Sequence analysis of the entire mitochondrial genome in Parkinson's disease

The pathogenesis of Parkinson's disease (PD) is largely unknown. Indirect evidence suggests that mutations in mitochondrial DNA (mtDNA) might play a role, but previous studies have not consistently associated any specific mutations with PD. However, these studies have generally been confined to limited areas of the mitochondrial genome. We therefore sequenced the entire mitochondrial genome from substantia nigra of 8 PD and 9 control subjects. Several sequence variants were distributed differently between PD and control subjects, but all were previously reported polymorphisms. Several secondary LHON mutations were found, as well as a number of novel missense mutations, but all were rare and did not differ between PD and control subjects. Finally, PD and control subjects did not differ in the total number of all mutations, nor the total number of missense mutations. Thus, mtDNA involvement in PD, if any, is likely to be complex and should be reconsidered carefully.     

根际微生物与植物再植病的发生发展关系

植物再植病严重危害农作物的生长发育,形成原因复杂,最新的研究显示它与根际微生态的变化相关,其中主要涉及根际微生物菌群结构的变化。现代高通量测序技术的迅速推广使得根际微生物和再植病间关系成为新的研究热点;本文根据国内外植物再植病病因、根际土壤微生物多样性以及它们与植物生长发育关系等多方面的研究成果,综合分析植物再植病与根际微生物间的关系,以期从根际微生物种群动态变化的角度认识植物再植病的病因并为防治提供新的思路。     

外显子组测序技术在人类疾病研究中的应用

外显子组测序是针对基因组中的蛋白质编码区,靶向富集外显子区域测序,以发现疾病相关遗传变异的技术。该技术近年越来越多地应用于发现人类基因组低频变异、鉴定单基因遗传病致病基因和肿瘤等复杂疾病易感基因研究,成为人类疾病相关变异研究的重要工具。综述了外显子组测序技术的基本原理及其在人类疾病相关基因研究中的应用。     

外显子组测序在人类疾病中的应用

据估计,约85%的人类遗传变异集中在蛋白编码区,因此对全部的蛋白编码区（外显子组）进行重测序,可以快速、有效地鉴定人类疾病遗传变异。以往鉴定孟德尔遗传病的致病基因多采用连锁分析结合候选定位克隆的方法,不仅耗时长,而且成功率低。2009年,科学家第一次应用外显子组测序在4名弗里曼谢尔登综合征（常染色体显性遗传病）中发现了位于MYH3中的点突变,显示出外显子组测序在孟德尔遗传病致病基因鉴定中的强大功效。就复杂疾病而言,传统的关联研究,包括全基因组关联研究（GWAS）,虽然鉴定了大量的常见变异,但对低频变异和罕见变异的检测能力十分有限;深度测序的发展为解决上述问题提供了良好的契机。本文就外显子组测序在人类疾病中的应用作一简要综述。     

Alternations in the gut microbiota and metabolome with newly diagnosed unstable angina

Gut microbiota plays an important role in coronary heart disease, but its compositional and functional changes in unstable angina (UA) remain unexplored. We performed metagenomic sequencing of 133 newly diagnosed UA patients and 133 sex- and age-matched controls, and profiled the fecal and plasma metabolomes in 30 case-control pairs. The alpha diversity of gut microbiota was increased in UA patients: the adjusted odds ratios (ORs) per standard deviation increase in Shannon and Simpson indices were 1.30 (95% confidence interval, 1.01–1.70) and 1.36 (1.05–1.81), respectively. Two common species (depleted Klebsiella pneumoniae and enriched Streptococcus parasanguinis; P ≤ 0.002) and three rare species (depleted Weissella confusa, enriched Granulicatella adiacens and Erysipelotrichaceae bacterium 6_1_45; P ≤ 0.005) were associated with UA. The UA-associated gut microbiota was depleted in the pathway of L-phenylalanine degradation (P = 0.001), primarily contributed by Klebsiella pneumoniae. Consistently, we found increased circulating phenylalanine in UA patients (OR = 2.76 [1.17–8.16]). Moreover, Streptococcus parasanguinis was negatively correlated with fecal citrulline (Spearman's r_s = ?0.470, P = 0.009), a metabolite depleted in UA patients (OR = 0.26 [0.08–0.63]). These findings are informative to help understand the metabolic connection between gut microbiota and UA.     

Whole-genome sequencing as a first-tier diagnostic framework for rare genetic diseases

Rare diseases affect nearly 300 million people globally with most patients aged five or less. Traditional diagnostic approaches have provided much of the diagnosis; however, there are limitations. For instance, simply inadequate and untimely diagnosis adversely affects both the patient and their families. This review advocates the use of whole genome sequencing in clinical settings for diagnosis of rare genetic diseases by showcasing five case studies. These examples specifically describe the utilization of whole genome sequencing, which helped in providing relief to patients via correct diagnosis followed by use of precision medicine.     

三株新城疫病毒强毒株的生物学特性及全基因组序列分析

2009～2011年从北方发病鸡群和鸭群中分离出3株新城疫病毒（Newcastle disease virus,NDV）。通过致病性指数测定及交叉血凝抑制试验初步分析了3个毒株的毒力和相互之间的同源性。选取鸡源分离株SDLY01与新城疫疫苗株（LaSota）进行了交叉保护试验,选取鸭源毒株SD03对樱桃谷鸭进行攻毒实验,同时设计引物对3个毒株进行了全基因组测序,并与36株NDV参考株进行了分子进化分析。结果表明3个分离株F蛋白裂解位点的氨基酸序列均为112R-R-Q-K-R-F117符合强毒株的序列特征,并与致病性指数测定结果相符。交叉血凝抑制试验发现3个分离株与疫苗株LaSota 的抗原同源性较低为82.5%～89.4%,两个鸡源分离株间的抗原同源性为90%,而鸭源毒株SD03与鸡源毒株SDSG01同源性为100%。交叉保护试验和攻毒实验结果显示传统的LaSota疫苗能对SDLY01流行株提供100%免疫保护,但第5天仍检测到排毒;鸭源毒株SD03对樱桃谷鸭不致病,但能检出排毒,排毒期最长为5d。全基因组测序与分析表明3个毒株基因组长度均为15192bp,属于基因Ⅶd型毒株,与同期流行的鹅源及鸭源NDV毒株之间全基因组核苷酸序列具有高度的同源性,揭示鸭源、鹅源NDV与鸡源NDV在遗传学和流行病学上密切相关。     

Elucidating cryptic dynamics of Theileria communities in African buffalo using a high‐throughput sequencing informatics approach

1. Increasing access to next‐generation sequencing (NGS) technologies is revolutionizing the life sciences. In disease ecology, NGS‐based methods have the potential to provide higher‐resolution data on communities of parasites found in individual hosts as well as host populations.
2. Here, we demonstrate how a novel analytical method, utilizing high‐throughput sequencing of PCR amplicons, can be used to explore variation in blood‐borne parasite (Theileria—Apicomplexa: Piroplasmida) communities of African buffalo at higher resolutions than has been obtained with conventional molecular tools.
3. Results reveal temporal patterns of synchronized and opposite fluctuations of prevalence and relative abundance of Theileria spp. within the host population, suggesting heterogeneous transmission across taxa. Furthermore, we show that the community composition of Theileria spp. and their subtypes varies considerably between buffalo, with differences in composition reflected in mean and variance of overall parasitemia, thereby showing potential to elucidate previously unexplained contrasts in infection outcomes for host individuals.
4. Importantly, our methods are generalizable as they can be utilized to describe blood‐borne parasite communities in any host species. Furthermore, our methodological framework can be adapted to any parasite system given the appropriate genetic marker.
5. The findings of this study demonstrate how a novel NGS‐based analytical approach can provide fine‐scale, quantitative data, unlocking opportunities for discovery in disease ecology.

     

养殖牙鲆淋巴囊肿病病原的研究

近几年我国海水养殖鱼发生病毒性传染病,病鱼鳍、鳃及体表皮肤出现乳头瘤样赘生物。将发病牙鲆（Ｐａｒａｌｉｃｈｔｈｙｓ ｏｌｉｖａｃｅｕｓ）表皮瘤组织进行超薄切片,电镜下在病变组织明显肥大的细胞胞浆内一球状病毒,呈二十面体对称,具包膜,直径约２１０ｎｍ。根据虹彩病毒主要衣壳蛋白（ｍａｊｏｒ ｃａｐｓｉｄ ｐｒｏｓｅｉｎ,ＭＣＰ）基因中间保守区序列设计简并引物,应用ＰＣＲ方法从病变组织中扩增出长６３６ｂ     

宏基因组测序在传染性疾病中的研究进展

微生物和人类已经共同进化几百万年了,微生物在维持宿主健康方面起到了非常重要的作用。随着下一代测序技术的进步,可以获得人体在不同环境下微生物群落的特征。本文综述了当前感染各种不同病原菌时复杂微生物群落的变化,病原菌包括HIV、乙肝病毒、流感病毒和结核分支杆菌,以及不同的身体部位。我们相信,增加对传染性疾病和微生物群落变化之间关系的认识,能够更好地管理疾病进展。然而,将来的研究可能需要更加整体化,通过分析人体宿主微生物与传染性疾病的发生机制之间的关系,以建立疾病的确切因果关系。     

基因诊断中测序技术的应用及优缺点

基因突变是当今生命科学研究的热点之一,其检测方法和诊断技术发展迅猛。测序技术在基因病的确诊、分型等方面发挥着不可或缺的作用。文章重点围绕第一~三代测序技术的研究进展、优缺点及其在基因诊断中的应用进行评述 ,并对未来的发展趋势进行了预测和展望。     

单细胞转录组测序技术在心脏发育、疾病以及医学中的 应用

单细胞转录组测序(Single-cell RNA sequencing,scRNA-seq)可以在单细胞水平描绘出每个细胞同一基因的表达量在不同细胞间的表达水平差异,使得在单细胞水平重新认识各种组织器官成为可能.目前对心脏的测序研究正从传统的普通转录组水平过渡到单细胞水平,对小鼠和人的心脏的测序陆续地发表出来.概述了s...     

慢性阻塞性肺疾病患者肠道微生物组成及基因功能分析

探讨慢性阻塞性肺疾病（COPD）患者肠道微生物组成、丰度及差异基因功能变化。

收集我院呼吸科10例确诊COPD患者（COPD组）和10例对照受试者（对照组）粪便样品进行宏基因组高通量测序分析。

PCA分析组间比较发现2组研究对象粪便样品微生物群落差异大,具有可比性。物种组成分析显示：对照组粪便样品中高度富集的菌科主要包括毛螺菌科、理研菌科、韦荣球菌科、优杆菌科、臭杆菌科、氨基酸球菌科、乳杆菌科等,属层面富集的主要包括罗斯菌属、胃瘤球菌属、小杆菌属、真杆菌属、拟杆菌属、粪球菌属、双歧杆菌属等;COPD组粪便样品富集菌群主要包括紫单胞菌科、Selenomonadaceae、巨单胞菌属和韦荣球菌属。物种多样性分析与对照组相比,COPD组多样性更低,差异有统计学意义（W = 87,P = 0.0039）。物种相关性网络图分析显示与其他门类菌群相比,拟杆菌门、厚壁菌门、变形菌门与其他物种的关联性最强（size = 52.07、18.87、14.01）。组间差异基因进行GO功能显著性富集分析,差异基因均富集在如细胞学过程、刺激应答、生物学黏附及调节、代谢过程及信号通路等。KEGG功能显著性富集分析显示差异基因主要富集途径包括代谢途径、次生代谢物合成、淀粉和蔗糖的合成、抗生素的合成等。

COPD患者肠道内存在菌群及基因功能失调。

     

A universal protocol to generate consensus level genome sequences for foot-and-mouth disease virus and other positive-sense polyadenylated RNA viruses using the Illumina MiSeq

Background

Next-Generation Sequencing (NGS) is revolutionizing molecular epidemiology by providing new approaches to undertake whole genome sequencing (WGS) in diagnostic settings for a variety of human and veterinary pathogens. Previous sequencing protocols have been subject to biases such as those encountered during PCR amplification and cell culture, or are restricted by the need for large quantities of starting material. We describe here a simple and robust methodology for the generation of whole genome sequences on the Illumina MiSeq. This protocol is specific for foot-and-mouth disease virus (FMDV) or other polyadenylated RNA viruses and circumvents both the use of PCR and the requirement for large amounts of initial template.

Results

The protocol was successfully validated using five FMDV positive clinical samples from the 2001 epidemic in the United Kingdom, as well as a panel of representative viruses from all seven serotypes. In addition, this protocol was successfully used to recover 94% of an FMDV genome that had previously been identified as cell culture negative. Genome sequences from three other non-FMDV polyadenylated RNA viruses (EMCV, ERAV, VESV) were also obtained with minor protocol amendments. We calculated that a minimum coverage depth of 22 reads was required to produce an accurate consensus sequence for FMDV O. This was achieved in 5 FMDV/O/UKG isolates and the type O FMDV from the serotype panel with the exception of the 5′ genomic termini and area immediately flanking the poly(C) region.

Conclusions

We have developed a universal WGS method for FMDV and other polyadenylated RNA viruses. This method works successfully from a limited quantity of starting material and eliminates the requirement for genome-specific PCR amplification. This protocol has the potential to generate consensus-level sequences within a routine high-throughput diagnostic environment.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-828) contains supplementary material, which is available to authorized users.     

基于新一代高通量技术的人类疾病组学研究策略

近年来,包括第二代测序技术和蛋白质谱技术等在内的新一代高通量技术越来越多的应用于解决生物学问题尤其是人类疾病的研究。这种以数据为导向,大规模、工业化的研究模式,使得从基因组水平、转录组水平、蛋白质组水平等角度对疾病展开全方位、多层次的研究成为可能。文章综述了新一代高通量技术在DNA、RNA、表观遗传、宏基因组和蛋白质组水平的人类疾病研究进展以及在转化医学领域的应用。在基因组水平上,外显子组测序是近年来持续的研究热点,随着测序成本的不断降低,全基因组重测序也越来越凸显了其在全基因组范围内检测大型结构变异的优势,并使得个人基因组引领的个体化医疗逐渐成为可能。在转录组水平,如小RNA测序技术可用来检测已知小RNA和预测新的小RNA,这些小RNA不仅可以作为疾病诊断和预后的分子标志物,在疾病治疗方面也具有无限潜力。在蛋白质组水平,如目标蛋白质组学可以有目标地测定可能与疾病相关的特定蛋白质或多肽,能够很好地应用于疾病的临床分期分型。文章进一步阐述了跨组学研究在疾病研究领域中的应用和发展趋势,借助生物信息学分析方法进行多组学整合研究,能更加系统地阐释疾病的发生及发展机理,为疾病的诊断治疗提供强有力的工具。     

Identification of A Novel SBF2 Frameshift Mutation in Charcot–Marie–Tooth Disease Type 4B2 Using Whole-exome Sequencing

Abstract Charcot-Marie-Tooth disease type 4B2 with early-onset glaucoma （CMT4B2, OMIM 604563） is a genetically-heterogeneous childhood-onset neuromuscular disorder. Here, we report the case of a 15-year-old male adolescent with lower extremity weakness, gait abnormalities, foot deformities and early-onset glaucoma. Since clinical diagnosis alone was insufficient for providing pathogenetic evidence to indicate that the condition belonged to a consanguineous family, we applied whole-exome sequencing to samples from the patient, his parents and his younger brother, assuming that the patient＇s condition is transmitted in an autosomal recessive pattern. A frame-shift mutation, c.4571delG （P.Gly1524Glufs＊42）, was revealed in the CMT4B2-related gene SBF2 （also known as MTMR13, MIM 607697）, and this mutation was found to be homozygous in the proband and heterozygous in his parents and younger brother. Together with the results of clinical diagnosis, this case was diagnosed as CMT4B2. Our finding further demonstrates the use of whole-exome sequencing in the diagnosis and treatment of rare diseases.