我国首次完成辛德毕斯病毒全基因组测序

我国首株辛德毕斯病毒 (ＸＪ - 16 0病毒株 )全基因组序列测定 ,现已由中国预防医学科学院病毒学研究所研究员梁国栋等完成 ,并已为国际基因库所录入 .此研究首次从分子学水平上证明了我国存在引起发热甚至脑炎的新病原 ,并查明了其传入路线 .辛德毕斯病毒是 195 2年首先在中东地区分离到的一种虫媒病毒 ,通过蚊虫叮咬感染人体 .我国 1990年夏从新疆伊梨捕获的按蚊中首次分离到这种病毒的毒株 .为阐明该病毒基因组与国外病毒株的差异 ,了解其来源、分类及致病的分子基础 ,在国家自然科学基金资助下 ,科研人员近两年来对该毒株展开全基因组核…     

单细胞全基因组测序分析体外受精三原核胚胎染色体

目的 应用单细胞全基因组测序分析体外受精(IVF)三原核(3PN)胚胎染色体.方法 选择2019年9月-2020年12月在本中心行IVF助孕的患者,治疗周期中废弃的3PN受精卵发育为胚胎,应用单细胞全基因组测序分析3PN胚胎染色体及拷贝数变异(CNV).结果 对3PN受精卵发育的11枚胚胎进行染色体核型和CNV检测,染...     

全基因组测序在环境样本新型冠状病毒检测中的应用

目的:通过对环境样本进行全基因组测序,为全基因组测序在检测环境标本中SARS-CoV-2的应用提供实践基础,为新冠肺炎疫情的溯源和防控工作提供科学依据.方法:采用新冠病毒全基因组靶向扩增结合Illumina二代测序的技术对5例SARS-CoV-2核酸阳性环境样本进行全基因组测序,应用多种分析软件及在线病毒变异分析平台,...     

快速低成本全基因组DNA测序技术

人类个体的全基因组序列信息，是最为重要的生物医学信息，是实现未来个体化医疗的基础；实现个体全基因组DNA序列的快速低成本检测，是人类基因组计划完成后又一个重要的技术挑战。对该技术当前的发展现状进行回顾和分析，预计在不长的时间内人类个体全基因组的测序成本能够降低到1万元人民币以下。     

宏基因组测序在微生物耐药研究中的应用进展

伴随广谱抗生素的过度使用,临床上多重耐药病原菌和超级细菌不断出现,为临床抗感染治疗带来了严峻挑战,传统微生物培养后药敏实验以及针对于特定耐药基因的基因扩增等方法已经无法满足现有需要,因而迫切需要新的研究方法的出现。宏基因组测序技术以其无偏差、高覆盖为微生物耐药研究提供了又一有利工具,本文对宏基因组测序在肠道、皮肤、组织和重症下呼吸道感染中微生物耐药研究的最新应用做一综述,以期为临床微生物耐药研究提供帮助。     

全基因组测序用于肾脏异常胎儿的产前诊断CSCD

目的应用全基因组测序技术对4例肾脏异常胎儿进行遗传学检测,以寻找其可能的遗传学病因。方法对4例肾脏异常胎儿的孕妇抽取羊水及胎儿父母外周血进行DNA提取,行全基因组测序检测,根据美国医学遗传学与基因组学学会(American College of Medical Genetics and Genomics,ACMG)原则对数据进行判定,拷贝数变异结果与SNP-array结果进行比对,致病性点变异行Sanger测序验证。结果全基因组测序技术检测2例胎儿为拷贝数变异致病,分别为染色体17q12缺失1.45 Mb和1q21.1-21.2重复1.85 Mb;2例胎儿为基因变异致病,分别为PKHD1 c.8301del(p.Asn2768Thr fs*18)和c.4481del(p.Asn1494Thrfs*6)复合杂合变异和BBS12:c.1372dup(p.Thr458Asnfs*5)纯合变异。SNP-array和Sanger测序结果与全基因组测序数据一致。根据ACMG遗传变异分类标准与指南,PKHD1 c.8301del(p.Asn2768Thr fs*18)和c.4481del(p.Asn1494Thrfs*6)变异均为致病性变异(PVS1+PM2+PM3+PP4),BBS12:c.1372dup(p.Thr458Asnfs*5)变异判定为可能致病性变异(PVS1+PM2)。结论全基因组测序技术可以高效快速的为产前肾脏异常胎儿提供遗传学诊断,并为遗传咨询提供依据。     

全基因组外显子测序及其在遗传病研究中的应用

全基因组外显子测序是指利用序列捕获技术将全基因组外显子区域DNA捕捉并富集,然后进行高通量测序的基因组分析方法.与传统测序方式相比,该技术具有耗时短、成本低、通量大,对样品的要求量少等特点,已经成为人们研究某些疾病致病基因的重要方法.此文就全基因组外显子测序的优缺点及其在遗传病中的应用作简要综述.     

我国首例输入性裂谷热病例病毒全基因组测序分析

目的 对我国首例输入性裂谷热病例病毒进行全基因组测定,分析其进化来源及潜在变异.方法 提取样本核酸,非特异性反转录扩增病毒基因组RNA,使用Ion Torrent二代测序仪进行病毒全基因组测定.对获得的基因组数据进行序列拼接、比对、进化树构建和关键位点分析.结果 通过测定获得了病毒全基因组11 979nt,该测定病毒属E基因分支,序列与先前南非分离株Kakamas相似度最高(＞98％).病毒Gn蛋白C端信号肽区存在1个氨基酸突变.结论 本研究分析测定的裂谷热病毒全基因组与目前非洲流行株高度相似,病毒基因特征未出现明显变异.     

基于高通量测序技术的发热伴血小板减少综合征病毒全基因组多重PCR测序方法的建立与评价

目的基于二代测序(next generation sequencing, NGS)技术建立一种发热伴血小板减少综合征病毒(severe fever with thrombocytopenia syndrome virus, SFTSV)基因组的测序方法。方法提取SFTSV阳性血清样本病毒核酸, 利用Primer5.0软件设计SFTSV特异性引物, 构建多重PCR方法对病毒核苷酸序列进行特异性扩增, 利用二代测序仪进行全基因组测序。结果 28份SFTSV阳性血清样本均能进行特异性扩增并获得全序, 覆盖度均超过94%, A型病毒株最多(20株)、其次为B型(4株)、E型(3株)。结论本研究基于多重PCR建立了针对SFTSV的特异性强、质量好的高通量测序方法, 提高了测序效率。     

Individual identification by DNA polymorphism using formalin-fixed placenta with whole genome amplification

Polymerase chain reaction (PCR) amplification using formalin-fixed material is very limited. In the present study the use of 6 week formalin-fixed placenta for individual identification was examined based on DNA analyses. The objective of the examination was to prove whether the placenta was from a woman who had just given birth. DNA extraction was carried out from the maternal blood sample and from the formalin-fixed placental samples composed of three parts: maternal side, infant side and umbilical cord. One minisatellite (D1S80), 12 short tandem repeat (STR) polymorphisms and amelogenin X, Y were investigated. All the polymorphic systems were detected in the maternal blood sample. The majority of the DNA isolated from the placental tissues had molecular weights of approximately 500 bp, and only two to four STR loci were amplified using the DNA. In order to amplify more DNA polymorphic markers from the formalin-fixed tissues, whole genome amplification was performed. After amplification by degenerate oligonucleotide-primed PCR (DOP-PCR), the products contained DNA with increased molecular weight up to >10 kbp. More DNA loci were typed using the DOP-PCR products. Furthermore, large molecular size fragments were purified from the DOP-PCR products by agarose electrophoresis, and then the D1S80 locus and 12 STR loci were successfully amplified using these fragments.     

Insights on biology and evolution from microbial genome sequencing

No field of research has embraced and applied genomic technology more than the field of microbiology. Comparative analysis of nearly 300 microbial species has demonstrated that the microbial genome is a dynamic entity shaped by multiple forces. Microbial genomics has provided a foundation for a broad range of applications, from understanding basic biological processes, host-pathogen interactions, and protein-protein interactions, to discovering DNA variations that can be used in genotyping or forensic analyses, the design of novel antimicrobial compounds and vaccines, and the engineering of microbes for industrial applications. Most recently, metagenomics approaches are allowing us to begin to probe complex microbial communities for the first time, and they hold great promise in helping to unravel the relationships between microbial species.     

Next-generation sequencing technology in clinical virology

Recent advances in nucleic acid sequencing technologies, referred to as ‘next-generation’ sequencing (NGS), have produced a true revolution and opened new perspectives for research and diagnostic applications, owing to the high speed and throughput of data generation. So far, NGS has been applied to metagenomics-based strategies for the discovery of novel viruses and the characterization of viral communities. Additional applications include whole viral genome sequencing, detection of viral genome variability, and the study of viral dynamics. These applications are particularly suitable for viruses such as human immunodeficiency virus, hepatitis B virus, and hepatitis C virus, whose error-prone replication machinery, combined with the high replication rate, results, in each infected individual, in the formation of many genetically related viral variants referred to as quasi-species. The viral quasi-species, in turn, represents the substrate for the selective pressure exerted by the immune system or by antiviral drugs. With traditional approaches, it is difficult to detect and quantify minority genomes present in viral quasi-species that, in fact, may have biological and clinical relevance. NGS provides, for each patient, a dataset of clonal sequences that is some order of magnitude higher than those obtained with conventional approaches. Hence, NGS is an extremely powerful tool with which to investigate previously inaccessible aspects of viral dynamics, such as the contribution of different viral reservoirs to replicating virus in the course of the natural history of the infection, co-receptor usage in minority viral populations harboured by different cell lineages, the dynamics of development of drug resistance, and the re-emergence of hidden genomes after treatment interruptions. The diagnostic application of NGS is just around the corner.     

The efficiency of single genome amplification and sequencing is improved by quantitation and use of a bioinformatics tool

Typically, population-based sequencing of HIV does not detect minority variants present at levels below 20–30%. Single genome amplification (SGA) and sequencing improves detection, but it requires many PCRs to find the optimal terminal dilution to use. A novel method for guiding the selection of a terminal dilution was developed and compared to standard methods.A quantitative real-time PCR (qRT-PCR) protocol was developed. HIV RNA was extracted, reverse transcribed, and quantitated. A bioinformatics web-based application was created for calculating the optimal concentration of cDNA to use based on results of a trial PCR using the dilution suggested by the qRT-PCR results. This method was compared to the standard.Using the standard protocol, the mean number of PCRs giving an average of 30 (26–34, SD = 3) SGA per sample was 245 (218–266, SD = 20) after an average of 8 trial dilutions. Using this method, 135 PCRs (135–135, SD = 0) produced 30 (27–30, SD = 1) SGA using exactly two dilutions. This new method reduced turnaround time from 8 to 2 days.Standard methods of SGA sequencing can be costly and both time- and labor-intensive. By choosing a terminal dilution concentration with the proposed method, the number of PCRs required is decreased and efficiency improved.     

Public views on participating in newborn screening using genome sequencing

Growing discussion on the use of whole-genome or exome sequencing (WG/ES) in newborn screening (NBS) has raised concerns regarding the generation of incidental information on millions of infants annually. It is unknown whether integrating WG/ES would alter public expectations regarding participation in universal NBS. We assessed public willingness to participate in NBS using WG/ES compared with current NBS. Our secondary objective was to assess the public''s beliefs regarding a parental responsibility to participate in WG/ES-based NBS compared with current NBS. We examined self-reported attitudes regarding willingness to participate in NBS using a cross-sectional national survey of Canadian residents recruited through an internet panel, reflective of the Canadian population by age, gender and region. Our results showed that fewer respondents would be willing to participate in NBS using WG/ES compared with NBS using current technologies (80 vs 94%, P<0.001), or perceived a parental responsibility to participate in WG/ES-based NBS vs current NBS (30 vs 48%, P<0.001). Our findings suggest that integrating WG/ES into NBS might reduce participation, and challenge the moral authority that NBS programmes rely upon to ensure population benefits. These findings point to the need for caution in the untargeted use of WG/ES in public health contexts.     

Personal genome sequencing: current approaches and challenges

The revolution in DNA sequencing technologies has now made it feasible to determine the genome sequences of many individuals; i.e., “personal genomes.” Genome sequences of cells and tissues from both normal and disease states have been determined. Using current approaches, whole human genome sequences are not typically assembled and determined de novo, but, instead, variations relative to a reference sequence are identified. We discuss the current state of personal genome sequencing, the main steps involved in determining a genome sequence (i.e., identifying single-nucleotide polymorphisms [SNPs] and structural variations [SVs], assembling new sequences, and phasing haplotypes), and the challenges and performance metrics for evaluating the accuracy of the reconstruction. Finally, we consider the possible individual and societal benefits of personal genome sequences.     

Effects of informed consent for individual genome sequencing on relevant knowledge

Kaphingst KA, Facio FM, Cheng M‐R, Brooks S, Eidem H, Linn A, Biesecker BB, Biesecker LG. Effects of informed consent for individual genome sequencing on relevant knowledge. Increasing availability of individual genomic information suggests that patients will need knowledge about genome sequencing to make informed decisions, but prior research is limited. In this study, we examined genome sequencing knowledge before and after informed consent among 311 participants enrolled in the ClinSeq? sequencing study. An exploratory factor analysis of knowledge items yielded two factors (sequencing limitations knowledge; sequencing benefits knowledge). In multivariable analysis, high pre‐consent sequencing limitations knowledge scores were significantly related to education [odds ratio (OR): 8.7, 95% confidence interval (CI): 2.45–31.10 for post‐graduate education, and OR: 3.9; 95% CI: 1.05, 14.61 for college degree compared with less than college degree] and race/ethnicity (OR: 2.4, 95% CI: 1.09, 5.38 for non‐Hispanic Whites compared with other racial/ethnic groups). Mean values increased significantly between pre‐ and post‐consent for the sequencing limitations knowledge subscale (6.9–7.7, p < 0.0001) and sequencing benefits knowledge subscale (7.0–7.5, p < 0.0001); increase in knowledge did not differ by sociodemographic characteristics. This study highlights gaps in genome sequencing knowledge and underscores the need to target educational efforts toward participants with less education or from minority racial/ethnic groups. The informed consent process improved genome sequencing knowledge. Future studies could examine how genome sequencing knowledge influences informed decision making.