Construction of SNP genetic maps based on targeted next-generation sequencing and QTL mapping of vital agronomic traits in faba bean(Vicia faba L.)

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.

Study on pathogenic genes of dwarfism disease by next-generation sequencing

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.

Application of metagenomic next-generation sequencing in the diagnosis of infectious diseases of the central nervous system after empirical treatment

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.

Detection of a novel panel of 24 genes with high frequencies of mutation in gastric cancer based on next-generation sequencing

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.

Comprehensive analysis of genetic variations in strictly-defined Leber congenital amaurosis with whole-exome sequencing in Chinese

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.

Prevalence of gene mutations in a Chinese 46,XY disorders of sex development cohort detected by targeted next-generation sequencing

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.

The Role of Quality Control in Targeted Next-generation Sequencing Library Preparation

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.

A genomic study of adult-onset idiopathic hypoparathyroidism in Chinese by targeted next-generation sequencing

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.

病原体靶向测序技术在疑似肺部感染患者中的应用价值分析

目的 探讨病原体靶向测序(tNGS)技术在疑似肺部感染患者中的应用价值。方法 回顾性分析2021年1月-2023年7月湖北省第三人民医院呼吸与危重症医学科收治的80例疑似肺部感染患者的临床资料,所有患者均行支气管肺泡灌洗液(BALF)tNGS及常规病原学检测。分析患者人口学特征,比较t NGS与常规方法检测的病原体分布结果,并比较确诊肺部单一感染者与混合感染者的临床资料。结果 80例中,74例确诊感染。大部分感染者存在基础疾病,以慢性心脏疾病(42.5%)、慢性呼吸系统疾病(35.0%)和糖尿病(20.0%)为主。t NGS检测结果致35例患者(43.8%)的治疗策略改变。共检出45种病原体,其中tNGS检出病原体169株,常规方法检出病原体63株;两者联合检测出的病原体以细菌较多。tNGS检出的病原体株数排序:细菌>病毒>真菌>非典型病原体>人结核分枝杆菌。常规方法检出的病原体株数排序:真菌>病毒>细菌>非典型病原体>人结核分枝杆菌。两种方法检测结果的一致性较差(Kappa值0.172,P=0.020)。t NGS检出病原菌阳性比例及在细菌、病毒、非典型病原体的阳性检出率均明显高于常规方法(P<0.05),但在真菌、人结核分枝杆菌的阳性检出率与常规方法比较差异无统计学意义(P>0.05)。以临床诊断为金标准,tNGS检测的敏感度明显高于常规方法(P=0.026),而两者特异度比较差异无统计学意义(P>0.05)。在74例确诊肺部感染者中,6例无明确的病原体,单一感染者23例,混合感染者45例。在混合感染者中,常见的组合是细菌-病毒混合感染(12/45,26.7%)。混合感染者的病死率和住院天数均明显高于或长于单一感染者(P<0.05);两者性别、年龄、基础疾病、白细胞计数、中性粒细胞百分比等差异均无统计学意义(P>0.05)。结论 tNGS技术较常规方法的病原体检测敏感度高,尤其对细菌、病毒、非典型病原体及少见病原体更为敏感。此技术有利于识别混合感染,可作为临床常规病原学检测方法的补充。

靶向高通量测序技术(tNGS)在下呼吸道感染病原体检测中的诊断价值

目的探讨靶向高通量测序(Targeted Next-generation Sequencing,tNGS)对下呼吸道感染病原体检测的优势及指导治疗作用。方法选取2022年1月—2023年12月广西梧州人民医院收治的295例疑似下呼吸道感染患者为研究对象,所有患者均行痰或肺泡灌洗液tNGS与传统病原学检测(Traditional Pathogen Testing,CMTs),以CMTs为金标准,分析tNGS的灵敏度、准确度、特异度、阳性预测值及一致性。结果295例疑似下呼吸道感染患者的检出结果显示,tNGS检测的阳性检出结果为290例,CMTs的阳性检出结果为289例。tNGS的灵敏度为99.31%,准确度为98.98%,特异度为80.00%,阳性预测值为99.65%。kappa值为0.722,提示两种检测方法具有较高的一致性。结论tNGS技术在疑似下呼吸道感染患者中具有较高的诊断价值。

肺泡灌洗液病原靶向测序(BALF tNGS)对肺结核患者的临床应用价值研究

探究肺泡灌洗液病原靶向测序(BALF tNGS)对肺结核患者的临床应用价值。选取黔西南布依族苗族自治州人民医院2022年10月—2023年11月收治的90例肺结核患者作为研究对象,收集患者的肺泡灌洗液(BALF),分别进行BALF tNGS及抗酸杆菌涂片检查。比较病原靶向测序与常规涂片检查的肺结核检出情况,包括灵敏度、特异度、阳性率及阳性结果一致性。结果显示,90例患者经病原靶向测序检出肺结核患者82例,抗酸染色检查检出肺结核患者60例。BALF tNGS与抗酸染色涂片检查相比,其灵敏度为96.77%(60/62),特异度为21.43%(6/28);BALF tNGS病原体阳性率高于抗酸杆菌涂片检查,差异有统计学意义(P<0.05)。研究发现,BALF tNGS技术不仅可以检测到不同株系的肺结核病原体,还可以对其耐药性进行分析,为制订个体化治疗方案提供重要依据。

The impact of next-generation sequencing on genomics

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.

Newborn screening with targeted sequencing:a multicenter investigation and a pilot clinical study in China

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.

Hepatocellular carcinoma: Therapeutic advances in signaling,epigenetic and immune targets

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.

病原靶向二代测序在下呼吸道感染病原体诊断中应用价值研究进展

下呼吸道感染(LRTI)病原体复杂,对婴幼儿和老年人的健康产生严重威胁,早期明确病原体诊断是有效治疗的关键。传统LRTI诊断方法存在一定局限,包括低灵敏度、标本低特异度、周转时间较长,以及无法在单一标本中同时检测多种病原体,临床上迫切需要更精准的病原体检测技术。病原靶向二代测序(tNGS)不依赖传统微生物培养,将多重聚合酶链反应(PCR)和tNGS巧妙结合,采用多重PCR正向富集靶向病原体,提高检测的灵敏度,同时排除宿主核酸的影响,实现不同器官感染、不同标本类型等关键病原体的鉴定。该文对近年来国内外关于tNGS在LRTI病原体鉴定中的应用作一综述,分析其在不同病原体中的检测效能、优势及局限性,以确保tNGS在临床诊断中能够得到适当应用。

疑似肺炎患者BALF样本应用tNGS技术进行病原学诊断的价值研究

目的 探究靶向高通量测序(targeted next-generation sequencing,tNGS)技术在疑似肺炎患者支气管肺泡灌洗液(bronchoalveolar lavage fluid,BALF)样本中的病原学诊断价值。方法 选取2022年7月~2023年3月武汉亚心总医院收治的102例疑似肺炎患者的102份BALF样本,对其同时进行了tNGS检测和传统微生物学检测,并收集患者的一般临床资料。对纳入研究的102例患者的tNGS检测结果、微生物培养结果以及其他实验室检查结果进行回顾性分析。结果 tNGS法的总体阳性检出率(93.14%)高于传统方法(58.82%),差异有统计学意义(χ^(2)=32.903,P<0.001);t NGS法在细菌、病毒以及混合感染方面的检出率(65.69%,55.89%,69.61%)高于传统方法(21.57%,6.86%,11.76%),差异有统计学意义(χ^(2)=32.903,56.920,70.708,均P<0.001)。结论 tNGS技术在疑似肺炎患者BALF样本中获得了较高的病原体检出率,可以有效应用于呼吸道感染性疾病的病原学诊断。

Potential unreliability of ALK variant allele frequency in the efficacy prediction of targeted therapy in NSCLC

Anaplastic lymphoma kinase(ALK)is the most common fusion gene involved in non-small cell lung cancer(NSCLC),and remarkable response has been achieved with the use of ALK tyrosine kinase inhibitors(ALK-TKIs).However,the clinical efficacy is highly variable.Pre-existing intratumoral heterogeneity(ITH)has been proven to contribute to the poor treatment response and the resistance to targeted therapies.In this work,we investigated whether the variant allele frequencies(VAFs)of ALK fusions can help assess ITH and predict targeted therapy efficacy.Through the application of next-generation sequencing(NGS),7.2%(326/4548)of patients were detected to be ALK positive.On the basis of the adjusted VAF(adjVAF,VAF normalization for tumor purity)of four different threshold values(adjVAF<50%,40%,30%,or 20%),the association of ALK subclonality with crizotinib efficacy was assessed.Nonetheless,no statistical association was observed between median progression-free survival(PFS)and ALK subclonality assessed by adjVAF,and a poor correlation of adjVAF with PFS was found among the 85 patients who received first-line crizotinib.Results suggest that the ALK VAF determined by hybrid capture-based NGS is probably unreliable for ITH assessment and targeted therapy efficacy prediction in NSCLC.

Hereditary cancer syndromes

Hereditary cancer syndromes(HCSs)are arguably the most frequent category of Mendelian genetic diseases,as at least 2%of presumably healthy subjects carry highly-penetrant tumor-predisposing pathogenic variants(PVs).Hereditary breast-ovarian cancer and Lynch syndrome make the highest contribution to cancer morbidity;in addition,there are several dozen less frequent types of familial tumors.The development of the majority albeit not all hereditary malignancies involves two-hit mechanism,i.e.the somatic inactivation of the remaining copy of the affected gene.Earlier studies on cancer families suggested nearly fatal penetrance for the majority of HCS genes;however,population-based investigations and especially large-scale next-generation sequencing data sets demonstrate that the presence of some highly-penetrant PVs is often compatible with healthy status.Hereditary cancer research initially focused mainly on cancer detection and prevention.Recent studies identified multiple HCS-specific drug vulnerabilities,which translated into the development of highly efficient therapeutic options.

Integration of molecular testing for the personalized management of patients with diffuse large B-cell lymphoma and follicular lymphoma

Diffuse large B-cell lymphoma(DLBCL)and follicular lymphoma(FL)are the most common forms of aggressive and indolent lymphoma,respectively.The majority of patients are cured by standard R-CHOP immunochemotherapy,but 30%–40%of DLBCL and 20%of FL patients relapse or are refractory(R/R).DLBCL and FL are phenotypically and genetically hereterogenous B-cell neoplasms.To date,the diagnosis of DLBCL and FL has been based on morphology,immunophenotyping and cytogenetics.However,next-generation sequencing(NGS)is widening our understanding of the genetic basis of the B-cell lymphomas.In this review we will discuss how integrating the NGS-based characterization of somatic gene mutations with diagnostic or prognostic value in DLBCL and FL could help refine B-cell lymphoma classification as part of a multidisciplinary pathology work-up.We will also discuss how molecular testing can identify candidates for clinical trials with targeted therapies and help predict therapeutic outcome to currently available treatments,including chimeric antigen receptor T-cell,as well as explore the application of circulating cell-free DNA,a non-invasive method for patient monitoring.We conclude that molecular analyses can drive improvements in patient outcomes due to an increased understanding of the different pathogenic pathways affected by each DLBCL subtype and indolent FL vs R/R FL.

古代病原微生物基因组的研究进展

古代病原微生物基因组研究对病理学、微生物学、考古学等领域均具有重要的价值。在过去的十年里,高通量测序和靶向富集技术的发展和应用使古代微生物基因组的获取成为可能,通过对古代人群样本中获取的宏基因组进行筛查,使得引发古代疫情的相关病原体的基因组得以重建,为研究人类传染病的起源、传播和演化提供了一个独特的窗口。在当今全球化的背景下,新发及再发传染性疾病的出现频率促使我们回顾过去,以便更好地了解现代病原菌出现和古代病原菌重新出现的过程和生态环境。在这篇文章中,我们总结了近十年古代病原微生物基因组水平的研究进展,并提出了这项研究所面临的挑战以及未来的研究前景和方向。