Objective:Autosomal recessive bestrophinopathy(ARB),a retinal degenerative disease,is characterized by central visual loss,yellowish multifocal diffuse subretinal deposits,and a dramatic decrease in the light peak on ...Objective:Autosomal recessive bestrophinopathy(ARB),a retinal degenerative disease,is characterized by central visual loss,yellowish multifocal diffuse subretinal deposits,and a dramatic decrease in the light peak on electrooculogram.The potential pathogenic mechanism involves mutations in the BEST1 gene,which encodes Ca2+-activated Cl−channels in the retinal pigment epithelium(RPE),resulting in degeneration of RPE and photoreceptor.In this study,the complete clinical characteristics of two Chinese ARB families were summarized.Methods:Pacific Biosciences(PacBio)single-molecule real-time(SMRT)sequencing was performed on the probands to screen for disease-causing gene mutations,and Sanger sequencing was applied to validate variants in the patients and their family members.Results:Two novel mutations,c.202T>C(chr11:61722628,p.Y68H)and c.867+97G>A,in the BEST1 gene were identified in the two Chinese ARB families.The novel missense mutation BEST1 c.202T>C(p.Y68H)resulted in the substitution of tyrosine with histidine in the N-terminal region of transmembrane domain 2 of bestrophin-1.Another novel variant,BEST1 c.867+97G>A(chr11:61725867),located in intron 7,might be considered a regulatory variant that changes allele-specific binding affinity based on motifs of important transcriptional regulators.Conclusion:Our findings represent the first use of third-generation sequencing(TGS)to identify novel BEST1 mutations in patients with ARB,indicating that TGS can be a more accurate and efficient tool for identifying mutations in specific genes.The novel variants identified further broaden the mutation spectrum of BEST1 in the Chinese population.展开更多
AIM:To estimate if nanopore targeted sequencing(NTS)could identify pathogens causing postoperative endophthalmitis and further determine the feasibility of clinical application of NTS.METHODS:A total of 55 patients(55...AIM:To estimate if nanopore targeted sequencing(NTS)could identify pathogens causing postoperative endophthalmitis and further determine the feasibility of clinical application of NTS.METHODS:A total of 55 patients(55 eyes)with postoperative endophthalmitis were retrospectively included in this study with their medical records.Intraocular fluid samples were examined by NTS and microbial culture.All included patients had undergone examinations including measurement of best corrected visual acuity(BCVA)and intraocular pressure(IOP),slit-lamp biomicroscopy,and indirect ophthalmoscopy;additionally,they underwent B-ultrasound,anterior segment photography,and fundus photography if necessary.RESULTS:Among 55 patients with postoperative endophthalmitis,the age was 65.25±15.04y and there were 30 female(54.54%)patients.Forty-one(74.54%)vitreous humor samples and fourteen(25.45%)aqueous humor samples were sent for both NTS and microbial culture.NTS had a notable higher detection rate than microbial culture in detecting pathogens(90.91%vs 38.18%,χ^(2)=33.409,P<0.001).NTS exhibited high sensitivity of pathogen detection in both microbial culture positive and negative samples(100%and 85.29%,respectively).In 16 of 21(76.19%)patients who showed culture-positivity,their results corresponded with those of NTS.Moreover,in two patients(9.52%),NTS showed a better species resolution than microbial culture;in three patients(14.28%),NTS identified additional pathogens.As for fungus,the positive detection rate of NTS was significantly higher than that of microbial culture(20%vs 3.64%,χ^(2)=7.066,P=0.008).Also,NTS could detect multi-infection by bacteria and fungi than microbial culture(32.73%vs 0,χ^(2)=21.522,P<0.001).NTS could detect bacteria as well as fungi simultaneously within 48h in all patients.Meanwhile,NTS had a shorter detection time than microbial culture(1.13±0.34 vs 2.67±0.55d,Z=-9.218,P<0.001).After the NTS results were obtained,15 patients received additional intravitreal/intracameral anti-infection treatment.At follow-up,there was a statistically significant improvement in the visual acuity relative to the baseline(Z=−5.222,P<0.001).CONCLUSION:NTS can provide rapid identification and highly sensitive detection of pathogens among patients with postoperative endophthalmitis,which can guide anti-infection treatment and improve visual prognosis.展开更多
The monkeypox virus(MPXV)has triggered a current outbreak globally.Genome sequencing of MPXV and rapid tracing of genetic variants will benefit disease diagnosis and control.It is a significant challenge but necessary...The monkeypox virus(MPXV)has triggered a current outbreak globally.Genome sequencing of MPXV and rapid tracing of genetic variants will benefit disease diagnosis and control.It is a significant challenge but necessary to optimize the strategy and application of rapid full-length genome identification and to track variations of MPXV in clinical specimens with low viral loads,as it is one of the DNA viruses with the largest genome and the most AT-biased,and has a significant number of tandem repeats.Here we evaluated the performance of metagenomic and amplicon sequencing techniques,and three sequencing platforms in MPXV genome sequencing based on multiple clinical specimens of five mpox cases in Chinese mainland.We rapidly identified the full-length genome of MPXV with the assembly of accurate tandem repeats in multiple clinical specimens.Amplicon sequencing enables cost-effective and rapid sequencing of clinical specimens to obtain high-quality MPXV genomes.Third-generation sequencing facilitates the assembly of the terminal tandem repeat regions in the monkeypox virus genome and corrects a common misassembly in published sequences.Besides,several intra-host single nucleotide variations were identified in the first imported mpox case.This study offers an evaluation of various strategies aimed at identifying the complete genome of MPXV in clinical specimens.The findings of this study will significantly enhance the surveillance of MPXV.展开更多
Over the past decade,nanopore sequencing has experienced significant advancements and changes,transitioning from an initially emerging technology to a significant instrument in the field of genomic sequencing.However,...Over the past decade,nanopore sequencing has experienced significant advancements and changes,transitioning from an initially emerging technology to a significant instrument in the field of genomic sequencing.However,as advancements in next-generation sequencing technology persist,nanopore sequencing also improves.This paper reviews the developments,applications,and outlook on nanopore sequencing technology.Currently,nanopore sequencing supports both DNA and RNA sequencing,making it widely applicable in areas such as telomere-to-telomere(T2T)genome assembly,direct RNA sequencing(DRS),and metagenomics.The openness and versatility of nanopore sequencing have established it as a preferred option for an increasing number of research teams,signaling a transformative influence on life science research.As the nanopore sequencing technology advances,it provides a faster,more costeffective approach with extended read lengths,demonstrating the significant potential for complex genome assembly,pathogen detection,environmental monitoring,and human disease research,offering a fresh perspective in sequencing technologies.展开更多
BACKGROUND Infectious diseases are still one of the greatest threats to human health,and the etiology of 20%of cases of clinical fever is unknown;therefore,rapid identification of pathogens is highly important.Traditi...BACKGROUND Infectious diseases are still one of the greatest threats to human health,and the etiology of 20%of cases of clinical fever is unknown;therefore,rapid identification of pathogens is highly important.Traditional culture methods are only able to detect a limited number of pathogens and are time-consuming;serologic detection has window periods,false-positive and false-negative problems;and nucleic acid molecular detection methods can detect several known pathogens only once.Three-generation nanopore sequencing technology provides new options for identifying pathogens.CASE SUMMARY Case 1:The patient was admitted to the hospital with abdominal pain for three days and cessation of defecation for five days,accompanied by cough and sputum.Nanopore sequencing of the drainage fluid revealed the presence of orallike bacteria,leading to a clinical diagnosis of bronchopleural fistula.Cefoperazone sodium sulbactam treatment was effective.Case 2:The patient was admitted to the hospital with fever and headache,and CT revealed lung inflammation.Antibiotic treatment for Streptococcus pneumoniae,identified through nanopore sequencing of cerebrospinal fluid,was effective.Case 3:The patient was admitted to our hospital with intermittent fever and an enlarged neck mass that had persisted for more than six months.Despite antibacterial treatment,her symptoms worsened.The nanopore sequencing results indicate that voriconazole treatment is effective for Aspergillus brookii.The patient was diagnosed with mixed cell type classical Hodgkin's lymphoma with infection.CONCLUSION Three-generation nanopore sequencing technology allows for rapid and accurate detection of pathogens in human infectious diseases.展开更多
Human leukocyte antigen(HLA)genes in the major histocompatibility complex(MHC)region are crucial for immunity and are associated with numerous diseases and phenotypes.The MHC region’s complexity and high genetic dive...Human leukocyte antigen(HLA)genes in the major histocompatibility complex(MHC)region are crucial for immunity and are associated with numerous diseases and phenotypes.The MHC region’s complexity and high genetic diversity make it challenging to analyze using short-read sequencing(SRS)technology.We sequence the MHC region of 100 Han Chinese individuals using both long-read sequencing(LRS)and SRS platforms at approximately 30X coverage to study genetic alterations and their potential functional impacts.LRS provides significantly greater coverage of the MHC region and eight classical HLA genes,particularly at the HLA-DRB1 locus,compared with SRS.We detect 78,249 single nucleotide polymorphisms(SNPs)using LRS,with 26.0%undetectable by SRS.Based on SNP and inferred HLA allele types,we construct an LRS-based MHC reference panel for the Han Chinese,containing approximately 2.6 times more genetic variants than the SRS-based Han-MHC reference panel.A phenome-wide association study assessing 26,024 phenotypes across 15 categories identifies significant associations for 7,879 independent variants(including 809 LRS-specific SNPs)with 409 phenotypes in nine categories.This analysis reveals 24 unreported HLA allele associations in the bioelectric and cellular categories.The conditional analysis identifies 530 independent signals across the 409 phenotypes,including 28 previously unreported signals of eight classical HLA genes associated with 33 phenotypes.Of the top-associated SNPs,191 are detected by LRS only.Fine-mapping identifies 126 independent candidate causal SNPs for three immune-related cellular phenotypes,with 17 detected exclusively by LRS.Our study reveals previously unreported variants and their functional impacts in the MHC region,enhancing our understanding of genetic diversity and its potential biological implications in the Han Chinese population.展开更多
Neurodegenerative diseases cause great medical and economic burdens for both patients and society;however, the complex molecular mechanisms thereof are not yet well understood. With the development of high-coverage se...Neurodegenerative diseases cause great medical and economic burdens for both patients and society;however, the complex molecular mechanisms thereof are not yet well understood. With the development of high-coverage sequencing technology, researchers have started to notice that genomic repeat regions, previously neglected in search of disease culprits, are active contributors to multiple neurodegenerative diseases. In this review, we describe the association between repeat element variants and multiple degenerative diseases through genome-wide association studies and targeted sequencing. We discuss the identification of disease-relevant repeat element variants, further powered by the advancement of long-read sequencing technologies and their related tools, and summarize recent findings in the molecular mechanisms of repeat element variants in brain degeneration, such as those causing transcriptional silencing or RNA-mediated gain of toxic function. Furthermore, we describe how in silico predictions using innovative computational models, such as deep learning language models, could enhance and accelerate our understanding of the functional impact of repeat element variants. Finally, we discuss future directions to advance current findings for a better understanding of neurodegenerative diseases and the clinical applications of genomic repeat elements.展开更多
Background The RNA virosphere’s extensive diversity and its role in emerging infectious diseases underscore the importance of non-targeted sequencing for identifying unknown or rare pathogens,including co-infections....Background The RNA virosphere’s extensive diversity and its role in emerging infectious diseases underscore the importance of non-targeted sequencing for identifying unknown or rare pathogens,including co-infections.However,enriching low-abundance viral sequences in RNA metaviromics,particularly in the preparation of cDNA libraries and their compatibility with next-generation sequencing(NGS)and third-generation sequencing(TGS),remains challenging.Therefore,our objective is to develop and systematically assess a practical RNA metavirome methodology specifically tailored for the enrichment of low-abundance viral sequences within samples.Methods We developed the SMART-RNA-Metavirome platform,integrating SMART-9n library preparation with NGS and TGS technologies.Total RNA was extracted from two field-collected wild Aedes albopictus pools,along with one laboratory-infected Ae.albopictus pool harboring dengue virus(DENV).This RNA was subjected to reverse transcription using both this optimized protocol and random primer-based methods,followed by high-throughput sequencing on Illumina,Oxford Nanopore,and QitanTech Nanopore technologies.Welch’s t-test was employed for comparative analysis of the subsequent RNA metavirome data,specifically to evaluate differences in viral species composition and abundance of viral reads between experimental groups.Furthermore,the effectiveness of this platform was systematically validated via RT-qPCR and SMART-RNA-Metavirome-based Oxford Nanopore sequencing across multiple sample types,including mosquito specimens from DENV-infected Ae.albopictus,serum samples from dengue patients and viral isolates of Japanese encephalitis virus(JEV)and Zika virus(ZIKV).Results The SMART-RNA-Metavirome platform has been systematically validated to excel in enriching the composition and diversity of the RNA virome(P=0.04),providing sufficient coverage for the complete reconstruction of viral genomes.When employed in the detection of DENV-infected Ae.albopictus,clinical serum samples,and viral isolates of JEV and ZIKV,this technique exhibits a robust correlation with RT-qPCR(r2>0.95).Notably,it demonstrates exceptional sensitivity,ensuring sufficient coverage even in samples of DENV-infected Ae.albopictus with a Ct-value of 35.3,attaining an impressive 99.88%genome coverage.Furthermore,this platform possesses the capability to identify virus species and determine their serotypes.Conclusions In our study,the SMART-RNA-Metavirome platform outperforms traditional methods,enriching RNA virome composition and diversity,enabling practical compatibility with both NGS and TGS technologies.It demonstrates significant proficiency in detecting both known and unknown arboviruses,even in low-titer samples such as those from wild mosquitoes and clinical sera.This platform facilitates comprehensive monitoring,risk assessment,and early warning of RNA virus transmissions,enhancing our understanding of RNA virome diversity and ecological patterns.展开更多
Recent developments in PacBio high-fidelity(HiFi)sequencing technologies have transformed genomic research,with circular consensus se-quencing now achieving 99.9%accuracy for long(up to 25 kb)single-molecule reads.Thi...Recent developments in PacBio high-fidelity(HiFi)sequencing technologies have transformed genomic research,with circular consensus se-quencing now achieving 99.9%accuracy for long(up to 25 kb)single-molecule reads.This method circumvents biases intrinsic to amplification-based approaches,enabling thorough analysis of complex genomic regions including tandem repeats,segmental duplications,ribosomal DNA(rDNA)arrays,and centromeresl as well as direct detection of base modifications,furnishing both sequence and epigenetic data concurrently.This has streamlined a number of tasks including genome assembly,variant detection,and full-length transcript analysis.This review provides a comprehensive overview of the applications and challenges of HiFi sequencing across various fields,including genomics,transcriptomics,and epigenetics.By delineating the evolving landscape of HiFi sequencing in multi-omics research,we highlight its potential to deepen our under-standingofgeneticmechanisms and to advance precision medicine.展开更多
Single-molecule, real-time sequencing developed by Pacific BioSciences offers longer read lengths than the second-generation sequencing （SGS） technologies, making it well-suited for unsolved problems in genome, tran...Single-molecule, real-time sequencing developed by Pacific BioSciences offers longer read lengths than the second-generation sequencing （SGS） technologies, making it well-suited for unsolved problems in genome, transcriptome, and epigenetics research. The highly-contiguous de novo assemblies using PacBio sequencing can close gaps in current reference assemblies and characterize structural variation （SV） in personal genomes. With longer reads, we can sequence through extended repetitive regions and detect mutations, many of which are associated with dis- eases. Moreover, PacBio transcriptome sequencing is advantageous for the identification of gene isoforms and facilitates reliable discoveries of novel genes and novel isoforms of annotated genes, due to its ability to sequence full-length transcripts or fragments with significant lengths. Addition- ally, PacBio＇s sequencing technique provides information that is useful for the direct detection of base modifications, such as methylation. In addition to using PacBio sequencing alone, many hybrid sequencing strategies have been developed to make use of more accurate short reads in conjunction with PacBio long reads. In general, hybrid sequencing strategies are more affordable and scalable especially for small-size laboratories than using PacBio Sequencing alone. The advent of PacBio sequencing has made available much information that could not be obtained via SGS alone.展开更多
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 prog...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.展开更多
Single-cell omics sequencingwas first achieved for the transcriptome in 2009,whichwas followed by fast development of technologies for profiling the genome,DNA methylome,3D genome architecture,chromatin accessibility,...Single-cell omics sequencingwas first achieved for the transcriptome in 2009,whichwas followed by fast development of technologies for profiling the genome,DNA methylome,3D genome architecture,chromatin accessibility,histone modifications,etc.,in an individual cell.In this review we mainly focus on the recent progress in four topics in the single-cell omics field:single-cell epigenome sequencing,single-cell genome sequencing for lineage tracing,spatially resolved single-cell transcriptomics and third-generation sequencing platform-based single-cell omics sequencing.We also discuss the potential applications and future directions of these single-cell omics sequencing technologies for different biomedical systems,especially for the human stem cell field.展开更多
DNA barcodes,short and unique DNA sequences,play a crucial role in sample identification when processing many samples simultaneously,which helps reduce experimental costs.Nevertheless,the low quality of long-read sequ...DNA barcodes,short and unique DNA sequences,play a crucial role in sample identification when processing many samples simultaneously,which helps reduce experimental costs.Nevertheless,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 run.Here,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 demultiplexing.We 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 NP-complete.For 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 accuracy.Specifically,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 rates.The 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 tool.When 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.展开更多
High-quality DNA extraction is a crucial step in metagenomic studies.Bias by different isolation kits impairs the comparison across datasets.A trending topic is,however,the analysis of multiple metagenomes from the sa...High-quality DNA extraction is a crucial step in metagenomic studies.Bias by different isolation kits impairs the comparison across datasets.A trending topic is,however,the analysis of multiple metagenomes from the same patients to draw a holistic picture of microbiota associated with diseases.We thus collected bile,stool,saliva,plaque,sputum,and conjunctival swab samples and performed DNA extraction with three commercial kits.For each combination of the specimen type and DNA extraction kit,20-gigabase(Gb)metagenomic data were generated using short-read sequencing.While profiles of the specimen types showed close proximity to each other,we observed notable differences in the alpha diversity and composition of the microbiota depending on the DNA extraction kits.No kit outperformed all selected kits on every specimen.We reached consistently good results using the Qiagen QiAamp DNA Microbiome Kit.Depending on the specimen,our data indicate that over 10 Gb of sequencing data are required to achieve sufficient resolution,but DNA-based identification is superior to identification by mass spectrometry.Finally,longread nanopore sequencing confirmed the results(correlation coefficient>0.98).Our results thus suggest using a strategy with only one kit for studies aiming for a direct comparison of multiple microbiotas from the same patients.展开更多
Endophthalmitis is a serious ophthalmic disease characterized by changes in the eye's posterior segment,such as hypopyon and intraocular inflammation,vitritis being a hallmark.Infection-caused endophthalmitis can ...Endophthalmitis is a serious ophthalmic disease characterized by changes in the eye's posterior segment,such as hypopyon and intraocular inflammation,vitritis being a hallmark.Infection-caused endophthalmitis can lead to irreversible vision loss,accompanied by eye pain or eye distention,and in the most severe cases the removal of the eyeball.Microorganisms such as bacteria,fungi,viruses,and parasites typically account for the disease and the entry pathways of the microbial can be divided into either endogenous or exogenous approaches,according to the origin of the etiological agents.Exogenous endophthalmitis can be derived from various occasions(such as postoperative complications or trauma)while endogenous endophthalmitis results from the bloodstream which carries pathogens to the eye.This review aims to summarize the application of new technology in pathogen identification of endophthalmitis so as to prevent the disease and better guide clinical diagnosis and treatment.展开更多
The Human Genome Project opened an era of(epi)genomic research,and also provided a platform for the development of new sequencing technologies.During and after the project,several sequencing technologies continue to d...The Human Genome Project opened an era of(epi)genomic research,and also provided a platform for the development of new sequencing technologies.During and after the project,several sequencing technologies continue to dominate nucleic acid sequencing markets.Currently,Illumina(short-read),PacBio(long-read),and Oxford Nanopore(longread)are the most popular sequencing technologies.Unlike 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 molecules.Nanopore 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 bases.In 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 perspective.We also address the problems researchers may encounter applying these approaches in their research topics,and how to resolve them.展开更多
Background:Oxford Nanopore long-read sequencing technology addresses current limitations for DNA methylation detection that are inherent in short-read bisulfite sequencing or methylation microarrays.A number of analyt...Background:Oxford Nanopore long-read sequencing technology addresses current limitations for DNA methylation detection that are inherent in short-read bisulfite sequencing or methylation microarrays.A number of analytical tools,such as Nanopolish,Guppy/Tombo and DeepMod,have been developed to detect DNA methylation on Nanopore data.However,additional improvements can be made in computational efficiency,prediction accuracy,and contextual interpretation on complex genomics regions(such as repetitive regions,low GC density regions).Method:In the current study,we apply Transformer architecture to detect DNA methylation on ionic signals from Oxford Nanopore sequencing data.Transformer is an algorithm that adopts self-attention architecture in the neural networks and has been widely used in natural language processing.Results:Compared to traditional deep-learning method such as convolutional neural network(CNN)and recurrent neural network(RNN),Transformer may have specific advantages in DNA methylation detection,because the self-attention mechanism can assist the relationship detection between bases that are far from each other and pay more attention to important bases that carry characteristic methylation-specific signals within a specific sequence context.Conclusion:We demonstrated the ability of Transformers to detect methylation on ionic signal data.展开更多
[Objectives]This study was conducted to investigate the effects of adding compound probiotics on the growth performance and intestinal flora of Kunming mice.[Methods]Twelve healthy 2-week-old Kunming male mice with bo...[Objectives]This study was conducted to investigate the effects of adding compound probiotics on the growth performance and intestinal flora of Kunming mice.[Methods]Twelve healthy 2-week-old Kunming male mice with body weight of(11.09±0.43)g were selected.They were randomly divided into two treatment groups,namely blank control group(NC)and compound probiotics group(CB+LR+BS),with six mice in each group.The two groups were fed with commercial basal diet,and the compound probiotic experimental group was fed with basal diet supplemented with compound probiotics,in which the contents of Clostridium butyricum spores,Lactobacillus reuteri and Bacillus subtilis spores were 1×1010,1×1011 and 1×1010 CUF/kg,respectively.The body weight,feed intake and water intake of mice were counted every 4 d,and the experimental period was 13 d.On the 13 th day,the cecal contents of the mice were collected for analysis.[Results]There was no significant change in body weight and feed intake when compound probiotics were added to the diet.However,the addition of compound probiotics reduced the abundance of harmful bacteria such as Escherichia coli,urease-negative Helicobacter typhlonius and Salmonella enterica,while increasing the abundance of beneficial bacteria such as Anaerostipes hadrus,and the contents of IgG and IgM increased significantly(P<0.05).[Conclusions]In summary,the addition of compound probiotics could significantly improve the structure of intestinal microbial flora,increase the quantity of beneficial bacteria,reduce the quantity of harmful bacteria,and improve the immune function of mice.展开更多
Anther is a key male reproductive organ that is essential for the plant life cycle,from the sporophyte to the gametophyte generation.To explore the isoform-level transcriptional landscape of developing anthers in maiz...Anther is a key male reproductive organ that is essential for the plant life cycle,from the sporophyte to the gametophyte generation.To explore the isoform-level transcriptional landscape of developing anthers in maize(Zea mays L.),we analyzed Iso-Seq data from anthers collected at 10 developmental stages,together with strand-specific RNA-seq,CAGE-seq,and PAS-seq data.Of the 152,026 high-confidence full-length isoforms identified,68.8%have not been described;these include 22,365 isoforms that originate from previously unannotated loci and 82,167 novel isoforms that originate from annotated protein-coding genes.Using our newly developed strategy to detect dynamic expression patterns of isoforms,we identify 13,899 differentially variable regions(DVRs);surprisingly,1275 genes contain more than two DVRs,revealing highly efficient utilization of limited genic regions.We identify 7876 long non-coding RNAs(lncRNAs)from 4098 loci,most of which were preferentially expressed during cell differentiation and meiosis.We also detected 371 long-range interactions involving intergenic lncRNAs(lincRNAs);interestingly,243 were lincRNA-gene ones,and the interacting genes were highly expressed in anthers,suggesting that many potential lncRNA regulators of key genes are required for anther development.This study provides valuable resources and fundamental information for studying the essential transcripts of key genes during anther development.展开更多
Over the past decade,high-throughput RNA sequencing(RNA-seq)has vastly expanded our understanding of transcriptome dynamics in human physiology and disease.As a powerful tool for investigating systematic changes in RN...Over the past decade,high-throughput RNA sequencing(RNA-seq)has vastly expanded our understanding of transcriptome dynamics in human physiology and disease.As a powerful tool for investigating systematic changes in RNA biology,RNA-seq has facilitated the discovery of novel functional RNA species.Mature RNA transcripts,which transmit genetic information from DNA to proteins,undergo intricate transcriptional and post-transcriptional regulation.This process allows a single gene to produce multiple RNA transcripts,each performing specific functions depending on the physiological or pathological context.Specific RNA transcripts(SRTs)are uniquely expressed in particular tissues or tumors and are closely associated with tissue-specific functions or disease states,particularly cancer.This review explores the generation of SRTs through key mechanisms,such as alternative splicing(AS),transcriptional regulation,polyadenylation(polyA),and the influence of transposable elements(TEs).We also examine their critical roles in normal tissue development and diseases,with an emphasis on their relevance to cancer.Furthermore,the potential applications of SRTs in diagnosing and treating diseases,especially malignancies,are discussed.By serving as diagnostic markers and therapeutic targets,SRTs hold significant promise in the development of personalized medicine and precision therapies.This review aims to provide new insights into the importance of SRTs in advancing the understanding and treatment of human diseases.展开更多
文摘Objective:Autosomal recessive bestrophinopathy(ARB),a retinal degenerative disease,is characterized by central visual loss,yellowish multifocal diffuse subretinal deposits,and a dramatic decrease in the light peak on electrooculogram.The potential pathogenic mechanism involves mutations in the BEST1 gene,which encodes Ca2+-activated Cl−channels in the retinal pigment epithelium(RPE),resulting in degeneration of RPE and photoreceptor.In this study,the complete clinical characteristics of two Chinese ARB families were summarized.Methods:Pacific Biosciences(PacBio)single-molecule real-time(SMRT)sequencing was performed on the probands to screen for disease-causing gene mutations,and Sanger sequencing was applied to validate variants in the patients and their family members.Results:Two novel mutations,c.202T>C(chr11:61722628,p.Y68H)and c.867+97G>A,in the BEST1 gene were identified in the two Chinese ARB families.The novel missense mutation BEST1 c.202T>C(p.Y68H)resulted in the substitution of tyrosine with histidine in the N-terminal region of transmembrane domain 2 of bestrophin-1.Another novel variant,BEST1 c.867+97G>A(chr11:61725867),located in intron 7,might be considered a regulatory variant that changes allele-specific binding affinity based on motifs of important transcriptional regulators.Conclusion:Our findings represent the first use of third-generation sequencing(TGS)to identify novel BEST1 mutations in patients with ARB,indicating that TGS can be a more accurate and efficient tool for identifying mutations in specific genes.The novel variants identified further broaden the mutation spectrum of BEST1 in the Chinese population.
基金Supported by Open Project of Key Laboratory of Hubei Province(No.2023KFZZ026).
文摘AIM:To estimate if nanopore targeted sequencing(NTS)could identify pathogens causing postoperative endophthalmitis and further determine the feasibility of clinical application of NTS.METHODS:A total of 55 patients(55 eyes)with postoperative endophthalmitis were retrospectively included in this study with their medical records.Intraocular fluid samples were examined by NTS and microbial culture.All included patients had undergone examinations including measurement of best corrected visual acuity(BCVA)and intraocular pressure(IOP),slit-lamp biomicroscopy,and indirect ophthalmoscopy;additionally,they underwent B-ultrasound,anterior segment photography,and fundus photography if necessary.RESULTS:Among 55 patients with postoperative endophthalmitis,the age was 65.25±15.04y and there were 30 female(54.54%)patients.Forty-one(74.54%)vitreous humor samples and fourteen(25.45%)aqueous humor samples were sent for both NTS and microbial culture.NTS had a notable higher detection rate than microbial culture in detecting pathogens(90.91%vs 38.18%,χ^(2)=33.409,P<0.001).NTS exhibited high sensitivity of pathogen detection in both microbial culture positive and negative samples(100%and 85.29%,respectively).In 16 of 21(76.19%)patients who showed culture-positivity,their results corresponded with those of NTS.Moreover,in two patients(9.52%),NTS showed a better species resolution than microbial culture;in three patients(14.28%),NTS identified additional pathogens.As for fungus,the positive detection rate of NTS was significantly higher than that of microbial culture(20%vs 3.64%,χ^(2)=7.066,P=0.008).Also,NTS could detect multi-infection by bacteria and fungi than microbial culture(32.73%vs 0,χ^(2)=21.522,P<0.001).NTS could detect bacteria as well as fungi simultaneously within 48h in all patients.Meanwhile,NTS had a shorter detection time than microbial culture(1.13±0.34 vs 2.67±0.55d,Z=-9.218,P<0.001).After the NTS results were obtained,15 patients received additional intravitreal/intracameral anti-infection treatment.At follow-up,there was a statistically significant improvement in the visual acuity relative to the baseline(Z=−5.222,P<0.001).CONCLUSION:NTS can provide rapid identification and highly sensitive detection of pathogens among patients with postoperative endophthalmitis,which can guide anti-infection treatment and improve visual prognosis.
基金supported by the National Key Research and Development Program of China(2022YFC2303401,2022YFC2304100,2016YFD0500301,2021YFC0863300)the Beijing Science and Technology Plan(Z211100002521017)the National Natural Science Foundation of China(82241080)。
文摘The monkeypox virus(MPXV)has triggered a current outbreak globally.Genome sequencing of MPXV and rapid tracing of genetic variants will benefit disease diagnosis and control.It is a significant challenge but necessary to optimize the strategy and application of rapid full-length genome identification and to track variations of MPXV in clinical specimens with low viral loads,as it is one of the DNA viruses with the largest genome and the most AT-biased,and has a significant number of tandem repeats.Here we evaluated the performance of metagenomic and amplicon sequencing techniques,and three sequencing platforms in MPXV genome sequencing based on multiple clinical specimens of five mpox cases in Chinese mainland.We rapidly identified the full-length genome of MPXV with the assembly of accurate tandem repeats in multiple clinical specimens.Amplicon sequencing enables cost-effective and rapid sequencing of clinical specimens to obtain high-quality MPXV genomes.Third-generation sequencing facilitates the assembly of the terminal tandem repeat regions in the monkeypox virus genome and corrects a common misassembly in published sequences.Besides,several intra-host single nucleotide variations were identified in the first imported mpox case.This study offers an evaluation of various strategies aimed at identifying the complete genome of MPXV in clinical specimens.The findings of this study will significantly enhance the surveillance of MPXV.
基金financially supported by the Natural Science Foundation of China(32470055 and U23A20148)the China Postdoctoral Science Foundation(2024M753580)the Agricultural Science and Technology Innovation Program(CAAS-ZDRW202308)。
文摘Over the past decade,nanopore sequencing has experienced significant advancements and changes,transitioning from an initially emerging technology to a significant instrument in the field of genomic sequencing.However,as advancements in next-generation sequencing technology persist,nanopore sequencing also improves.This paper reviews the developments,applications,and outlook on nanopore sequencing technology.Currently,nanopore sequencing supports both DNA and RNA sequencing,making it widely applicable in areas such as telomere-to-telomere(T2T)genome assembly,direct RNA sequencing(DRS),and metagenomics.The openness and versatility of nanopore sequencing have established it as a preferred option for an increasing number of research teams,signaling a transformative influence on life science research.As the nanopore sequencing technology advances,it provides a faster,more costeffective approach with extended read lengths,demonstrating the significant potential for complex genome assembly,pathogen detection,environmental monitoring,and human disease research,offering a fresh perspective in sequencing technologies.
基金Supported by Research and Development Funding for Medical and Health Institutions,No.2021YL007.
文摘BACKGROUND Infectious diseases are still one of the greatest threats to human health,and the etiology of 20%of cases of clinical fever is unknown;therefore,rapid identification of pathogens is highly important.Traditional culture methods are only able to detect a limited number of pathogens and are time-consuming;serologic detection has window periods,false-positive and false-negative problems;and nucleic acid molecular detection methods can detect several known pathogens only once.Three-generation nanopore sequencing technology provides new options for identifying pathogens.CASE SUMMARY Case 1:The patient was admitted to the hospital with abdominal pain for three days and cessation of defecation for five days,accompanied by cough and sputum.Nanopore sequencing of the drainage fluid revealed the presence of orallike bacteria,leading to a clinical diagnosis of bronchopleural fistula.Cefoperazone sodium sulbactam treatment was effective.Case 2:The patient was admitted to the hospital with fever and headache,and CT revealed lung inflammation.Antibiotic treatment for Streptococcus pneumoniae,identified through nanopore sequencing of cerebrospinal fluid,was effective.Case 3:The patient was admitted to our hospital with intermittent fever and an enlarged neck mass that had persisted for more than six months.Despite antibacterial treatment,her symptoms worsened.The nanopore sequencing results indicate that voriconazole treatment is effective for Aspergillus brookii.The patient was diagnosed with mixed cell type classical Hodgkin's lymphoma with infection.CONCLUSION Three-generation nanopore sequencing technology allows for rapid and accurate detection of pathogens in human infectious diseases.
基金supported by the National Natural Science Foundation of China(32370686)the National Key Research and Development Program of China(2021YFC2500202)+2 种基金the 111 Project(B13016)Shanghai Municipal Science and Technology(2017SHZDZX01)supported by the Human Phenome Data Center at Fudan University.
文摘Human leukocyte antigen(HLA)genes in the major histocompatibility complex(MHC)region are crucial for immunity and are associated with numerous diseases and phenotypes.The MHC region’s complexity and high genetic diversity make it challenging to analyze using short-read sequencing(SRS)technology.We sequence the MHC region of 100 Han Chinese individuals using both long-read sequencing(LRS)and SRS platforms at approximately 30X coverage to study genetic alterations and their potential functional impacts.LRS provides significantly greater coverage of the MHC region and eight classical HLA genes,particularly at the HLA-DRB1 locus,compared with SRS.We detect 78,249 single nucleotide polymorphisms(SNPs)using LRS,with 26.0%undetectable by SRS.Based on SNP and inferred HLA allele types,we construct an LRS-based MHC reference panel for the Han Chinese,containing approximately 2.6 times more genetic variants than the SRS-based Han-MHC reference panel.A phenome-wide association study assessing 26,024 phenotypes across 15 categories identifies significant associations for 7,879 independent variants(including 809 LRS-specific SNPs)with 409 phenotypes in nine categories.This analysis reveals 24 unreported HLA allele associations in the bioelectric and cellular categories.The conditional analysis identifies 530 independent signals across the 409 phenotypes,including 28 previously unreported signals of eight classical HLA genes associated with 33 phenotypes.Of the top-associated SNPs,191 are detected by LRS only.Fine-mapping identifies 126 independent candidate causal SNPs for three immune-related cellular phenotypes,with 17 detected exclusively by LRS.Our study reveals previously unreported variants and their functional impacts in the MHC region,enhancing our understanding of genetic diversity and its potential biological implications in the Han Chinese population.
基金supported by the National Natural Science Foundation of China, No.61932008Natural Science Foundation of Shanghai, No.21ZR1403200 (both to JC)。
文摘Neurodegenerative diseases cause great medical and economic burdens for both patients and society;however, the complex molecular mechanisms thereof are not yet well understood. With the development of high-coverage sequencing technology, researchers have started to notice that genomic repeat regions, previously neglected in search of disease culprits, are active contributors to multiple neurodegenerative diseases. In this review, we describe the association between repeat element variants and multiple degenerative diseases through genome-wide association studies and targeted sequencing. We discuss the identification of disease-relevant repeat element variants, further powered by the advancement of long-read sequencing technologies and their related tools, and summarize recent findings in the molecular mechanisms of repeat element variants in brain degeneration, such as those causing transcriptional silencing or RNA-mediated gain of toxic function. Furthermore, we describe how in silico predictions using innovative computational models, such as deep learning language models, could enhance and accelerate our understanding of the functional impact of repeat element variants. Finally, we discuss future directions to advance current findings for a better understanding of neurodegenerative diseases and the clinical applications of genomic repeat elements.
基金supported by Guangdong S&T Program(2022B1111030002)National Natural Science Foundation of China(82072311)+1 种基金National Key R&D Program of China(2020YFC120104,and 2016YFC1200500)National Parasitic Resources Center,and the Ministry of Science and Technology fund(NPRC-2019-194-30).
文摘Background The RNA virosphere’s extensive diversity and its role in emerging infectious diseases underscore the importance of non-targeted sequencing for identifying unknown or rare pathogens,including co-infections.However,enriching low-abundance viral sequences in RNA metaviromics,particularly in the preparation of cDNA libraries and their compatibility with next-generation sequencing(NGS)and third-generation sequencing(TGS),remains challenging.Therefore,our objective is to develop and systematically assess a practical RNA metavirome methodology specifically tailored for the enrichment of low-abundance viral sequences within samples.Methods We developed the SMART-RNA-Metavirome platform,integrating SMART-9n library preparation with NGS and TGS technologies.Total RNA was extracted from two field-collected wild Aedes albopictus pools,along with one laboratory-infected Ae.albopictus pool harboring dengue virus(DENV).This RNA was subjected to reverse transcription using both this optimized protocol and random primer-based methods,followed by high-throughput sequencing on Illumina,Oxford Nanopore,and QitanTech Nanopore technologies.Welch’s t-test was employed for comparative analysis of the subsequent RNA metavirome data,specifically to evaluate differences in viral species composition and abundance of viral reads between experimental groups.Furthermore,the effectiveness of this platform was systematically validated via RT-qPCR and SMART-RNA-Metavirome-based Oxford Nanopore sequencing across multiple sample types,including mosquito specimens from DENV-infected Ae.albopictus,serum samples from dengue patients and viral isolates of Japanese encephalitis virus(JEV)and Zika virus(ZIKV).Results The SMART-RNA-Metavirome platform has been systematically validated to excel in enriching the composition and diversity of the RNA virome(P=0.04),providing sufficient coverage for the complete reconstruction of viral genomes.When employed in the detection of DENV-infected Ae.albopictus,clinical serum samples,and viral isolates of JEV and ZIKV,this technique exhibits a robust correlation with RT-qPCR(r2>0.95).Notably,it demonstrates exceptional sensitivity,ensuring sufficient coverage even in samples of DENV-infected Ae.albopictus with a Ct-value of 35.3,attaining an impressive 99.88%genome coverage.Furthermore,this platform possesses the capability to identify virus species and determine their serotypes.Conclusions In our study,the SMART-RNA-Metavirome platform outperforms traditional methods,enriching RNA virome composition and diversity,enabling practical compatibility with both NGS and TGS technologies.It demonstrates significant proficiency in detecting both known and unknown arboviruses,even in low-titer samples such as those from wild mosquitoes and clinical sera.This platform facilitates comprehensive monitoring,risk assessment,and early warning of RNA virus transmissions,enhancing our understanding of RNA virome diversity and ecological patterns.
基金supported by the National Key R&D Program of China(Grant Nos.2022YFC3400300 and 2023YFF0613300)the National Natural Science Foundation of China(Grant Nos.32200510,32400509,32125009,32070663,32371516,and 32400520).
文摘Recent developments in PacBio high-fidelity(HiFi)sequencing technologies have transformed genomic research,with circular consensus se-quencing now achieving 99.9%accuracy for long(up to 25 kb)single-molecule reads.This method circumvents biases intrinsic to amplification-based approaches,enabling thorough analysis of complex genomic regions including tandem repeats,segmental duplications,ribosomal DNA(rDNA)arrays,and centromeresl as well as direct detection of base modifications,furnishing both sequence and epigenetic data concurrently.This has streamlined a number of tasks including genome assembly,variant detection,and full-length transcript analysis.This review provides a comprehensive overview of the applications and challenges of HiFi sequencing across various fields,including genomics,transcriptomics,and epigenetics.By delineating the evolving landscape of HiFi sequencing in multi-omics research,we highlight its potential to deepen our under-standingofgeneticmechanisms and to advance precision medicine.
基金supported by the institutional fund of the Department of Internal Medicine, University of Iowa, USA
文摘Single-molecule, real-time sequencing developed by Pacific BioSciences offers longer read lengths than the second-generation sequencing （SGS） technologies, making it well-suited for unsolved problems in genome, transcriptome, and epigenetics research. The highly-contiguous de novo assemblies using PacBio sequencing can close gaps in current reference assemblies and characterize structural variation （SV） in personal genomes. With longer reads, we can sequence through extended repetitive regions and detect mutations, many of which are associated with dis- eases. Moreover, PacBio transcriptome sequencing is advantageous for the identification of gene isoforms and facilitates reliable discoveries of novel genes and novel isoforms of annotated genes, due to its ability to sequence full-length transcripts or fragments with significant lengths. Addition- ally, PacBio＇s sequencing technique provides information that is useful for the direct detection of base modifications, such as methylation. In addition to using PacBio sequencing alone, many hybrid sequencing strategies have been developed to make use of more accurate short reads in conjunction with PacBio long reads. In general, hybrid sequencing strategies are more affordable and scalable especially for small-size laboratories than using PacBio Sequencing alone. The advent of PacBio sequencing has made available much information that could not be obtained via SGS alone.
基金supported by the Wellcome Trust,the United Kingdom
文摘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.
基金This work was supported by the National Key Research and Development Program of China(Grant No.2018YFA0107601).
文摘Single-cell omics sequencingwas first achieved for the transcriptome in 2009,whichwas followed by fast development of technologies for profiling the genome,DNA methylome,3D genome architecture,chromatin accessibility,histone modifications,etc.,in an individual cell.In this review we mainly focus on the recent progress in four topics in the single-cell omics field:single-cell epigenome sequencing,single-cell genome sequencing for lineage tracing,spatially resolved single-cell transcriptomics and third-generation sequencing platform-based single-cell omics sequencing.We also discuss the potential applications and future directions of these single-cell omics sequencing technologies for different biomedical systems,especially for the human stem cell field.
文摘DNA barcodes,short and unique DNA sequences,play a crucial role in sample identification when processing many samples simultaneously,which helps reduce experimental costs.Nevertheless,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 run.Here,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 demultiplexing.We 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 NP-complete.For 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 accuracy.Specifically,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 rates.The 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 tool.When 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.
文摘High-quality DNA extraction is a crucial step in metagenomic studies.Bias by different isolation kits impairs the comparison across datasets.A trending topic is,however,the analysis of multiple metagenomes from the same patients to draw a holistic picture of microbiota associated with diseases.We thus collected bile,stool,saliva,plaque,sputum,and conjunctival swab samples and performed DNA extraction with three commercial kits.For each combination of the specimen type and DNA extraction kit,20-gigabase(Gb)metagenomic data were generated using short-read sequencing.While profiles of the specimen types showed close proximity to each other,we observed notable differences in the alpha diversity and composition of the microbiota depending on the DNA extraction kits.No kit outperformed all selected kits on every specimen.We reached consistently good results using the Qiagen QiAamp DNA Microbiome Kit.Depending on the specimen,our data indicate that over 10 Gb of sequencing data are required to achieve sufficient resolution,but DNA-based identification is superior to identification by mass spectrometry.Finally,longread nanopore sequencing confirmed the results(correlation coefficient>0.98).Our results thus suggest using a strategy with only one kit for studies aiming for a direct comparison of multiple microbiotas from the same patients.
文摘Endophthalmitis is a serious ophthalmic disease characterized by changes in the eye's posterior segment,such as hypopyon and intraocular inflammation,vitritis being a hallmark.Infection-caused endophthalmitis can lead to irreversible vision loss,accompanied by eye pain or eye distention,and in the most severe cases the removal of the eyeball.Microorganisms such as bacteria,fungi,viruses,and parasites typically account for the disease and the entry pathways of the microbial can be divided into either endogenous or exogenous approaches,according to the origin of the etiological agents.Exogenous endophthalmitis can be derived from various occasions(such as postoperative complications or trauma)while endogenous endophthalmitis results from the bloodstream which carries pathogens to the eye.This review aims to summarize the application of new technology in pathogen identification of endophthalmitis so as to prevent the disease and better guide clinical diagnosis and treatment.
基金supported by the Key-Areas Research and Development Program of Guangdong Province(2020B020220004)the Youth Innovation Promotion Association,Chinese Academy of Sciences(2017399)+2 种基金the Science and Technology Program of Guangzhou(202002030097)the Hong Kong Research Grants Council Area of Excellence Scheme(AoE/M-403/16),the ECS(27204518)TRS of the HKSAR government(T21-705/20-N).
文摘The Human Genome Project opened an era of(epi)genomic research,and also provided a platform for the development of new sequencing technologies.During and after the project,several sequencing technologies continue to dominate nucleic acid sequencing markets.Currently,Illumina(short-read),PacBio(long-read),and Oxford Nanopore(longread)are the most popular sequencing technologies.Unlike 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 molecules.Nanopore 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 bases.In 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 perspective.We also address the problems researchers may encounter applying these approaches in their research topics,and how to resolve them.
文摘Background:Oxford Nanopore long-read sequencing technology addresses current limitations for DNA methylation detection that are inherent in short-read bisulfite sequencing or methylation microarrays.A number of analytical tools,such as Nanopolish,Guppy/Tombo and DeepMod,have been developed to detect DNA methylation on Nanopore data.However,additional improvements can be made in computational efficiency,prediction accuracy,and contextual interpretation on complex genomics regions(such as repetitive regions,low GC density regions).Method:In the current study,we apply Transformer architecture to detect DNA methylation on ionic signals from Oxford Nanopore sequencing data.Transformer is an algorithm that adopts self-attention architecture in the neural networks and has been widely used in natural language processing.Results:Compared to traditional deep-learning method such as convolutional neural network(CNN)and recurrent neural network(RNN),Transformer may have specific advantages in DNA methylation detection,because the self-attention mechanism can assist the relationship detection between bases that are far from each other and pay more attention to important bases that carry characteristic methylation-specific signals within a specific sequence context.Conclusion:We demonstrated the ability of Transformers to detect methylation on ionic signal data.
基金Supported by China National University Student Innovation and Entrepreneurship Development Program(S202310553010)2023 Undergraduate Innovation and Entrepreneurship Training Program of Hunan University of Humanities,Science and Technology(14).
文摘[Objectives]This study was conducted to investigate the effects of adding compound probiotics on the growth performance and intestinal flora of Kunming mice.[Methods]Twelve healthy 2-week-old Kunming male mice with body weight of(11.09±0.43)g were selected.They were randomly divided into two treatment groups,namely blank control group(NC)and compound probiotics group(CB+LR+BS),with six mice in each group.The two groups were fed with commercial basal diet,and the compound probiotic experimental group was fed with basal diet supplemented with compound probiotics,in which the contents of Clostridium butyricum spores,Lactobacillus reuteri and Bacillus subtilis spores were 1×1010,1×1011 and 1×1010 CUF/kg,respectively.The body weight,feed intake and water intake of mice were counted every 4 d,and the experimental period was 13 d.On the 13 th day,the cecal contents of the mice were collected for analysis.[Results]There was no significant change in body weight and feed intake when compound probiotics were added to the diet.However,the addition of compound probiotics reduced the abundance of harmful bacteria such as Escherichia coli,urease-negative Helicobacter typhlonius and Salmonella enterica,while increasing the abundance of beneficial bacteria such as Anaerostipes hadrus,and the contents of IgG and IgM increased significantly(P<0.05).[Conclusions]In summary,the addition of compound probiotics could significantly improve the structure of intestinal microbial flora,increase the quantity of beneficial bacteria,reduce the quantity of harmful bacteria,and improve the immune function of mice.
基金supported by the Excellent Young Scientists Fund(Category B)(32422063)the National Key Research and Development Program of China(2022YFF1003500)the Zhengzhou University Qiushi Postdoctoral Research Funding Program.For open access,the authors have applied for a Creative Commons Attribution(CC BY)license for any Author Accepted Manuscript version arising from this submission.
文摘Anther is a key male reproductive organ that is essential for the plant life cycle,from the sporophyte to the gametophyte generation.To explore the isoform-level transcriptional landscape of developing anthers in maize(Zea mays L.),we analyzed Iso-Seq data from anthers collected at 10 developmental stages,together with strand-specific RNA-seq,CAGE-seq,and PAS-seq data.Of the 152,026 high-confidence full-length isoforms identified,68.8%have not been described;these include 22,365 isoforms that originate from previously unannotated loci and 82,167 novel isoforms that originate from annotated protein-coding genes.Using our newly developed strategy to detect dynamic expression patterns of isoforms,we identify 13,899 differentially variable regions(DVRs);surprisingly,1275 genes contain more than two DVRs,revealing highly efficient utilization of limited genic regions.We identify 7876 long non-coding RNAs(lncRNAs)from 4098 loci,most of which were preferentially expressed during cell differentiation and meiosis.We also detected 371 long-range interactions involving intergenic lncRNAs(lincRNAs);interestingly,243 were lincRNA-gene ones,and the interacting genes were highly expressed in anthers,suggesting that many potential lncRNA regulators of key genes are required for anther development.This study provides valuable resources and fundamental information for studying the essential transcripts of key genes during anther development.
基金supported by grants from the National Key Specialized Program of China(No.2024YFC3405903)the National Natural Science Foundation of China(Nos.82472641 and 8217293).
文摘Over the past decade,high-throughput RNA sequencing(RNA-seq)has vastly expanded our understanding of transcriptome dynamics in human physiology and disease.As a powerful tool for investigating systematic changes in RNA biology,RNA-seq has facilitated the discovery of novel functional RNA species.Mature RNA transcripts,which transmit genetic information from DNA to proteins,undergo intricate transcriptional and post-transcriptional regulation.This process allows a single gene to produce multiple RNA transcripts,each performing specific functions depending on the physiological or pathological context.Specific RNA transcripts(SRTs)are uniquely expressed in particular tissues or tumors and are closely associated with tissue-specific functions or disease states,particularly cancer.This review explores the generation of SRTs through key mechanisms,such as alternative splicing(AS),transcriptional regulation,polyadenylation(polyA),and the influence of transposable elements(TEs).We also examine their critical roles in normal tissue development and diseases,with an emphasis on their relevance to cancer.Furthermore,the potential applications of SRTs in diagnosing and treating diseases,especially malignancies,are discussed.By serving as diagnostic markers and therapeutic targets,SRTs hold significant promise in the development of personalized medicine and precision therapies.This review aims to provide new insights into the importance of SRTs in advancing the understanding and treatment of human diseases.
