore,a complete understanding of this new technology will help promote the clinical application of mNGS to infectious diseases.This review briefly introduces the history of next-generation sequencing,mainstream sequencing platforms,and mNGS workflow,and discusses the clinical applications of mNGS to infectious diseases and its advantages and disadvantages.

High quality genome sequences of thirteen Hypoxylaceae(Ascomycota)strengthen the phylogenetic family backbone and enable the discovery of new taxa

The Hypoxylaceae(Xylariales,Ascomycota)is a diverse family of mainly saprotrophic fungi,which commonly occur in angiosperm-dominated forests around the world.Despite their importance in forest and plant ecology as well as a prolific source of secondary metabolites and enzymes,genome sequences of related taxa are scarce and usually derived from envi-ronmental isolates.To address this lack of knowledge thirteen taxonomically well-defined representatives of the family and one member of the closely related Xylariaceae were genome sequenced using combinations of Illumina and Oxford nanopore technologies or PacBio sequencing.The workflow leads to high quality draft genome sequences with an average N50 of 3.0 Mbp.A backbone phylogenomic tree was calculated based on the amino acid sequences of 4912 core genes reflecting the current accepted taxonomic concept of the Hypoxylaceae.A Percentage of Conserved Proteins(POCP)analysis revealed that 70%of the proteins are conserved within the family,a value with potential application for the definition of family boundaries within the order Xylariales.Also,Hypomontagnella spongiphila is proposed as a new marine derived lineage of Hypom.monticulosa based on in-depth genomic comparison and morphological differences of the cultures.The results showed that both species share 95%of their genes corresponding to more than 700 strain-specific proteins.This difference is not reflected by standard taxonomic assessments(morphology of sexual and asexual morph,chemotaxonomy,phylogeny),preventing species delimitation based on traditional concepts.Genetic changes are likely to be the result of environmental adaptations and selective pressure,the driving force of speciation.These data provide an important starting point for the establishment of a stable phylogeny of the Xylariales;they enable studies on evolution,ecological behavior and biosynthesis of natural products;and they significantly advance the taxonomy of fungi.