In rice fields,rice plants usually grow alongside wild weeds and are attacked by various invertebrate species.Viruses are abundant in plants and invertebrates,playing crucial ecological roles in controlling microbial ...In rice fields,rice plants usually grow alongside wild weeds and are attacked by various invertebrate species.Viruses are abundant in plants and invertebrates,playing crucial ecological roles in controlling microbial abundance and maintaining community structures.To date,only 16 rice viruses have been documented in rice-growing regions.These viruses pose serious threats to rice production and have traditionally been identified only from rice plants and insect vectors by isolation techniques.Advances in next-generation sequencing(NGS)have made it feasible to discover viruses on a global scale.Recently,numerous viruses have been identified in plants and invertebrates using NGS technologies.In this review,we discuss viral studies in rice plants,invertebrate species,and weeds in rice fields.Many novel viruses have been discovered in rice ecosystems through NGS technologies,with some also detected using metatranscriptomic and small RNA sequencing.These analyses greatly expand our understanding of viruses in rice fields and provide valuable insights for developing efficient strategies to manage insect pests and virus-mediated rice diseases.展开更多
This study investigates the diversity of gut microbiota in Metaphire peguana,an earthworm species commonly found in agricultural areas of Thailand.Earthworms play a critical role in soil ecosystems by supporting nutri...This study investigates the diversity of gut microbiota in Metaphire peguana,an earthworm species commonly found in agricultural areas of Thailand.Earthworms play a critical role in soil ecosystems by supporting nutrient cycling and breaking down organic matter.Understanding the microbial diversity in their gut is essential for exploring their ecological contributions.Using Next Generation Sequencing(NGS),we analyzed the mycobiome in the gut of M.peguana.Our findings revealed a high diversity of fungal species,primarily belonging to two major phyla:Ascomycota and Basidiomycota.Ascomycota was the most abundant phylum,comprising 40.1% of the total fungal species identified.A total of 33 distinct fungal species were identified,which underscores the richness of microbial life within the earthworm gut.This study successfully created the first genetic database of the microbial community in M.peguana,providing a foundation for future research in agricultural applications.The microbial species identified,particularly siderophoreproducing fungi,could have significant implications for improving soil fertility and promoting sustainable agricultural practices.The use of NGS technology has enabled comprehensive profiling of microbial communities,allowing for precise identification of fungi that may play essential roles in soil health.Furthermore,the study paves the way for future studies on the potential applications of earthworm gut microbiomes in biotechnology,especially in enhancing soil nutrient availability and plant growth.The findings of this research contribute to the broader understanding of the ecological roles of earthworms and their microbiomes in soil ecosystems.展开更多
Objective and Background Early and accurate diagnosis of spinal infections,including spinal tuberculosis,is pivotal for effective treatment but remains challenging.This study aims to assess the diagnostic yield of met...Objective and Background Early and accurate diagnosis of spinal infections,including spinal tuberculosis,is pivotal for effective treatment but remains challenging.This study aims to assess the diagnostic yield of metagenomic next-generation sequencing(mNGS)compared with that of conventional microbiological tests(CMTs)in identifying pathogens associated with spinal pathologies,with a special focus on infections leading to surgical interventions.Methods We enrolled 85 patients who underwent spinal surgery,comprising 63 patients with clinically diagnosed spinal infections,including patients with spinal tuberculosis,and 22 patients with noninfectious spinal conditions.The procedures involved irrigation and debridement for persistent wound drainage,with subsequent DNA extraction from plasma and joint fluid for mNGS and CMT analysis.Results Significantly increased C-reactive protein(CRP)levels were observed in patients with infections.The mNGS approach showed greater diagnostic sensitivity(92.06%)for detecting pathogens,including Mycobacterium tuberculosis,than did CMTs(36.51%).Despite its low specificity,mNGS had considerable negative predictive value(70.59%),underscoring its utility in ruling out infections.Conclusions The mNGS offers superior sensitivity over CMTs in the diagnosis of a variety of spinal infections,notably spinal tuberculosis.This study highlights the potential of mNGS in enhancing the diagnosis of complex spinal infections,thereby informing targeted treatment strategies.展开更多
BACKGROUND Leuconostoc garlicum is commonly found in fermented foods and very few infected patients have been reported,who typically present symptoms such as fever and fatigue.Conventional clinical examinations often ...BACKGROUND Leuconostoc garlicum is commonly found in fermented foods and very few infected patients have been reported,who typically present symptoms such as fever and fatigue.Conventional clinical examinations often struggle to identify this bacterium,and routine anti-infective treatments are generally ineffective.Both diagnostic challenges and therapeutic limitations pose significant difficulties for clinicians.CASE SUMMARY We report a patient ultimately diagnosed with Leuconostoc garlicum infection.The primary manifestations included persistent fever,cough and fatigue.These symptoms lasted for 2 months.He received anti-infective treatment at a community hospital,but this was ineffective.After inquiring about the patient's medical history and conducting a physical examination,the patient underwent laboratory tests.Complete blood count tests revealed that the patient had a high proportion of neutrophils,C-reactive protein level was 235.9 mg/L,erythrocyte sedimentation rate was 67 mm/h,respiratory pathogen testing was negative,and he was then thought to have an infectious disease.However,conventional anti-infective treatments were ineffective.After excluding infectious neurological diseases,urologic diseases and digestive problems,we ultimately focused our attention on the lungs.A lung computed tomography scan indicated pulmonary inflammation.Bronchoalveolar lavage fluid for next-generation sequencing suggested lung infection with Leuconostoc garlicum.The patient's symptoms gradually improved following treatment with piperacillin tazobactam and linezolid.During the follow-up period,the patient's temperature remained normal.CONCLUSION For patients with suspected bacterial infection and experiencing fever,conventional anti-infective treatment can be ineffective in controlling their symptoms,and an infection due to rare bacteria or drug-resistant bacteria should be considered.Next-generation sequencing enables rapid and precise identification of infection-related pathogens in febrile patients.展开更多
The improvement of soybean seed carotenoid contents is very important due to the beneficial role of carotenoids in human health and nutrition. However, the genetic architecture underlying soybean carotenoid biosynthes...The improvement of soybean seed carotenoid contents is very important due to the beneficial role of carotenoids in human health and nutrition. However, the genetic architecture underlying soybean carotenoid biosynthesis remains largely unknown. In the present study, we employed next generation sequencing-based bulked-segregant analysis to identify new genomic regions governing seed carotenoids in 1,551 natural soybean accessions. The genomic DNA samples of individual plants with extreme phenotypes were pooled to form two bulks with high(50 accessions) and low(50 accessions) carotenoid contents for Illumina sequencing. A total of 125.09 Gb of clean bases and 89.82% of Q30 were obtained, and the average alignment efficiency was 99.45% with an average coverage depth of 62.20× and 99.75% genome coverage. Based on the G prime statistic algorithm(G') method analysis, 16 candidate genomic loci with a total length 20.41 Mb were found to be related to the trait. Of these loci, the most significant regions displaying the highest elevated G' values were found in chromosome 06 at a position of 18.53–22.67 Mb, and chromosome 19 at genomic region intervals of 8.36–10.94, 12.06–13.79 and 18.45–20.26 Mb. These regions were then used to identify the key candidate genes. In these regions, 250 predicted genes were found and analyzed to obtain 90 significantly enriched(P<0.05) Gene Ontology(GO) terms. Based on ANNOVAR analysis, 50 genes with non-synonymous and stopgained mutations were preferentially selected as potential candidate genes. Of those 50 genes, following their gene annotation functions and high significant haplotype variations in various environments,five genes were identified as the most promising candidate genes regulating soybean seed carotenoid accumulation, and they should be investigated in further functional validation studies. Collectively, understanding the genetic basis of carotenoid pigments and identifying genes underpinning carotenoid accumulation via a bulked-segregant analysis-based sequencing(BSA-seq) approach provide new insights for exploring future molecular breeding efforts to produce soybean cultivars with high carotenoid content.展开更多
In this study,an amine-reactive poly(pentafluorophenyl acrylate)(PPFPA)platform was developed for advanced surface engineering of next-generation sequencing(NGS)chips.Through post-polymerization modification,PPFPA was...In this study,an amine-reactive poly(pentafluorophenyl acrylate)(PPFPA)platform was developed for advanced surface engineering of next-generation sequencing(NGS)chips.Through post-polymerization modification,PPFPA was functionalized with dual moieties:azide groups for covalent immobilization of DBCO-modified DNA primers via click chemistry and tunable hydrophilic side chains to optimize biocompatibility and surface properties.Systematic screening revealed that hydrophobic azide carriers combined with neutral hydroxyl groups maximized the DNA immobilization efficacy,approaching the performance of commercial polyacrylamide-based polymers.The negatively charged carboxyl groups severely impede DNA primer attachment.Higher molecular weight derivatives further enhance the efficacy of DNA immobilization.In NGS validation,optimized surface modification polymers achieved robust surface density of clustered DNA and high sequencing accuracy,surpassing quality benchmarks and comparable to those of conventional analogs.This platform demonstrates significant potential for tailoring high-sensitivity surfaces for genomic applications,advancing clinical diagnostics,and personalized medicine.展开更多
Global brain ischemia and neurological deficit are consequences of cardiac arrest that lead to high mortality.Despite advancements in resuscitation science,our limited understanding of the cellular and molecular mecha...Global brain ischemia and neurological deficit are consequences of cardiac arrest that lead to high mortality.Despite advancements in resuscitation science,our limited understanding of the cellular and molecular mechanisms underlying post-cardiac arrest brain injury have hindered the development of effective neuroprotective strategies.Previous studies primarily focused on neuronal death,potentially overlooking the contributions of non-neuronal cells and intercellular communication to the pathophysiology of cardiac arrest-induced brain injury.To address these gaps,we hypothesized that single-cell transcriptomic analysis could uncover previously unidentified cellular subpopulations,altered cell communication networks,and novel molecular mechanisms involved in post-cardiac arrest brain injury.In this study,we performed a single-cell transcriptomic analysis of the hippocampus from pigs with ventricular fibrillation-induced cardiac arrest at 6 and 24 hours following the return of spontaneous circulation,and from sham control pigs.Sequencing results revealed changes in the proportions of different cell types,suggesting post-arrest disruption in the blood-brain barrier and infiltration of neutrophils.These results were validated through western blotting,quantitative reverse transcription-polymerase chain reaction,and immunofluorescence staining.We also identified and validated a unique subcluster of activated microglia with high expression of S100A8,which increased over time following cardiac arrest.This subcluster simultaneously exhibited significant M1/M2 polarization and expressed key functional genes related to chemokines and interleukins.Additionally,we revealed the post-cardiac arrest dysfunction of oligodendrocytes and the differentiation of oligodendrocyte precursor cells into oligodendrocytes.Cell communication analysis identified enhanced post-cardiac arrest communication between neutrophils and microglia that was mediated by neutrophil-derived resistin,driving pro-inflammatory microglial polarization.Our findings provide a comprehensive single-cell map of the post-cardiac arrest hippocampus,offering potential novel targets for neuroprotection and repair following cardiac arrest.展开更多
Cystic echinococcosis (CE) is a prevalent zoonotic disease caused by Echinococcus granulosus, with a cosmopolitan distribution. The parasite is transmitted cyclically between canines and numerous intermediate herbivor...Cystic echinococcosis (CE) is a prevalent zoonotic disease caused by Echinococcus granulosus, with a cosmopolitan distribution. The parasite is transmitted cyclically between canines and numerous intermediate herbivorous livestock animals. Also, other Taeniid tapeworms could infect domestic dogs and they pose significant veterinary and public health concerns worldwide. This study aimed to develop a sensitive molecular method for detecting Echinococcus spp. DNA in dog fecal samples using next-generation sequencing (NGS). A set of PCR primers targeting conserved regions of Taeniid tapeworms’ 18s rRNA genes was designed and tested for amplifying genomic DNA from various tapeworm species. The PCR system demonstrated high sensitivity, amplifying DNA from all tested tapeworm species, with differences observed in amplified band sizes. The primers were adapted for NGS analysis by adding forward and reverse adapters, enabling the sequencing of amplified DNA fragments. Application of the developed PCR system to dog fecal samples collected from Yatta town, Palestine, revealed the presence of E. granulosus DNA in five out of 50 samples. NGS analysis confirmed the specificity of the amplified DNA fragments, showing 98% - 99% similarity with the 18s rDNA gene of E. granulosus. This study demonstrates the utility of NGS-based molecular methods for accurate and sensitive detection of Echinococcus spp. in dog fecal samples, providing valuable insights for epidemiological surveillance and control programs of echinococcosis in endemic regions.展开更多
This article reviews basic concepts, general applications, and the potential impact of next-generation sequencing （NGS） technologies on genomics, with particular reference to currently available and possible future ...This article reviews basic concepts, general applications, and the potential impact of next-generation sequencing （NGS） technologies on genomics, with particular reference to currently available and possible future platforms and bioinformatics. NGS technologies have demon- strated the capacity to sequence DNA at unprecedented speed, thereby enabling previously unimaginable scientific achievements and novel biological applications. But, the massive data produced by NGS also presents a significant challenge for data storage, analyses, and management solutions. Advanced bioinformatic tools are essential for the successful application of NGS technology. As evidenced throughout this review, NGS technologies will have a striking impact on genomic research and the entire biological field. With its ability to tackle the unsolved challenges unconquered by previous genomic technologies, NGS is likely to unravel the complexity of the human genome in terms of genetic variations, some of which may be confined to susceptible loci for some common human conditions. The impact of NGS technologies on genomics will be far reaching and likely change the field for years to come.展开更多
BACKGROUND: The study aims to investigate the performance of a metagenomic next-generationsequencing (NGS)-based diagnostic technique for the identifi cation of potential bacterial and viral infectionsand eff ects of ...BACKGROUND: The study aims to investigate the performance of a metagenomic next-generationsequencing (NGS)-based diagnostic technique for the identifi cation of potential bacterial and viral infectionsand eff ects of concomitant viral infection on the survival rate of intensive care unit (ICU) sepsis patients.METHODS: A total of 74 ICU patients with sepsis who were admitted to our institution from February1, 2018 to June 30, 2019 were enrolled. Separate blood samples were collected from patients for bloodcultures and metagenomic NGS when the patients’ body temperature was higher than 38 °C. Patients’demographic data, including gender, age, ICU duration, ICU scores, and laboratory results, were recorded.The correlations between pathogen types and sepsis severity and survival rate were evaluated.RESULTS: NGS produced higher positive results (105 of 118;88.98%) than blood cultures(18 of 118;15.25%) over the whole study period. Concomitant viral infection correlated closelywith sepsis severity and had the negative effect on the survival of patients with sepsis. However,correlation analysis indicated that the bacterial variety did not correlate with the severity of sepsis.CONCLUSIONS: Concurrent viral load correlates closely with the severity of sepsis and thesurvival rate of the ICU sepsis patients. This suggests that prophylactic administration of antiviraldrugs combined with antibiotics may be benefi cial to ICU sepsis patients.展开更多
Reliable and accurate pre-implantation genetic diagnosis （PGD） of patient＇s embryos by next-generation sequencing （NGS） is dependent on efficient whole genome amplification （WGA） of a representative biopsy samp...Reliable and accurate pre-implantation genetic diagnosis （PGD） of patient＇s embryos by next-generation sequencing （NGS） is dependent on efficient whole genome amplification （WGA） of a representative biopsy sample. However, the performance of the current state of the art WGA methods has not been evaluated for sequencing. Using low template DNA （15 pg） and single cells, we showed that the two PCR-based WGA systems SurePlex and MALBAC are superior to the REPLI-g WGA multiple displacement amplification （MDA） system in terms of consistent and reproducible genome coverage and sequence bias across the 24 chromosomes, allowing better normalization of test to reference sequencing data. When copy number variation sequencing （CNV-Seq） was applied to single cell WGA products derived by either SurePlex or MALBAC amplification, we showed that known disease CNVs in the range of 3-15 Mb could be reliably and accurately detected at the correct genomic positions. These findings indicate that our CNV-Seq pipeline incorporating either SurePlex or MALBAC as the key initial WGA step is a powerful methodology for clinical PGD to identify euploid embryos in a patient＇s cohort for uterine transplantation,展开更多
Gastric cancer(GC)is one of the most common malignancies and remains the second leading cause of cancer-related death worldwide.There is an increasing understanding of the roles that genetic and epigenetic alterations...Gastric cancer(GC)is one of the most common malignancies and remains the second leading cause of cancer-related death worldwide.There is an increasing understanding of the roles that genetic and epigenetic alterations play in GCs.Recent studies using nextgeneration sequencing(NGS)have revealed a number of potential cancer-driving genes in GC.Whole-exome sequencing of GC has identified recurrent somatic mutations in the chromatin remodeling gene ARID1A and alterations in the cell adhesion gene FAT4,a member of the cadherin gene family.Mutations in chromatin remodeling genes(ARID1A,MLL3 and MLL)have been found in 47%of GCs.Whole-genome sequencing and whole-transcriptome sequencing analyses have also discovered novel alterations in GC.Recent studies of cancer epigenetics have revealed widespread alterations in genes involved in the epigenetic machinery,such as DNA methylation,histone modifications,nucleosome positioning,noncoding RNAs and microRNAs.Recent advances in molecular research on GC have resulted in the introduction of new diagnostic and therapeutic strategies into clinical settings.The antihuman epidermal growth receptor 2(HER2)antibody trastuzumab has led to an era of personalized therapy in GC.In addition,ramucirumab,a monoclonal antibody targeting vascular endothelial growth factor receptor(VEGFR)-2,is the first biological treatment that showed survival benefits as a single-agent therapy in patients with advanced GC who progressed after firstline chemotherapy.Using NGS to systematically identify gene alterations in GC is a promising approach with remarkable potential for investigating the pathogenesis of GC and identifying novel therapeutic targets,as well as useful biomarkers.In this review,we will summarize the recent advances in the understanding of the molecular pathogenesis of GC,focusing on the potential use of these genetic and epigenetic alterations as diagnostic biomarkers and novel therapeutic targets.展开更多
Border-associated macrophages are located at the interface between the brain and the periphery, including the perivascular spaces, choroid plexus, and meninges. Until recently, the functions of border-associated macro...Border-associated macrophages are located at the interface between the brain and the periphery, including the perivascular spaces, choroid plexus, and meninges. Until recently, the functions of border-associated macrophages have been poorly understood and largely overlooked. However, a recent study reported that border-associated macrophages participate in stroke-induced inflammation, although many details and the underlying mechanisms remain unclear. In this study, we performed a comprehensive single-cell analysis of mouse border-associated macrophages using sequencing data obtained from the Gene Expression Omnibus(GEO) database(GSE174574 and GSE225948). Differentially expressed genes were identified, and enrichment analysis was performed to identify the transcription profile of border-associated macrophages. CellChat analysis was conducted to determine the cell communication network of border-associated macrophages. Transcription factors were predicted using the ‘pySCENIC' tool. We found that, in response to hypoxia, borderassociated macrophages underwent dynamic transcriptional changes and participated in the regulation of inflammatory-related pathways. Notably, the tumor necrosis factor pathway was activated by border-associated macrophages following ischemic stroke. The pySCENIC analysis indicated that the activity of signal transducer and activator of transcription 3(Stat3) was obviously upregulated in stroke, suggesting that Stat3 inhibition may be a promising strategy for treating border-associated macrophages-induced neuroinflammation. Finally, we constructed an animal model to investigate the effects of border-associated macrophages depletion following a stroke. Treatment with liposomes containing clodronate significantly reduced infarct volume in the animals and improved neurological scores compared with untreated animals. Taken together, our results demonstrate comprehensive changes in border-associated macrophages following a stroke, providing a theoretical basis for targeting border-associated macrophages-induced neuroinflammation in stroke treatment.展开更多
Over the past decade, there has been a growing realization that studying the small RNA transcriptome is essential for understanding the complexity of transcriptional regulation. With an increased throughput and a redu...Over the past decade, there has been a growing realization that studying the small RNA transcriptome is essential for understanding the complexity of transcriptional regulation. With an increased throughput and a reduced cost, next-generation sequencing technology has provided an unprecedented opportunity to measure the extent and complexity of small RNA transcriptome. Meanwhile, the large amount of obtained data and varied technology platforms have also posed multiple challenges for effective data analysis and mining. To provide some insight into the small RNA transcriptome investigation, this review describes the major small RNA classes, experimental methods to identify small RNAs, and available bioinformatics tools and databases.展开更多
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 un...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.展开更多
A large proportion of patients with idiopathic spermatogenic failure(SPGF;oligozoospermia or nonobstructive azoospermia[NOA])do not receive a diagnosis despite an extensive diagnostic workup.Recent evidence has shown ...A large proportion of patients with idiopathic spermatogenic failure(SPGF;oligozoospermia or nonobstructive azoospermia[NOA])do not receive a diagnosis despite an extensive diagnostic workup.Recent evidence has shown that the etiology remains undefined in up to 75%of these patients.A number of genes involved in germ-cell proliferation,spermatocyte meiotic divisions,and spermatid development have been called into play in the pathogenesis of idiopathic oligozoospermia or NOA.However,this evidence mainly comes from case reports.Therefore,this study was undertaken to identify the molecular causes of SPGF.To accomplish this,15 genes(USP9Y,NR5A1,KLHL10,ZMYND15,PLK4,TEX15,TEX11,MEIOB,SOHLH1,HSF2,SYCP3,TAF4B,NANOS1,SYCE1,and RHOXF2)involved in idiopathic SPGF were simultaneously analyzed in a cohort of 25 patients with idiopathic oligozoospermia or NOA,accurately selected after a thorough diagnostic workup.After next-generation sequencing(NGS)analysis,we identified the presence of rare variants in the NR5A1 and TEX11 genes with a pathogenic role in 3/25(12.0%)patients.Seventeen other different variants were identified,and among them,13 have never been reported before.Eleven out of 17 variants were likely pathogenic and deserve functional or segregation studies.The genes most frequently mutated were MEIOB,followed by USP9Y,KLHL10,NR5A1,and SOHLH1.No alterations were found in the SYCP3,TAF4B,NANOS1,SYCE1,or RH0XF2 genes.In conclusion,NGS technology,by screening a specific custom-made panel of genes,could help increase the diagnostic rate in patients with idiopathic oligozoospermia or NOA.展开更多
Next-generation sequencing(NGS) technology is capable of sequencing millions or billions of DNA molecules simultaneously.Therefore, it represents a promising tool for the analysis of molecular targets for the initial ...Next-generation sequencing(NGS) technology is capable of sequencing millions or billions of DNA molecules simultaneously.Therefore, it represents a promising tool for the analysis of molecular targets for the initial diagnosis of disease, monitoring of disease progression, and identifying the mechanism of drug resistance. On behalf of the Tumor Biomarker Committee of the Chinese Society of Clinical Oncology(CSCO) and the China Actionable Genome Consortium(CAGC), the present expert group hereby proposes advisory guidelines on clinical applications of NGS technology for the analysis of cancer driver genes for precision cancer therapy. This group comprises an assembly of laboratory cancer geneticists, clinical oncologists, bioinformaticians,pathologists, and other professionals. After multiple rounds of discussions and revisions, the expert group has reached a preliminary consensus on the need of NGS in clinical diagnosis, its regulation, and compliance standards in clinical sample collection. Moreover, it has prepared NGS criteria, the sequencing standard operation procedure(SOP), data analysis, report, and NGS platform certification and validation.展开更多
BACKGROUND Visceral leishmaniasis(VL)is a parasitic disease caused by Leishmania and transmitted by infected sand flies.VL has a low incidence in China,and its clinical presentation is complex and atypical.This diseas...BACKGROUND Visceral leishmaniasis(VL)is a parasitic disease caused by Leishmania and transmitted by infected sand flies.VL has a low incidence in China,and its clinical presentation is complex and atypical.This disease is easily misdiagnosed and can become life-threatening within a short period of time.Therefore,early,rapid and accurate diagnosis and treatment of the disease are essential.CASE SUMMARY A 25-year-old male patient presented with the clinical manifestations of irregular fever,hepatosplenomegaly,increased polyclonal globulin,and pancytopenia.The first bone marrow puncture biopsy did not provide a clear diagnosis.In order to relieve the pressure and discomfort of the organs caused by the enlarged spleen and to confirm the diagnosis,splenectomy was performed,and hemophagocytic syndrome was diagnosed by pathological examination of the spleen biopsy.Following bone marrow and spleen pathological re-diagnosis and metagenomic next-generation sequencing(mNGS)technology detection,the patient was finally diagnosed with VL.After treatment with liposomal amphotericin B,the body temperature quickly returned to normal and the hemocytes recovered gradually.Post-treatment re-examination of the bone marrow puncture and mNGS data showed that Leishmania was not detected.CONCLUSION As a fast and accurate detection method,mNGS can diagnose and evaluate the efficacy of treatment in suspicious cases of leishmaniasis.展开更多
The application of next-generation sequencing （NGS） technology in cancer is influenced by the quality and purity of tissue samples. This issue is especially critical for patient-derived xenograft （PDX） models, whi...The application of next-generation sequencing （NGS） technology in cancer is influenced by the quality and purity of tissue samples. This issue is especially critical for patient-derived xenograft （PDX） models, which have proven to be by far the best preclinical tool for investigating human tumor biology, because the sensitivity and specificity of NGS analysis in xenograft samples would be compromised by the contamination of mouse DNA and RNA. This definitely affects downstream analyses by causing inaccurate mutation calling and gene expression estimates. The reliability of NGS data analysis for cancer xenograft samples is therefore highly dependent on whether the sequencing reads derived from the xenograft could be distinguished from those originated from the host. That is, each sequence read needs to be accurately assigned to its original species. Here, we review currently available methodologies in this field, including Xenome, Disambiguate, bamcmp and pdxBlacklist, and provide guidelines for users.展开更多
46,XY disorders of sex development(DSD)is characterized by incomplete masculinization genitalia,with gonadal dysplasia and with/without the presence of Mullerian structures.At least 30 genes related to 46,XY DSD have ...46,XY disorders of sex development(DSD)is characterized by incomplete masculinization genitalia,with gonadal dysplasia and with/without the presence of Mullerian structures.At least 30 genes related to 46,XY DSD have been found.However,the clinical phenotypes of patients with different gene mutations overlap,and accurate diagnosis relies on gene sequencing technology.Therefore,this study aims to determine the prevalence of pathogenic mutations in a Chinese cohort with 46,XY DSD by the targeted nextgeneration sequencing(NGS)technology.Eighty-seven 46,XY DSD patients were enrolled from the Peking Union Medical College Hospital(Beijing,China).A total of fifty-four rare variants were identified in 60 patients with 46,XY DSD.The incidence of these rare variants was approximately 69.0%(60/87).Twenty-five novel variants and 29 reported variants were identified.Based on the American College of Medical Genetics and Genomics(ACMG)guidelines,thirty-three variants were classified as pathogenic or likely pathogenic variants and 21 variants were assessed as variants of uncertain significance.The overall diagnostic rate was about 42.5%based on the pathogenic and likely pathogenic variants.Androgen receptor{AR),steroid 5-alpha-reductase 2(SRD5A2)and nuclear receptor subfamily 5 Group A member 1(NR5A1)gene variants were identified in 21,13 and 13 patients,respectively.The incidence of these three gene variants was about 78.3%(47/60)in patients with rare variants.It is concluded that targeted NGS is an effective method to detect pathogenic mutations in 46,XY DSD patients and AR,SRD5A2,and NR5A1 genes were the most common pathogenic genes in our cohort.展开更多
基金supported by the National Natural Science Foundation of China(Grant Nos.31972983 and 32072487)the Key Technology R&D Program of Zhejiang Province,China(Grant No.2021C02006)the Zhejiang Provincial Natural Science Foundation of China(Grant No.LY23C140001).
文摘In rice fields,rice plants usually grow alongside wild weeds and are attacked by various invertebrate species.Viruses are abundant in plants and invertebrates,playing crucial ecological roles in controlling microbial abundance and maintaining community structures.To date,only 16 rice viruses have been documented in rice-growing regions.These viruses pose serious threats to rice production and have traditionally been identified only from rice plants and insect vectors by isolation techniques.Advances in next-generation sequencing(NGS)have made it feasible to discover viruses on a global scale.Recently,numerous viruses have been identified in plants and invertebrates using NGS technologies.In this review,we discuss viral studies in rice plants,invertebrate species,and weeds in rice fields.Many novel viruses have been discovered in rice ecosystems through NGS technologies,with some also detected using metatranscriptomic and small RNA sequencing.These analyses greatly expand our understanding of viruses in rice fields and provide valuable insights for developing efficient strategies to manage insect pests and virus-mediated rice diseases.
文摘This study investigates the diversity of gut microbiota in Metaphire peguana,an earthworm species commonly found in agricultural areas of Thailand.Earthworms play a critical role in soil ecosystems by supporting nutrient cycling and breaking down organic matter.Understanding the microbial diversity in their gut is essential for exploring their ecological contributions.Using Next Generation Sequencing(NGS),we analyzed the mycobiome in the gut of M.peguana.Our findings revealed a high diversity of fungal species,primarily belonging to two major phyla:Ascomycota and Basidiomycota.Ascomycota was the most abundant phylum,comprising 40.1% of the total fungal species identified.A total of 33 distinct fungal species were identified,which underscores the richness of microbial life within the earthworm gut.This study successfully created the first genetic database of the microbial community in M.peguana,providing a foundation for future research in agricultural applications.The microbial species identified,particularly siderophoreproducing fungi,could have significant implications for improving soil fertility and promoting sustainable agricultural practices.The use of NGS technology has enabled comprehensive profiling of microbial communities,allowing for precise identification of fungi that may play essential roles in soil health.Furthermore,the study paves the way for future studies on the potential applications of earthworm gut microbiomes in biotechnology,especially in enhancing soil nutrient availability and plant growth.The findings of this research contribute to the broader understanding of the ecological roles of earthworms and their microbiomes in soil ecosystems.
基金supported by the Hubei Provincial Natural Science Foundation of China(No.2023AFB646)Knowledge Innovation Program of Wuhan(No.2023020201010155)Educational Research Program of Huazhong University of Science and Technology(No.2022135).
文摘Objective and Background Early and accurate diagnosis of spinal infections,including spinal tuberculosis,is pivotal for effective treatment but remains challenging.This study aims to assess the diagnostic yield of metagenomic next-generation sequencing(mNGS)compared with that of conventional microbiological tests(CMTs)in identifying pathogens associated with spinal pathologies,with a special focus on infections leading to surgical interventions.Methods We enrolled 85 patients who underwent spinal surgery,comprising 63 patients with clinically diagnosed spinal infections,including patients with spinal tuberculosis,and 22 patients with noninfectious spinal conditions.The procedures involved irrigation and debridement for persistent wound drainage,with subsequent DNA extraction from plasma and joint fluid for mNGS and CMT analysis.Results Significantly increased C-reactive protein(CRP)levels were observed in patients with infections.The mNGS approach showed greater diagnostic sensitivity(92.06%)for detecting pathogens,including Mycobacterium tuberculosis,than did CMTs(36.51%).Despite its low specificity,mNGS had considerable negative predictive value(70.59%),underscoring its utility in ruling out infections.Conclusions The mNGS offers superior sensitivity over CMTs in the diagnosis of a variety of spinal infections,notably spinal tuberculosis.This study highlights the potential of mNGS in enhancing the diagnosis of complex spinal infections,thereby informing targeted treatment strategies.
文摘BACKGROUND Leuconostoc garlicum is commonly found in fermented foods and very few infected patients have been reported,who typically present symptoms such as fever and fatigue.Conventional clinical examinations often struggle to identify this bacterium,and routine anti-infective treatments are generally ineffective.Both diagnostic challenges and therapeutic limitations pose significant difficulties for clinicians.CASE SUMMARY We report a patient ultimately diagnosed with Leuconostoc garlicum infection.The primary manifestations included persistent fever,cough and fatigue.These symptoms lasted for 2 months.He received anti-infective treatment at a community hospital,but this was ineffective.After inquiring about the patient's medical history and conducting a physical examination,the patient underwent laboratory tests.Complete blood count tests revealed that the patient had a high proportion of neutrophils,C-reactive protein level was 235.9 mg/L,erythrocyte sedimentation rate was 67 mm/h,respiratory pathogen testing was negative,and he was then thought to have an infectious disease.However,conventional anti-infective treatments were ineffective.After excluding infectious neurological diseases,urologic diseases and digestive problems,we ultimately focused our attention on the lungs.A lung computed tomography scan indicated pulmonary inflammation.Bronchoalveolar lavage fluid for next-generation sequencing suggested lung infection with Leuconostoc garlicum.The patient's symptoms gradually improved following treatment with piperacillin tazobactam and linezolid.During the follow-up period,the patient's temperature remained normal.CONCLUSION For patients with suspected bacterial infection and experiencing fever,conventional anti-infective treatment can be ineffective in controlling their symptoms,and an infection due to rare bacteria or drug-resistant bacteria should be considered.Next-generation sequencing enables rapid and precise identification of infection-related pathogens in febrile patients.
基金financially supported by the National Natural Science Foundation of China (32161143033, 32272178, and 32001574)National Key Research and Development Program of China (2021YFD1201605)the Agricultural Science and Technology Innovation Project of CAAS。
文摘The improvement of soybean seed carotenoid contents is very important due to the beneficial role of carotenoids in human health and nutrition. However, the genetic architecture underlying soybean carotenoid biosynthesis remains largely unknown. In the present study, we employed next generation sequencing-based bulked-segregant analysis to identify new genomic regions governing seed carotenoids in 1,551 natural soybean accessions. The genomic DNA samples of individual plants with extreme phenotypes were pooled to form two bulks with high(50 accessions) and low(50 accessions) carotenoid contents for Illumina sequencing. A total of 125.09 Gb of clean bases and 89.82% of Q30 were obtained, and the average alignment efficiency was 99.45% with an average coverage depth of 62.20× and 99.75% genome coverage. Based on the G prime statistic algorithm(G') method analysis, 16 candidate genomic loci with a total length 20.41 Mb were found to be related to the trait. Of these loci, the most significant regions displaying the highest elevated G' values were found in chromosome 06 at a position of 18.53–22.67 Mb, and chromosome 19 at genomic region intervals of 8.36–10.94, 12.06–13.79 and 18.45–20.26 Mb. These regions were then used to identify the key candidate genes. In these regions, 250 predicted genes were found and analyzed to obtain 90 significantly enriched(P<0.05) Gene Ontology(GO) terms. Based on ANNOVAR analysis, 50 genes with non-synonymous and stopgained mutations were preferentially selected as potential candidate genes. Of those 50 genes, following their gene annotation functions and high significant haplotype variations in various environments,five genes were identified as the most promising candidate genes regulating soybean seed carotenoid accumulation, and they should be investigated in further functional validation studies. Collectively, understanding the genetic basis of carotenoid pigments and identifying genes underpinning carotenoid accumulation via a bulked-segregant analysis-based sequencing(BSA-seq) approach provide new insights for exploring future molecular breeding efforts to produce soybean cultivars with high carotenoid content.
基金financially supported by the Science and Technology Commission of Shanghai Municipality(No.24ZR1401400)Shenzhen Salus Bio Med Company for their strong support in this study。
文摘In this study,an amine-reactive poly(pentafluorophenyl acrylate)(PPFPA)platform was developed for advanced surface engineering of next-generation sequencing(NGS)chips.Through post-polymerization modification,PPFPA was functionalized with dual moieties:azide groups for covalent immobilization of DBCO-modified DNA primers via click chemistry and tunable hydrophilic side chains to optimize biocompatibility and surface properties.Systematic screening revealed that hydrophobic azide carriers combined with neutral hydroxyl groups maximized the DNA immobilization efficacy,approaching the performance of commercial polyacrylamide-based polymers.The negatively charged carboxyl groups severely impede DNA primer attachment.Higher molecular weight derivatives further enhance the efficacy of DNA immobilization.In NGS validation,optimized surface modification polymers achieved robust surface density of clustered DNA and high sequencing accuracy,surpassing quality benchmarks and comparable to those of conventional analogs.This platform demonstrates significant potential for tailoring high-sensitivity surfaces for genomic applications,advancing clinical diagnostics,and personalized medicine.
基金supported by the National Science Foundation of China,Nos.82325031(to FX),82030059(to YC),82102290(to YG),U23A20485(to YC)Noncommunicable Chronic Diseases-National Science and Technology Major Project,No.2023ZD0505504(to FX),2023ZD0505500(to YC)the Key R&D Program of Shandong Province,No.2022ZLGX03(to YC).
文摘Global brain ischemia and neurological deficit are consequences of cardiac arrest that lead to high mortality.Despite advancements in resuscitation science,our limited understanding of the cellular and molecular mechanisms underlying post-cardiac arrest brain injury have hindered the development of effective neuroprotective strategies.Previous studies primarily focused on neuronal death,potentially overlooking the contributions of non-neuronal cells and intercellular communication to the pathophysiology of cardiac arrest-induced brain injury.To address these gaps,we hypothesized that single-cell transcriptomic analysis could uncover previously unidentified cellular subpopulations,altered cell communication networks,and novel molecular mechanisms involved in post-cardiac arrest brain injury.In this study,we performed a single-cell transcriptomic analysis of the hippocampus from pigs with ventricular fibrillation-induced cardiac arrest at 6 and 24 hours following the return of spontaneous circulation,and from sham control pigs.Sequencing results revealed changes in the proportions of different cell types,suggesting post-arrest disruption in the blood-brain barrier and infiltration of neutrophils.These results were validated through western blotting,quantitative reverse transcription-polymerase chain reaction,and immunofluorescence staining.We also identified and validated a unique subcluster of activated microglia with high expression of S100A8,which increased over time following cardiac arrest.This subcluster simultaneously exhibited significant M1/M2 polarization and expressed key functional genes related to chemokines and interleukins.Additionally,we revealed the post-cardiac arrest dysfunction of oligodendrocytes and the differentiation of oligodendrocyte precursor cells into oligodendrocytes.Cell communication analysis identified enhanced post-cardiac arrest communication between neutrophils and microglia that was mediated by neutrophil-derived resistin,driving pro-inflammatory microglial polarization.Our findings provide a comprehensive single-cell map of the post-cardiac arrest hippocampus,offering potential novel targets for neuroprotection and repair following cardiac arrest.
文摘Cystic echinococcosis (CE) is a prevalent zoonotic disease caused by Echinococcus granulosus, with a cosmopolitan distribution. The parasite is transmitted cyclically between canines and numerous intermediate herbivorous livestock animals. Also, other Taeniid tapeworms could infect domestic dogs and they pose significant veterinary and public health concerns worldwide. This study aimed to develop a sensitive molecular method for detecting Echinococcus spp. DNA in dog fecal samples using next-generation sequencing (NGS). A set of PCR primers targeting conserved regions of Taeniid tapeworms’ 18s rRNA genes was designed and tested for amplifying genomic DNA from various tapeworm species. The PCR system demonstrated high sensitivity, amplifying DNA from all tested tapeworm species, with differences observed in amplified band sizes. The primers were adapted for NGS analysis by adding forward and reverse adapters, enabling the sequencing of amplified DNA fragments. Application of the developed PCR system to dog fecal samples collected from Yatta town, Palestine, revealed the presence of E. granulosus DNA in five out of 50 samples. NGS analysis confirmed the specificity of the amplified DNA fragments, showing 98% - 99% similarity with the 18s rDNA gene of E. granulosus. This study demonstrates the utility of NGS-based molecular methods for accurate and sensitive detection of Echinococcus spp. in dog fecal samples, providing valuable insights for epidemiological surveillance and control programs of echinococcosis in endemic regions.
基金supported by NINDS/NIH(JZ),Coldwell Foundation(JZ) and TTUHSC(JZ)
文摘This article reviews basic concepts, general applications, and the potential impact of next-generation sequencing （NGS） technologies on genomics, with particular reference to currently available and possible future platforms and bioinformatics. NGS technologies have demon- strated the capacity to sequence DNA at unprecedented speed, thereby enabling previously unimaginable scientific achievements and novel biological applications. But, the massive data produced by NGS also presents a significant challenge for data storage, analyses, and management solutions. Advanced bioinformatic tools are essential for the successful application of NGS technology. As evidenced throughout this review, NGS technologies will have a striking impact on genomic research and the entire biological field. With its ability to tackle the unsolved challenges unconquered by previous genomic technologies, NGS is likely to unravel the complexity of the human genome in terms of genetic variations, some of which may be confined to susceptible loci for some common human conditions. The impact of NGS technologies on genomics will be far reaching and likely change the field for years to come.
基金supported by grants from Scienceand Technology Committee of Shanghai (18411951400)KeyClinical Medical Specialties Project in Shanghai Pudong NewArea (PWZzk2017-22)+1 种基金Science and Technology Action Plan(19495810200)Leading Talent Project in Shanghai Pudong NewArea Health System (PWRl2018-08).
文摘BACKGROUND: The study aims to investigate the performance of a metagenomic next-generationsequencing (NGS)-based diagnostic technique for the identifi cation of potential bacterial and viral infectionsand eff ects of concomitant viral infection on the survival rate of intensive care unit (ICU) sepsis patients.METHODS: A total of 74 ICU patients with sepsis who were admitted to our institution from February1, 2018 to June 30, 2019 were enrolled. Separate blood samples were collected from patients for bloodcultures and metagenomic NGS when the patients’ body temperature was higher than 38 °C. Patients’demographic data, including gender, age, ICU duration, ICU scores, and laboratory results, were recorded.The correlations between pathogen types and sepsis severity and survival rate were evaluated.RESULTS: NGS produced higher positive results (105 of 118;88.98%) than blood cultures(18 of 118;15.25%) over the whole study period. Concomitant viral infection correlated closelywith sepsis severity and had the negative effect on the survival of patients with sepsis. However,correlation analysis indicated that the bacterial variety did not correlate with the severity of sepsis.CONCLUSIONS: Concurrent viral load correlates closely with the severity of sepsis and thesurvival rate of the ICU sepsis patients. This suggests that prophylactic administration of antiviraldrugs combined with antibiotics may be benefi cial to ICU sepsis patients.
基金supported by grants awarded to Yuanqing Yao by the Key Program of the "Twelfth Five-year plan" of People’s liberation Army(No.BWS11J058)the National High Technology Research and Development Program(SS2015AA020402)
文摘Reliable and accurate pre-implantation genetic diagnosis （PGD） of patient＇s embryos by next-generation sequencing （NGS） is dependent on efficient whole genome amplification （WGA） of a representative biopsy sample. However, the performance of the current state of the art WGA methods has not been evaluated for sequencing. Using low template DNA （15 pg） and single cells, we showed that the two PCR-based WGA systems SurePlex and MALBAC are superior to the REPLI-g WGA multiple displacement amplification （MDA） system in terms of consistent and reproducible genome coverage and sequence bias across the 24 chromosomes, allowing better normalization of test to reference sequencing data. When copy number variation sequencing （CNV-Seq） was applied to single cell WGA products derived by either SurePlex or MALBAC amplification, we showed that known disease CNVs in the range of 3-15 Mb could be reliably and accurately detected at the correct genomic positions. These findings indicate that our CNV-Seq pipeline incorporating either SurePlex or MALBAC as the key initial WGA step is a powerful methodology for clinical PGD to identify euploid embryos in a patient＇s cohort for uterine transplantation,
基金Supported by Grants-in-Aid for Scientific Research from the Ministry of Education,Culture,Sports,Science and Technology of Japan
文摘Gastric cancer(GC)is one of the most common malignancies and remains the second leading cause of cancer-related death worldwide.There is an increasing understanding of the roles that genetic and epigenetic alterations play in GCs.Recent studies using nextgeneration sequencing(NGS)have revealed a number of potential cancer-driving genes in GC.Whole-exome sequencing of GC has identified recurrent somatic mutations in the chromatin remodeling gene ARID1A and alterations in the cell adhesion gene FAT4,a member of the cadherin gene family.Mutations in chromatin remodeling genes(ARID1A,MLL3 and MLL)have been found in 47%of GCs.Whole-genome sequencing and whole-transcriptome sequencing analyses have also discovered novel alterations in GC.Recent studies of cancer epigenetics have revealed widespread alterations in genes involved in the epigenetic machinery,such as DNA methylation,histone modifications,nucleosome positioning,noncoding RNAs and microRNAs.Recent advances in molecular research on GC have resulted in the introduction of new diagnostic and therapeutic strategies into clinical settings.The antihuman epidermal growth receptor 2(HER2)antibody trastuzumab has led to an era of personalized therapy in GC.In addition,ramucirumab,a monoclonal antibody targeting vascular endothelial growth factor receptor(VEGFR)-2,is the first biological treatment that showed survival benefits as a single-agent therapy in patients with advanced GC who progressed after firstline chemotherapy.Using NGS to systematically identify gene alterations in GC is a promising approach with remarkable potential for investigating the pathogenesis of GC and identifying novel therapeutic targets,as well as useful biomarkers.In this review,we will summarize the recent advances in the understanding of the molecular pathogenesis of GC,focusing on the potential use of these genetic and epigenetic alterations as diagnostic biomarkers and novel therapeutic targets.
基金supported by Qingdao Key Medical and Health Discipline ProjectThe Intramural Research Program of the Affiliated Hospital of Qingdao University,No. 4910Qingdao West Coast New Area Science and Technology Project,No. 2020-55 (all to SW)。
文摘Border-associated macrophages are located at the interface between the brain and the periphery, including the perivascular spaces, choroid plexus, and meninges. Until recently, the functions of border-associated macrophages have been poorly understood and largely overlooked. However, a recent study reported that border-associated macrophages participate in stroke-induced inflammation, although many details and the underlying mechanisms remain unclear. In this study, we performed a comprehensive single-cell analysis of mouse border-associated macrophages using sequencing data obtained from the Gene Expression Omnibus(GEO) database(GSE174574 and GSE225948). Differentially expressed genes were identified, and enrichment analysis was performed to identify the transcription profile of border-associated macrophages. CellChat analysis was conducted to determine the cell communication network of border-associated macrophages. Transcription factors were predicted using the ‘pySCENIC' tool. We found that, in response to hypoxia, borderassociated macrophages underwent dynamic transcriptional changes and participated in the regulation of inflammatory-related pathways. Notably, the tumor necrosis factor pathway was activated by border-associated macrophages following ischemic stroke. The pySCENIC analysis indicated that the activity of signal transducer and activator of transcription 3(Stat3) was obviously upregulated in stroke, suggesting that Stat3 inhibition may be a promising strategy for treating border-associated macrophages-induced neuroinflammation. Finally, we constructed an animal model to investigate the effects of border-associated macrophages depletion following a stroke. Treatment with liposomes containing clodronate significantly reduced infarct volume in the animals and improved neurological scores compared with untreated animals. Taken together, our results demonstrate comprehensive changes in border-associated macrophages following a stroke, providing a theoretical basis for targeting border-associated macrophages-induced neuroinflammation in stroke treatment.
基金supported by the National High Technology Research and Development Program of China(No. 2006AA02A304)Major State Basic Research Development Program of China(No.2007CB512302)
文摘Over the past decade, there has been a growing realization that studying the small RNA transcriptome is essential for understanding the complexity of transcriptional regulation. With an increased throughput and a reduced cost, next-generation sequencing technology has provided an unprecedented opportunity to measure the extent and complexity of small RNA transcriptome. Meanwhile, the large amount of obtained data and varied technology platforms have also posed multiple challenges for effective data analysis and mining. To provide some insight into the small RNA transcriptome investigation, this review describes the major small RNA classes, experimental methods to identify small RNAs, and available bioinformatics tools and databases.
基金supported by the Medicine and Health,Science and Technology Plan Project of Zhejiang(Nos.2020KY1009 and 2021KY387)the Jinhua Science and Technology Planning Project Social Development Key Project(No.2021-3-072),China.
文摘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.
文摘A large proportion of patients with idiopathic spermatogenic failure(SPGF;oligozoospermia or nonobstructive azoospermia[NOA])do not receive a diagnosis despite an extensive diagnostic workup.Recent evidence has shown that the etiology remains undefined in up to 75%of these patients.A number of genes involved in germ-cell proliferation,spermatocyte meiotic divisions,and spermatid development have been called into play in the pathogenesis of idiopathic oligozoospermia or NOA.However,this evidence mainly comes from case reports.Therefore,this study was undertaken to identify the molecular causes of SPGF.To accomplish this,15 genes(USP9Y,NR5A1,KLHL10,ZMYND15,PLK4,TEX15,TEX11,MEIOB,SOHLH1,HSF2,SYCP3,TAF4B,NANOS1,SYCE1,and RHOXF2)involved in idiopathic SPGF were simultaneously analyzed in a cohort of 25 patients with idiopathic oligozoospermia or NOA,accurately selected after a thorough diagnostic workup.After next-generation sequencing(NGS)analysis,we identified the presence of rare variants in the NR5A1 and TEX11 genes with a pathogenic role in 3/25(12.0%)patients.Seventeen other different variants were identified,and among them,13 have never been reported before.Eleven out of 17 variants were likely pathogenic and deserve functional or segregation studies.The genes most frequently mutated were MEIOB,followed by USP9Y,KLHL10,NR5A1,and SOHLH1.No alterations were found in the SYCP3,TAF4B,NANOS1,SYCE1,or RH0XF2 genes.In conclusion,NGS technology,by screening a specific custom-made panel of genes,could help increase the diagnostic rate in patients with idiopathic oligozoospermia or NOA.
基金supported by grants from Guangdong Provincial Key Lab of Translational Medicine in Lung Cancer (Grant No. 2017B030314120)General Research Project of Guangzhou Science and Technology Bureau (Grant No. 201607010391)+1 种基金National Key Research and Development Program of China (Grant No. 2016YFC1303800)Guangdong Provincial Applied S&T R&D Program (Grant No. 2016B020237006)
文摘Next-generation sequencing(NGS) technology is capable of sequencing millions or billions of DNA molecules simultaneously.Therefore, it represents a promising tool for the analysis of molecular targets for the initial diagnosis of disease, monitoring of disease progression, and identifying the mechanism of drug resistance. On behalf of the Tumor Biomarker Committee of the Chinese Society of Clinical Oncology(CSCO) and the China Actionable Genome Consortium(CAGC), the present expert group hereby proposes advisory guidelines on clinical applications of NGS technology for the analysis of cancer driver genes for precision cancer therapy. This group comprises an assembly of laboratory cancer geneticists, clinical oncologists, bioinformaticians,pathologists, and other professionals. After multiple rounds of discussions and revisions, the expert group has reached a preliminary consensus on the need of NGS in clinical diagnosis, its regulation, and compliance standards in clinical sample collection. Moreover, it has prepared NGS criteria, the sequencing standard operation procedure(SOP), data analysis, report, and NGS platform certification and validation.
基金by National Nature Science Foundation of China,No.81401321Basic Public Welfare Research Project of Zhejiang Province,No.LGF19H080002+1 种基金Science Research Project of Medicine and Hygiene of Zhejiang Province,No.2018PY052Public Welfare Science and Technology Project of Ningbo,No.2019C50068。
文摘BACKGROUND Visceral leishmaniasis(VL)is a parasitic disease caused by Leishmania and transmitted by infected sand flies.VL has a low incidence in China,and its clinical presentation is complex and atypical.This disease is easily misdiagnosed and can become life-threatening within a short period of time.Therefore,early,rapid and accurate diagnosis and treatment of the disease are essential.CASE SUMMARY A 25-year-old male patient presented with the clinical manifestations of irregular fever,hepatosplenomegaly,increased polyclonal globulin,and pancytopenia.The first bone marrow puncture biopsy did not provide a clear diagnosis.In order to relieve the pressure and discomfort of the organs caused by the enlarged spleen and to confirm the diagnosis,splenectomy was performed,and hemophagocytic syndrome was diagnosed by pathological examination of the spleen biopsy.Following bone marrow and spleen pathological re-diagnosis and metagenomic next-generation sequencing(mNGS)technology detection,the patient was finally diagnosed with VL.After treatment with liposomal amphotericin B,the body temperature quickly returned to normal and the hemocytes recovered gradually.Post-treatment re-examination of the bone marrow puncture and mNGS data showed that Leishmania was not detected.CONCLUSION As a fast and accurate detection method,mNGS can diagnose and evaluate the efficacy of treatment in suspicious cases of leishmaniasis.
基金supported by the grants from the National Natural Science Foundation of China(Nos.,81672736 and 91529302)the Shanghai Industrial Technology Institute(17CXXF008)+3 种基金the Shanghai Sailing Program(16YF1408600)the Shanghai Municipal Commission of Science and Technology(14DZ2252000)the administrative committee of Shanghai Zhangjiang Hi-Teck Park(2016e08)the Medical engineering cross fund of Shanghai Jiao Tong University(YG2015QN27)
文摘The application of next-generation sequencing （NGS） technology in cancer is influenced by the quality and purity of tissue samples. This issue is especially critical for patient-derived xenograft （PDX） models, which have proven to be by far the best preclinical tool for investigating human tumor biology, because the sensitivity and specificity of NGS analysis in xenograft samples would be compromised by the contamination of mouse DNA and RNA. This definitely affects downstream analyses by causing inaccurate mutation calling and gene expression estimates. The reliability of NGS data analysis for cancer xenograft samples is therefore highly dependent on whether the sequencing reads derived from the xenograft could be distinguished from those originated from the host. That is, each sequence read needs to be accurately assigned to its original species. Here, we review currently available methodologies in this field, including Xenome, Disambiguate, bamcmp and pdxBlacklist, and provide guidelines for users.
基金This work was supported by the National Natural Science Foundation of China(No.81971375 and No.81771576)the National Key Research and Development Program of China(2016YFC0905100)+1 种基金the CAMS Innovation Fund for Medical Sciences(2016-I2M-1-002)the Nonprofit Central Research Institute Fund of the Chinese Academy of Medical Sciences(No.2017PT32020 and No.2018PT32001).
文摘46,XY disorders of sex development(DSD)is characterized by incomplete masculinization genitalia,with gonadal dysplasia and with/without the presence of Mullerian structures.At least 30 genes related to 46,XY DSD have been found.However,the clinical phenotypes of patients with different gene mutations overlap,and accurate diagnosis relies on gene sequencing technology.Therefore,this study aims to determine the prevalence of pathogenic mutations in a Chinese cohort with 46,XY DSD by the targeted nextgeneration sequencing(NGS)technology.Eighty-seven 46,XY DSD patients were enrolled from the Peking Union Medical College Hospital(Beijing,China).A total of fifty-four rare variants were identified in 60 patients with 46,XY DSD.The incidence of these rare variants was approximately 69.0%(60/87).Twenty-five novel variants and 29 reported variants were identified.Based on the American College of Medical Genetics and Genomics(ACMG)guidelines,thirty-three variants were classified as pathogenic or likely pathogenic variants and 21 variants were assessed as variants of uncertain significance.The overall diagnostic rate was about 42.5%based on the pathogenic and likely pathogenic variants.Androgen receptor{AR),steroid 5-alpha-reductase 2(SRD5A2)and nuclear receptor subfamily 5 Group A member 1(NR5A1)gene variants were identified in 21,13 and 13 patients,respectively.The incidence of these three gene variants was about 78.3%(47/60)in patients with rare variants.It is concluded that targeted NGS is an effective method to detect pathogenic mutations in 46,XY DSD patients and AR,SRD5A2,and NR5A1 genes were the most common pathogenic genes in our cohort.
