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
Objective This study aimed to explore the diagnostic value of novel technique-targeted next-generation sequencing(tNGS)of bronchoalveolar lavage fluid(BALF)in pulmonary mycobacterial infections.Methods This retrospect...Objective This study aimed to explore the diagnostic value of novel technique-targeted next-generation sequencing(tNGS)of bronchoalveolar lavage fluid(BALF)in pulmonary mycobacterial infections.Methods This retrospective study was conducted on patients who underwent bronchoscopy and tNGS,smear microscopy,and mycobacterial culture of BALF.Patients with positive Mycobacterium tuberculosis(MTB)culture or GeneXpert results were classified into the tuberculosis case group.Those diagnosed with nontuberculous mycobacteria(NTM)-pulmonary disease(NTM-PD)composed the case group of NTM-PD patients.The control group comprised patients without tuberculosis or NTM-PD.Sensitivity,specificity,and receiver operating characteristic(ROC)curves were used to evaluate the diagnostic performance.Results For tuberculosis patients with positive mycobacterial culture results,the areas under the ROC curves(AUCs)for tNGS,GeneXpert,and smear microscopy were 0.975(95%CI:0.935,1.000),0.925(95%CI:0.859,0.991),and 0.675(95%CI:0.563,0.787),respectively.For tuberculosis patients with positive GeneXpert results,the AUCs of tNGS,culture,and smear microscopy were 0.970(95%CI:0.931,1.000),0.850(95%CI:0.770,0.930),and 0.680(95%CI:0.579,0.781),respectively.For NTM-PD,the AUCs of tNGS,culture,and smear-positive but GeneXpert-negative results were 0.987(95%CI:0.967,1.000),0.750(95%CI:0.622,0.878),and 0.615(95%CI:0.479,0.752),respectively.The sensitivity and specificity of tNGS in NTM-PD patients were 100%and 97.5%,respectively.Conclusion tNGS demonstrated superior diagnostic efficacy in mycobacterial infection,indicating its potential for clinical application.展开更多
Owing to the limitation of a large genome size(~13 Gb),the genetic and gene mapping studies on faba bean(Vicia faba L.)are lagging far behind those for other legumes.In this study,we selected three purified faba bean ...Owing to the limitation of a large genome size(~13 Gb),the genetic and gene mapping studies on faba bean(Vicia faba L.)are lagging far behind those for other legumes.In this study,we selected three purified faba bean lines(Yundou 8137,H0003712,and H000572)as parents and constructed two F2 populations.These two F2 populations,namely 167 F2 plants in Pop1(Yundou 8137×H0003712)and 204 F2 plants in Pop2(H000572×Yundou 8137),were genotyped using a targeted next-generation sequencing(TNGS)genotyping platform,and two high-density single nucleotide polymorphisms(SNP)genetic linkage maps of faba bean were constructed.The map constructed from Pop1 contained 5103 SNPs with a length of 1333.31 cM and an average marker density of 0.26 cM.The map constructed from Pop2 contained 1904 SNPs with a greater length of 1610.61 cM.In these two F2 populations,QTL mapping identified 98 QTLs for 14 agronomic traits related to the flowers,pods,plant types and grains.The two maps were then merged into an integrated genetic linkage map containing 6895 SNPs,with a length of 3324.48 cM.These results not only lay the foundation for fine mapping and map-based cloning of related genes,but can also accelerate the molecular marker-assisted breeding of faba bean.展开更多
BACKGROUND There are many factors that lead to dwarfism,and the mechanism has not yet been elucidated.Next-generation sequencing may identify candidate-related gene mutations,which may clarify the molecular cause.AIM ...BACKGROUND There are many factors that lead to dwarfism,and the mechanism has not yet been elucidated.Next-generation sequencing may identify candidate-related gene mutations,which may clarify the molecular cause.AIM To analyze genetic variation by using a constructed panel related to dwarfism by utilizing next-generation sequencing platform sequencing analysis to screen candidate-related gene mutations.METHODS Physical and laboratory characteristics,including clinical examination,growth hormone drug challenge test,serum insulin-like growth factor-1(IGF-1),IGF binding protein 3,other related tests,imaging examination,and chromosome karyotyping,were analyzed.Next-generation sequencing was performed to analyze pathogenicity variability.RESULTS In the 39 dwarfism patients,10 had pathogenicity variability.Gene variation was found in the OBSL1,SLC26A2,PTPN11,COL27AI,HDAC6,CUL7,FGFR3,DYNC2H1,GH1,and ATP7B genes.Of the 10 patients with pathogenicity variability,the related physical characteristics included double breast development and growth hormone deficiency,enuresis and indirect inguinal hernia on the left,two finger distance of 70.2 cm,head circumference of 49.2 cm,ischium/lower body length of 1.8 cm,weak limb muscles,and partial growth hormone deficiency.After 6 mo of growth hormone therapy,the concentrations of IGF-1 and IGF binding protein 3 increased from 215.2±170.3 to 285.0±166.0 and 3.9±1.4 to 4.2±1.1,respectively.CONCLUSION OBSL1,SLC26A2,PTPN11,COL27AI,HDAC6,CUL7,FGFR3,DYNC2H1,GH1,and ATP7B genes may be related to the incidence of dwarfism,and more research needs to be performed to elucidate the mechanism.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
Different newborn screening(NBS) programs have been practiced in many countries since the 1960 s. It is of considerable interest whether next-generation sequencing is applicable in NBS. We have developed a panel of 46...Different newborn screening(NBS) programs have been practiced in many countries since the 1960 s. It is of considerable interest whether next-generation sequencing is applicable in NBS. We have developed a panel of 465 causative genes for 596 early-onset, relatively high incidence, and potentially actionable severe inherited diseases in our Newborn Screening with Targeted Sequencing(NESTS) program to screen 11,484 babies in 8 Women and Children’s hospitals nationwide in China retrospectively. The positive rate from preliminary screening of NESTS was 7.85%(902/11,484). With 45.89%(414/902) follow-up of preliminary positive cases, the overall clinically confirmative diagnosis rate of monogenic disorders was 12.07%(50/414), estimating an average of 0.95%(7.85% × 12.07%) clinical diagnosis rate, suggesting that monogenic disorders account for a considerable proportion of birth defects. The disease/gene spectrum varied in different regions of China. NESTS was implemented in a hospital by screening 3923 newborns to evaluate its clinical application. The turn-around time of a primary report, including the sequencing period of < 7 days, was within 11 days by our automatic interpretation pipeline. Our results suggest that NESTS is feasible and cost-effective as a first-tier NBS program, which will change the status of current clinical practice of NBS in China.展开更多
BACKGROUND The diagnostic value of metagenomic next-generation sequencing(mNGS)in central nervous system(CNS)infectious diseases after empirical treatment has not been reported.AIM To investigate the diagnostic value ...BACKGROUND The diagnostic value of metagenomic next-generation sequencing(mNGS)in central nervous system(CNS)infectious diseases after empirical treatment has not been reported.AIM To investigate the diagnostic value of mNGS of cerebrospinal fluid(CSF)in the empirically treated CNS infectious diseases.METHODS A total of 262 CSF samples from patients with suspected CNS infections were collected between August 2020 and December 2021.Both mNGS and conventional methods were used for testing.The conventional methods included microbial culture,smear,polymerase chain reaction,etc.RESULTS Among 262 suspected cases,183 cases(69.84%)were diagnosed as CNS infection,including 86 cases of virus infection(47.00%),70 cases of bacterial infection(38.25%)and 27 cases of fungal infection(14.76%).The sensitivity and specificity of mNGS were 65.6%(95%CI:58.2%-72.3%)and 89.6%(95%CI:79.1%-95.3%),respectively.The PPV of mNGS was 94.5%(95%CI:88.6%-97.6%),and the NPV was 48.8%(95%CI:39.7%–57.9%).The pathogen detective sensitivity and accuracy of mNGS were higher than those of conventional methods(Sensitivity:65.6%vs 37.2%;P<0.001;Accuracy:72.0%vs 50%,P<0.001).The results showed that compared with conventional methods,mNGS technology was a more sensitive method for the diagnosis of CNS infection after empirical treatment.CONCLUSION mNGS can be a better method applied in the diagnosis of CNS infection after empirical treatment.展开更多
BACKGROUND Gastric cancer is a leading cause of cancer-related mortality worldwide.Many somatic mutations have been identified based on next-generation sequencing;they likely play a vital role in cancer treatment sele...BACKGROUND Gastric cancer is a leading cause of cancer-related mortality worldwide.Many somatic mutations have been identified based on next-generation sequencing;they likely play a vital role in cancer treatment selection.However,nextgeneration sequencing has not been widely used to diagnose and treat gastric cancer in the clinic.AIM To test the mutant gene frequency as a guide for molecular diagnosis and personalized therapy in gastric cancer by use of next-generation sequencing.METHODS We constructed a panel of 24 mutant genes to detect somatic nucleotide variations and copy number variations based on a next-generation sequencing technique.Our custom panel included high-mutation frequency cancer driver and tumour suppressor genes.Mutated genes were also analyzed using the cBioPortal database.The clinical annotation of important variant mutation sites was evaluated in the ClinVar database.We searched for candidate drugs for targeted therapy and immunotherapy from the OncoKB database.RESULTS In our study,the top 16 frequently mutated genes were TP53(58%),ERBB2(28%),BRCA2(23%),NF1(19%),PIK3CA(14%),ATR(14%),MSH2(12%),FBXW7(12%),BMPR1A(12%),ERBB3(11%),ATM(9%),FGFR2(8%),MET(8%),PTEN(6%),CHD4(6%),and KRAS(5%).TP53 is a commonly mutated gene in gastric cancer and has a similar frequency to that in the cBioPortal database.33 gastric cancer patients(51.6%)with microsatellite stability and eight patients(12.5%)with microsatellite instability-high were investigated.Enrichment analyses demonstrated that high-frequency mutated genes had transmembrane receptor protein kinase activity.We discovered that BRCA2,PIK3CA,and FGFR2 gene mutations represent promising biomarkers in gastric cancer.CONCLUSION We developed a powerful panel of 24 genes with high frequencies of mutation that could detect common somatic mutations.The observed mutations provide potential targets for the clinical treatment of gastric cancer.展开更多
The expanding application of targeted next-generation sequencing(tNGS)in clinical diagnostics has led to an increasing frequency of Nocardia spp.detection in respiratory specimens;however,the clinical relevance and in...The expanding application of targeted next-generation sequencing(tNGS)in clinical diagnostics has led to an increasing frequency of Nocardia spp.detection in respiratory specimens;however,the clinical relevance and interpretation of these findings remain uncertain.This study describes the epidemiology of Nocardia pneumonia and colonization in patients at The University of Hong Kong–Shenzhen Hospital,China,and evaluates the diagnostic role of tNGS.The analysis was conducted from January 2023 through December 2024 and included 22 patients in whom Nocardia spp.were detected in respiratory specimens via tNGS.Among these patients,81.8%(18/22)had comorbidities,most commonly chronic lung disease(40.9%,9/22).Common clinical symptoms included cough,sputum and fever.Nocardia farcinica and Nocardia abscessus were the predominant species identified.Although the median number of se-quence reads of Nocardia were higher in infected patients than in colonized individuals(289,IQR:132.5–3747 vs.133.5,IQR:51.75–261.3),the difference was not statistically significant(P>0.05).In a comparison of diagnostic performance,tNGS identified Nocardia sequences in all 25 respiratory samples collected from the enrolled patients,whereas conventional aerobic culture success-fully isolated the pathogen in only two patients.Additionally,tNGS exhibited a significantly shorter median turnaround time than culture(38 h vs.376.5 h).These findings highlight tNGS as a rapid tool for Nocardia detection.However,the final diagnosis of nocardiosis can-not rely solely on sequence read thresholds.Clinical,radiological and laboratory integration remains critical in distinguishing true infection from colonization,ensuring accurate diagnosis and management of nocardiosis.展开更多
Background:Molecular subtyping is an essential complementarity after pathological analyses for targeted therapy.This study aimed to investigate the consistency of next-generation sequencing(NGS)results between circula...Background:Molecular subtyping is an essential complementarity after pathological analyses for targeted therapy.This study aimed to investigate the consistency of next-generation sequencing(NGS)results between circulating tumor DNA(ctDNA)-based and tissue-based in non-small cell lung cancer(NSCLC)and identify the patient characteristics that favor ctDNA testing.Methods:Patients who diagnosed with NSCLC and received both ctDNA-and cancer tissue-based NGS before surgery or systemic treatment in Lung Cancer Center,Sichuan University West China Hospital between December 2017 and August 2022 were enrolled.A 425-cancer panel with a HiSeq 4000 NGS platform was used for NGS.The unweighted Cohen’s kappa coefficient was employed to discriminate the high-concordance group from the low-concordance group with a cutoff value of 0.6.Six machine learning models were used to identify patient characteristics that relate to high concordance between ctDNA-based and tissue-based NGS.Results:A total of 85 patients were enrolled,of which 22.4%(19/85)had stage III disease and 56.5%(48/85)had stage IV disease.Forty-four patients(51.8%)showed consistent gene mutation types between ctDNA-based and tissue-based NGS,while one patient(1.2%)tested negative in both approaches.Patients with advanced diseases and metastases to other organs would be suitable for the ctDNA-based NGS,and the generalized linear model showed that T stage,M stage,and tumor mutation burden were the critical discriminators to predict the consistency of results between ctDNA-based and tissue-based NGS.Conclusion:ctDNA-based NGS showed comparable detection performance in the targeted gene mutations compared with tissue-based NGS,and it could be considered in advanced or metastatic NSCLC.展开更多
AIM：To make a comprehensive analysis of the potential pathogenic genes related with Leber congenital amaurosis（LCA） in Chinese.METHODS：LCA subjects and their families were retrospectively collected from 2013 to 20...AIM：To make a comprehensive analysis of the potential pathogenic genes related with Leber congenital amaurosis（LCA） in Chinese.METHODS：LCA subjects and their families were retrospectively collected from 2013 to 2015.Firstly,whole-exome sequencing was performed in patients who had underwent gene mutation screening with nothing found,and then homozygous sites was selected,candidate sites were annotated,and pathogenic analysis was conducted using softwares including Sorting Tolerant from Intolerant（SIFT）,Polyphen-2,Mutation assessor,Condel,and Functional Analysis through Hidden Markov Models（FATHMM）.Furthermore,Gene Ontology function and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of pathogenic genes were performed followed by co-segregation analysis using Fisher exact Test.Sanger sequencing was used to validate single-nucleotide variations（SNVs）.Expanded verification was performed in the rest patients.RESULTS：Totally 51 LCA families with 53 patients and24 family members were recruited.A total of 104 SNVs（66 LCA-related genes and 15 co-segregated genes）were submitted for expand verification.The frequencies of homozygous mutation of KRT12 and CYP1A1 were simultaneously observed in 3 families.Enrichment analysis showed that the potential pathogenic genes were mainly enriched in functions related to cell adhesion,biological adhesion,retinoid metabolic process,and eye development biological adhesion.Additionally,WFS7 and STAU2 had the highest homozygous frequencies.CONCLUSION：LCA is a highly heterogeneous disease.Mutations in KRT12,CVP1A1,WFS1,and STAU2 may be involved in the development of LCA.展开更多
Next-generation sequencing （NGS） is getting routinely used in the diagnosis of hereditary diseases, such as human cardiomyopathies. Hence. it is of utter importance to secure high quality sequencing data, enabling t...Next-generation sequencing （NGS） is getting routinely used in the diagnosis of hereditary diseases, such as human cardiomyopathies. Hence. it is of utter importance to secure high quality sequencing data, enabling the identification of disease-relevant mutations or the conclusion of negative test results. During the process of sample preparation, each protocol for target enrichment library preparation has its own requirements for quality control （QC）; however, there is little evi- dence on the actual impact of these guidelines on resulting data quality. In this study, we analyzed the impact of QC during the diverse library preparation steps of Agilent SureSelect XT target enrichment and lllumina sequencing. We quantified the parameters for a cohort of around 600 samples, which include starting amount of DNA, amount of sheared DNA, smallest and largest fragment size of the starting DNA; amount of DNA after the pre-PCR, and smallest and largest fragment size of the resulting DNA; as well as the amount of the final library, the corresponding smallest and largest fragment size, and the number of detected variants. Intriguingly, there is a high tolerance for variations in all QC steps, meaning that within the boundaries proposed in the current study, a considerable variance at each step of QC can be well tolerated without compromising NGS quality.展开更多
Pseudogenes are frequently encountered noncoding sequences with a high sequence similarity to their protein-coding paralogue.For this reason,their presence is often considered troublesome in molecular diagnostics.In p...Pseudogenes are frequently encountered noncoding sequences with a high sequence similarity to their protein-coding paralogue.For this reason,their presence is often considered troublesome in molecular diagnostics.In pseudoxanthoma elasticum(PXE),a disease predominantly caused by mutations in ATPbinding cassette family C member 6(ABCC6),the presence of two pseudogenes complicates the analysis of sequence data.With whole-exome sequencing(WES)becoming the standard of care in molecular diagnostics,we wanted to evaluate whether this technique is as reliable as gene-specific targeted enrichment analysis for the analysis of ABCC6.We established a PCR-based targeted enrichment and next-generation sequencing testing approach and demonstrated that the ABCC6-specific enrichment combined with the applied mapping algorithm overcomes the complication of ABCC6 pseudogene aspecificities,contrary to WES.We propose a time-and cost-efficient diagnostic strategy for comprehensive and accurate molecular genetic testing of PXE,which is highly automatable.展开更多
Objective To screen gene mutation in adult-onset hypoparathyroidism in Chinese through the targeted nextgeneration sequencing(NGS).Methods We recruited 17patients with adult-onset hypoparathyroidism who were regularly...Objective To screen gene mutation in adult-onset hypoparathyroidism in Chinese through the targeted nextgeneration sequencing(NGS).Methods We recruited 17patients with adult-onset hypoparathyroidism who were regularly followed or newly diagnosed at our centre during the past one year.Nine of them developed hypercalciuria during the treatment with calcium and vitamin D.展开更多
Hepatocellular carcinoma(HCC)remains a global medical burden with rising incidence due to chronic viral hepatitis and non-alcoholic fatty liver diseases.Treatment of advanced disease stages is still unsatisfying.Besid...Hepatocellular carcinoma(HCC)remains a global medical burden with rising incidence due to chronic viral hepatitis and non-alcoholic fatty liver diseases.Treatment of advanced disease stages is still unsatisfying.Besides first and second generation tyrosine kinase inhibitors,immune checkpoint inhibitors have become central for the treatment of HCC.New modalities like epigenetic therapy using histone deacetylase inhibitors(HDACi)and cell therapy approaches with chimeric antigen receptor T cells(CAR-T cells)are currently under investigation in clinical trials.Development of such novel drugs is closely linked to the availability and improvement of novel preclinical and animal models and the identification of predictive biomarkers.The current status of treatment options for advanced HCC,emerging novel therapeutic approaches and different preclinical models for HCC drug discovery and development are reviewed here.展开更多
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.
文摘Objective This study aimed to explore the diagnostic value of novel technique-targeted next-generation sequencing(tNGS)of bronchoalveolar lavage fluid(BALF)in pulmonary mycobacterial infections.Methods This retrospective study was conducted on patients who underwent bronchoscopy and tNGS,smear microscopy,and mycobacterial culture of BALF.Patients with positive Mycobacterium tuberculosis(MTB)culture or GeneXpert results were classified into the tuberculosis case group.Those diagnosed with nontuberculous mycobacteria(NTM)-pulmonary disease(NTM-PD)composed the case group of NTM-PD patients.The control group comprised patients without tuberculosis or NTM-PD.Sensitivity,specificity,and receiver operating characteristic(ROC)curves were used to evaluate the diagnostic performance.Results For tuberculosis patients with positive mycobacterial culture results,the areas under the ROC curves(AUCs)for tNGS,GeneXpert,and smear microscopy were 0.975(95%CI:0.935,1.000),0.925(95%CI:0.859,0.991),and 0.675(95%CI:0.563,0.787),respectively.For tuberculosis patients with positive GeneXpert results,the AUCs of tNGS,culture,and smear microscopy were 0.970(95%CI:0.931,1.000),0.850(95%CI:0.770,0.930),and 0.680(95%CI:0.579,0.781),respectively.For NTM-PD,the AUCs of tNGS,culture,and smear-positive but GeneXpert-negative results were 0.987(95%CI:0.967,1.000),0.750(95%CI:0.622,0.878),and 0.615(95%CI:0.479,0.752),respectively.The sensitivity and specificity of tNGS in NTM-PD patients were 100%and 97.5%,respectively.Conclusion tNGS demonstrated superior diagnostic efficacy in mycobacterial infection,indicating its potential for clinical application.
基金supported by the National Key R&D Program of China(2019YFD1001300 and 2019YFD1001303)the Construction of Molecular Database of Faba Bean and Pea and Identification of Maize Germplasm Project,Ministry of Agriculture and Rural Affairs,China(19200030)+3 种基金the Yunnan Key R&D Program,China(202202AE090003)the earmarked fund for China Agriculture Research System(CARS-08)the Crop Germplasm Resources Protection(2130135)the Major Agricultural Science and Technology Program of Chinese Academy of Agricultural Sciences(CAAS-XTCX20190025)。
文摘Owing to the limitation of a large genome size(~13 Gb),the genetic and gene mapping studies on faba bean(Vicia faba L.)are lagging far behind those for other legumes.In this study,we selected three purified faba bean lines(Yundou 8137,H0003712,and H000572)as parents and constructed two F2 populations.These two F2 populations,namely 167 F2 plants in Pop1(Yundou 8137×H0003712)and 204 F2 plants in Pop2(H000572×Yundou 8137),were genotyped using a targeted next-generation sequencing(TNGS)genotyping platform,and two high-density single nucleotide polymorphisms(SNP)genetic linkage maps of faba bean were constructed.The map constructed from Pop1 contained 5103 SNPs with a length of 1333.31 cM and an average marker density of 0.26 cM.The map constructed from Pop2 contained 1904 SNPs with a greater length of 1610.61 cM.In these two F2 populations,QTL mapping identified 98 QTLs for 14 agronomic traits related to the flowers,pods,plant types and grains.The two maps were then merged into an integrated genetic linkage map containing 6895 SNPs,with a length of 3324.48 cM.These results not only lay the foundation for fine mapping and map-based cloning of related genes,but can also accelerate the molecular marker-assisted breeding of faba bean.
基金Quanzhou Science and Technology Bureau,No.2018Z072。
文摘BACKGROUND There are many factors that lead to dwarfism,and the mechanism has not yet been elucidated.Next-generation sequencing may identify candidate-related gene mutations,which may clarify the molecular cause.AIM To analyze genetic variation by using a constructed panel related to dwarfism by utilizing next-generation sequencing platform sequencing analysis to screen candidate-related gene mutations.METHODS Physical and laboratory characteristics,including clinical examination,growth hormone drug challenge test,serum insulin-like growth factor-1(IGF-1),IGF binding protein 3,other related tests,imaging examination,and chromosome karyotyping,were analyzed.Next-generation sequencing was performed to analyze pathogenicity variability.RESULTS In the 39 dwarfism patients,10 had pathogenicity variability.Gene variation was found in the OBSL1,SLC26A2,PTPN11,COL27AI,HDAC6,CUL7,FGFR3,DYNC2H1,GH1,and ATP7B genes.Of the 10 patients with pathogenicity variability,the related physical characteristics included double breast development and growth hormone deficiency,enuresis and indirect inguinal hernia on the left,two finger distance of 70.2 cm,head circumference of 49.2 cm,ischium/lower body length of 1.8 cm,weak limb muscles,and partial growth hormone deficiency.After 6 mo of growth hormone therapy,the concentrations of IGF-1 and IGF binding protein 3 increased from 215.2±170.3 to 285.0±166.0 and 3.9±1.4 to 4.2±1.1,respectively.CONCLUSION OBSL1,SLC26A2,PTPN11,COL27AI,HDAC6,CUL7,FGFR3,DYNC2H1,GH1,and ATP7B genes may be related to the incidence of dwarfism,and more research needs to be performed to elucidate the mechanism.
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partially supported by grants from the Ministry of Science and Technology of China(2016YFC1000306)the Beijing Municipal Science and Technology Commission Foundation(Z181100001918003)+1 种基金the Beijing Municipal Commission of Health and Family Planning Foundation(2018-21141,2020-4-1144)Beihang University&Capital Medical University Advanced Innovation Center for Big Data-Based Precision Medicine Plan(BHME-201905)。
文摘Different newborn screening(NBS) programs have been practiced in many countries since the 1960 s. It is of considerable interest whether next-generation sequencing is applicable in NBS. We have developed a panel of 465 causative genes for 596 early-onset, relatively high incidence, and potentially actionable severe inherited diseases in our Newborn Screening with Targeted Sequencing(NESTS) program to screen 11,484 babies in 8 Women and Children’s hospitals nationwide in China retrospectively. The positive rate from preliminary screening of NESTS was 7.85%(902/11,484). With 45.89%(414/902) follow-up of preliminary positive cases, the overall clinically confirmative diagnosis rate of monogenic disorders was 12.07%(50/414), estimating an average of 0.95%(7.85% × 12.07%) clinical diagnosis rate, suggesting that monogenic disorders account for a considerable proportion of birth defects. The disease/gene spectrum varied in different regions of China. NESTS was implemented in a hospital by screening 3923 newborns to evaluate its clinical application. The turn-around time of a primary report, including the sequencing period of < 7 days, was within 11 days by our automatic interpretation pipeline. Our results suggest that NESTS is feasible and cost-effective as a first-tier NBS program, which will change the status of current clinical practice of NBS in China.
文摘BACKGROUND The diagnostic value of metagenomic next-generation sequencing(mNGS)in central nervous system(CNS)infectious diseases after empirical treatment has not been reported.AIM To investigate the diagnostic value of mNGS of cerebrospinal fluid(CSF)in the empirically treated CNS infectious diseases.METHODS A total of 262 CSF samples from patients with suspected CNS infections were collected between August 2020 and December 2021.Both mNGS and conventional methods were used for testing.The conventional methods included microbial culture,smear,polymerase chain reaction,etc.RESULTS Among 262 suspected cases,183 cases(69.84%)were diagnosed as CNS infection,including 86 cases of virus infection(47.00%),70 cases of bacterial infection(38.25%)and 27 cases of fungal infection(14.76%).The sensitivity and specificity of mNGS were 65.6%(95%CI:58.2%-72.3%)and 89.6%(95%CI:79.1%-95.3%),respectively.The PPV of mNGS was 94.5%(95%CI:88.6%-97.6%),and the NPV was 48.8%(95%CI:39.7%–57.9%).The pathogen detective sensitivity and accuracy of mNGS were higher than those of conventional methods(Sensitivity:65.6%vs 37.2%;P<0.001;Accuracy:72.0%vs 50%,P<0.001).The results showed that compared with conventional methods,mNGS technology was a more sensitive method for the diagnosis of CNS infection after empirical treatment.CONCLUSION mNGS can be a better method applied in the diagnosis of CNS infection after empirical treatment.
基金Supported by the Science and Technology Foundation of Guangzhou,No.201803010059the Natural Science Foundation of Bengbu Medical College,No.BYKY2019129ZD.
文摘BACKGROUND Gastric cancer is a leading cause of cancer-related mortality worldwide.Many somatic mutations have been identified based on next-generation sequencing;they likely play a vital role in cancer treatment selection.However,nextgeneration sequencing has not been widely used to diagnose and treat gastric cancer in the clinic.AIM To test the mutant gene frequency as a guide for molecular diagnosis and personalized therapy in gastric cancer by use of next-generation sequencing.METHODS We constructed a panel of 24 mutant genes to detect somatic nucleotide variations and copy number variations based on a next-generation sequencing technique.Our custom panel included high-mutation frequency cancer driver and tumour suppressor genes.Mutated genes were also analyzed using the cBioPortal database.The clinical annotation of important variant mutation sites was evaluated in the ClinVar database.We searched for candidate drugs for targeted therapy and immunotherapy from the OncoKB database.RESULTS In our study,the top 16 frequently mutated genes were TP53(58%),ERBB2(28%),BRCA2(23%),NF1(19%),PIK3CA(14%),ATR(14%),MSH2(12%),FBXW7(12%),BMPR1A(12%),ERBB3(11%),ATM(9%),FGFR2(8%),MET(8%),PTEN(6%),CHD4(6%),and KRAS(5%).TP53 is a commonly mutated gene in gastric cancer and has a similar frequency to that in the cBioPortal database.33 gastric cancer patients(51.6%)with microsatellite stability and eight patients(12.5%)with microsatellite instability-high were investigated.Enrichment analyses demonstrated that high-frequency mutated genes had transmembrane receptor protein kinase activity.We discovered that BRCA2,PIK3CA,and FGFR2 gene mutations represent promising biomarkers in gastric cancer.CONCLUSION We developed a powerful panel of 24 genes with high frequencies of mutation that could detect common somatic mutations.The observed mutations provide potential targets for the clinical treatment of gastric cancer.
基金supported by the Health Commission of Shenzhen Municipality under the Sanming Project of Medicine(SZSM201911014)in Shenzhenthe High Level-Hospital Program,Health Commission of Guangdong Province,China.
文摘The expanding application of targeted next-generation sequencing(tNGS)in clinical diagnostics has led to an increasing frequency of Nocardia spp.detection in respiratory specimens;however,the clinical relevance and interpretation of these findings remain uncertain.This study describes the epidemiology of Nocardia pneumonia and colonization in patients at The University of Hong Kong–Shenzhen Hospital,China,and evaluates the diagnostic role of tNGS.The analysis was conducted from January 2023 through December 2024 and included 22 patients in whom Nocardia spp.were detected in respiratory specimens via tNGS.Among these patients,81.8%(18/22)had comorbidities,most commonly chronic lung disease(40.9%,9/22).Common clinical symptoms included cough,sputum and fever.Nocardia farcinica and Nocardia abscessus were the predominant species identified.Although the median number of se-quence reads of Nocardia were higher in infected patients than in colonized individuals(289,IQR:132.5–3747 vs.133.5,IQR:51.75–261.3),the difference was not statistically significant(P>0.05).In a comparison of diagnostic performance,tNGS identified Nocardia sequences in all 25 respiratory samples collected from the enrolled patients,whereas conventional aerobic culture success-fully isolated the pathogen in only two patients.Additionally,tNGS exhibited a significantly shorter median turnaround time than culture(38 h vs.376.5 h).These findings highlight tNGS as a rapid tool for Nocardia detection.However,the final diagnosis of nocardiosis can-not rely solely on sequence read thresholds.Clinical,radiological and laboratory integration remains critical in distinguishing true infection from colonization,ensuring accurate diagnosis and management of nocardiosis.
基金supported by the grants from the Tianjin science and technology innovation system construction project(No.07SYSYSF05000)the Tianjin Science and Technology Support Plan Key Project(No.06YFSZSF05300)+2 种基金the National Natural Science Foundation of China(No.81572288)the Key Project of International Cooperation of Science and Technology Innovation between Governmentsthe National Key Research and Development Plan of China(No.2016YEE0103400).
文摘Background:Molecular subtyping is an essential complementarity after pathological analyses for targeted therapy.This study aimed to investigate the consistency of next-generation sequencing(NGS)results between circulating tumor DNA(ctDNA)-based and tissue-based in non-small cell lung cancer(NSCLC)and identify the patient characteristics that favor ctDNA testing.Methods:Patients who diagnosed with NSCLC and received both ctDNA-and cancer tissue-based NGS before surgery or systemic treatment in Lung Cancer Center,Sichuan University West China Hospital between December 2017 and August 2022 were enrolled.A 425-cancer panel with a HiSeq 4000 NGS platform was used for NGS.The unweighted Cohen’s kappa coefficient was employed to discriminate the high-concordance group from the low-concordance group with a cutoff value of 0.6.Six machine learning models were used to identify patient characteristics that relate to high concordance between ctDNA-based and tissue-based NGS.Results:A total of 85 patients were enrolled,of which 22.4%(19/85)had stage III disease and 56.5%(48/85)had stage IV disease.Forty-four patients(51.8%)showed consistent gene mutation types between ctDNA-based and tissue-based NGS,while one patient(1.2%)tested negative in both approaches.Patients with advanced diseases and metastases to other organs would be suitable for the ctDNA-based NGS,and the generalized linear model showed that T stage,M stage,and tumor mutation burden were the critical discriminators to predict the consistency of results between ctDNA-based and tissue-based NGS.Conclusion:ctDNA-based NGS showed comparable detection performance in the targeted gene mutations compared with tissue-based NGS,and it could be considered in advanced or metastatic NSCLC.
基金Supported by National Natural Science Foundation of China(No.81470642No.81271045)
文摘AIM：To make a comprehensive analysis of the potential pathogenic genes related with Leber congenital amaurosis（LCA） in Chinese.METHODS：LCA subjects and their families were retrospectively collected from 2013 to 2015.Firstly,whole-exome sequencing was performed in patients who had underwent gene mutation screening with nothing found,and then homozygous sites was selected,candidate sites were annotated,and pathogenic analysis was conducted using softwares including Sorting Tolerant from Intolerant（SIFT）,Polyphen-2,Mutation assessor,Condel,and Functional Analysis through Hidden Markov Models（FATHMM）.Furthermore,Gene Ontology function and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses of pathogenic genes were performed followed by co-segregation analysis using Fisher exact Test.Sanger sequencing was used to validate single-nucleotide variations（SNVs）.Expanded verification was performed in the rest patients.RESULTS：Totally 51 LCA families with 53 patients and24 family members were recruited.A total of 104 SNVs（66 LCA-related genes and 15 co-segregated genes）were submitted for expand verification.The frequencies of homozygous mutation of KRT12 and CYP1A1 were simultaneously observed in 3 families.Enrichment analysis showed that the potential pathogenic genes were mainly enriched in functions related to cell adhesion,biological adhesion,retinoid metabolic process,and eye development biological adhesion.Additionally,WFS7 and STAU2 had the highest homozygous frequencies.CONCLUSION：LCA is a highly heterogeneous disease.Mutations in KRT12,CVP1A1,WFS1,and STAU2 may be involved in the development of LCA.
基金supported by the German Centre for Cardiovascular Research (DZHK), the European Union (FP7 Inheritance and FP7 Best Ageing), and the German Ministry of Education and Research (BMBF)
文摘Next-generation sequencing （NGS） is getting routinely used in the diagnosis of hereditary diseases, such as human cardiomyopathies. Hence. it is of utter importance to secure high quality sequencing data, enabling the identification of disease-relevant mutations or the conclusion of negative test results. During the process of sample preparation, each protocol for target enrichment library preparation has its own requirements for quality control （QC）; however, there is little evi- dence on the actual impact of these guidelines on resulting data quality. In this study, we analyzed the impact of QC during the diverse library preparation steps of Agilent SureSelect XT target enrichment and lllumina sequencing. We quantified the parameters for a cohort of around 600 samples, which include starting amount of DNA, amount of sheared DNA, smallest and largest fragment size of the starting DNA; amount of DNA after the pre-PCR, and smallest and largest fragment size of the resulting DNA; as well as the amount of the final library, the corresponding smallest and largest fragment size, and the number of detected variants. Intriguingly, there is a high tolerance for variations in all QC steps, meaning that within the boundaries proposed in the current study, a considerable variance at each step of QC can be well tolerated without compromising NGS quality.
基金supported by a Methusalem grant(BOF08/01M01108)from Ghent Universityfunded by Ghent University+1 种基金FWOthe Flemish Government-department EWI。
文摘Pseudogenes are frequently encountered noncoding sequences with a high sequence similarity to their protein-coding paralogue.For this reason,their presence is often considered troublesome in molecular diagnostics.In pseudoxanthoma elasticum(PXE),a disease predominantly caused by mutations in ATPbinding cassette family C member 6(ABCC6),the presence of two pseudogenes complicates the analysis of sequence data.With whole-exome sequencing(WES)becoming the standard of care in molecular diagnostics,we wanted to evaluate whether this technique is as reliable as gene-specific targeted enrichment analysis for the analysis of ABCC6.We established a PCR-based targeted enrichment and next-generation sequencing testing approach and demonstrated that the ABCC6-specific enrichment combined with the applied mapping algorithm overcomes the complication of ABCC6 pseudogene aspecificities,contrary to WES.We propose a time-and cost-efficient diagnostic strategy for comprehensive and accurate molecular genetic testing of PXE,which is highly automatable.
文摘Objective To screen gene mutation in adult-onset hypoparathyroidism in Chinese through the targeted nextgeneration sequencing(NGS).Methods We recruited 17patients with adult-onset hypoparathyroidism who were regularly followed or newly diagnosed at our centre during the past one year.Nine of them developed hypercalciuria during the treatment with calcium and vitamin D.
文摘Hepatocellular carcinoma(HCC)remains a global medical burden with rising incidence due to chronic viral hepatitis and non-alcoholic fatty liver diseases.Treatment of advanced disease stages is still unsatisfying.Besides first and second generation tyrosine kinase inhibitors,immune checkpoint inhibitors have become central for the treatment of HCC.New modalities like epigenetic therapy using histone deacetylase inhibitors(HDACi)and cell therapy approaches with chimeric antigen receptor T cells(CAR-T cells)are currently under investigation in clinical trials.Development of such novel drugs is closely linked to the availability and improvement of novel preclinical and animal models and the identification of predictive biomarkers.The current status of treatment options for advanced HCC,emerging novel therapeutic approaches and different preclinical models for HCC drug discovery and development are reviewed here.
