微生物基因组序列测量及其重大意义

微生物基因组测序不仅可使人们更好地了解病原微生物的致病机制以及它们与宿主的相互关系,促进寻找更灵敏及特异的病毒原微生物的诊断,分型手段,而且为临床筛选有效的药物及发展疫苗提供参考,此外,它还能提高对人类相关基因功能的认识,为探讨人类遗传性疾病机制提供参考,本文就微生物序列测定重大的理论及应用价值作一简要概述。     

人腺病毒55型感染肺部病变与外周血淋巴细胞关系研究

目的:探讨人腺病毒55型感染肺部病变与外周血淋巴细胞改变的关系及致病意义。方法:以50例经胸部CT证实为腺病毒肺炎的患者为研究对象(肺炎组),调查其外周血细胞及T淋巴细胞亚群变化情况,并与同期腺病毒55型感染未出现肺部病变的患者30例(非肺炎组)对照;亚组分析50例肺炎组中多肺叶病变与单肺叶病变患者之间T淋巴细胞变化的差异。结果:与非肺炎组患者相比,肺炎组患者急性期外周血淋巴细胞比例明显降低、单核细胞比例明显升高(P0.01);肺炎组CD3+T淋巴细胞比例、CD3+CD4+T淋巴细胞比例及CD4/CD8比值较非肺炎组均有明显降低(P0.01)。亚组分析显示,肺炎组患者肺部病变范围不同,T淋巴细胞亚群的变化亦有差异,多肺叶病变组患者CD3+CD8+T淋巴细胞比例较单肺叶病变组明显升高、CD4/CD8比值较单肺叶病变组明显降低(P0.05)。结论:HAdv-55感染引起肺炎病变时,宿主存在明显的细胞免疫功能受损,且与肺部病变程度有一定正相关。     

腺病毒55型抗原表位分析及评价

目的:筛选腺病毒55型(Ad55)抗原表位,为研制腺病毒免疫诊断试剂提供基础。方法:采用生物信息学方法预测Ad55六邻体、纤突的B细胞抗原表位;利用大肠杆菌优势密码子获得相应基因,插入载体pBVIL-1克隆表达后获得重组Ad55表位抗原;用临床疑似腺病毒呼吸道感染患者血清对重组Ad55表位抗原活性进行检测,用ROC曲线分析重组Ad55抗原的诊断意义;采用ClustalX软件进行多序列比较。结果:Ad55六邻体含有6个主要抗原表位,纤突含有2个主要抗原表位;采用退火延伸法获得上述8种表位基因,片段长度为90~180 bp;获得上述8种重组基因工程抗原,相对分子质量为18×103~21×103;血清学检测的ROC曲线分析显示,270~320、410~460和135~165氨基酸残基抗原表位的AUC面积超过0.75,具有一定的诊断意义(P0.05);序列比较结果显示,上述3种抗原表位与腺病毒11型序列高度同源,与3型存在较大差异。结论:获得了3个Ad55主要抗原,对研制通用性呼吸道传播腺病毒免疫诊断试剂具有一定的意义。     

血清10型禽腺病毒AHFY19株全基因组序列测定与分析

禽腺病毒（Fowl Adenovirus,FAdV）是一组血清型众多及致病性多样的病原体,多数呈亚临诊性长期潜伏带毒,部分毒株可造成禽群包涵体肝炎、心包积液、肌胃糜烂症、产蛋下降、生长迟缓等病症,严重威胁养禽业健康发展。为分析血清10型禽腺病毒（FAdV-10）全基因组序列特征,本研究将分离自外表健康鸡泄殖腔拭子的FAdV-10 AHFY19株经卵黄囊途径接种8日龄SPF鸡胚扩繁,从死亡鸡胚肝组织中提取病毒基因组,采用illumina二代基因测序技术和特殊结构区二温式PCR扩增测定病毒全基因组并分析其遗传特征。结果显示,AHFY19株（103.1EID50/0.1 mL/枚）接种的鸡胚于18～21日龄全部死亡。全基因组测序显示,AHFY19株病毒基因组全长为45 757 bp(GenBank登录号：MN542422),其G+C含量为54.63%,末端重复序列（ITR）长为56 bp,共有52个开放阅读框（ORF）。基因组比对分析显示,AHFY19株属I群FAdV C种FAdV-10型,与C2-B-FAdV-10株（MK572851）全基因组同源性最高（99.91%）,但AHFY19株基...     

面向新一代基因组测序技术的序列拼接算法

随着新一代测序技术的发展,新的拼接算法应运而生。介绍了目前国际上广泛认可的几种新的拼接算法的基本原理与具体步骤,分析每种算法的优缺点以及适用范围。用Helicobacter acinonychis的Illumina 1G测序数据检测SSAKE,VCAKE,SHARCGS以及velvet的性能,并对未来拼接算法的研究提出展望。     

高通量测序技术在农作物全基因组序列测定中的应用概览

近几年飞速发展的高通量测序技术(next generation sequencing,NGS)在生命科学研究的各个领域充分展现了其低成本、高通量和应用面广等优势。在现代农业生物技术领域,利用高通量测序技术,科学家们不仅能更经济而高效对农作物、模式植物或不同栽培品种进行深入的全基因组测序、重测序,也可以对成百上千的栽培品种进行高效而准确的遗传差异分析、分子标记分析、连锁图谱分析、表观遗传学分析、转录组分析,进而改进农作物的育种技术,加快新品种的育种研究。其中,获得农作物的全基因组序列是其他研究和分析的基础。本文通过介绍近年来发表的一些利用高通量测序技术进行的农作物全基因组测定和组装的工作,展示高通量测序技术在现代农业生物技术领域的广泛前景以及其建立起来的研究基础。     

人腺病毒B、D亚种基因组中简单重复序列分析

本文以人腺病毒B亚种31条基因组序列及D亚种39条基因组序列为研究材料,利用ImperfectMicrosatelliteExtractor和DNAMAN软件对这些基因组序列中简单重复序列(SSR)的分布情况进行了系统性分析和比较。分析结果显示:人腺病毒B、D亚种基因组中简单重复序列的平均相对密度是十分接近的,但在不同类型SSR中分布情况又有所不同。D亚种中二型SSR明显高于B亚种,在两亚种一型SSR中(A)n、(T)n都是比较多的,而在两亚种二型SSR中的(CG/GC)n表现出了较高的偏好性。在同亚种多序列比对分析中,D亚种表现出了更高的稳定性。B、D亚种中SSR的这种特异性分布可能与它们的进化机制和致病性有关。     

我国四株埃可病毒6型全基因组序列及重组特征分析

埃可病毒6型(Echovirus 6,ECHO6)作为肠道病毒B组(Enterovirus B,EV-B)的一员,在临床上常导致无菌性脑膜炎(Aseptic meningitis,AM)、急性弛缓性麻痹(Acute flaccid paralysis,AFP)、手足口病(Hand,foot,and mouth disease,HFMD)等多种疾病。本文挑选四株中国大陆地区不同年份、地区及疾病严重程度的代表毒株进行全基因组测定分析,与GenBank上ECHO6全长代表株及EV-B组原型株比对分析,了解其进化特征,并筛选BLAST结果的EV-B组流行株,探究我国流行的ECHO6与其他血清型的基因重组特征。结果提示,本研究四株ECHO6毒株在非结构蛋白区与EV-B其他血清型均发生重组,其中死亡病例标本重组模式更为复杂,可能与疾病严重程度有关。通过本研究分析,可进一步揭示ECHO6的遗传特征,为肠道病毒血清型研究提供基础数据,对了解其进化变异及临床致病力影响具有重要意义。     

腺病毒4型E3区核苷酸序列测定及其基因结构与功能分析

本文将Ａｄ４基因组相当于７３．３－８９．２基因图谱单位的ＤＮＡ片段进行了序列测定及基因结构分析,它包括了Ａｄ４Ｅ３区全基因及该区两侧的部分序列。序列分析表明,Ａｄ４ Ｅ３区从ＴＡＴＡＡ ｂｏｘ起至该区基因结束共４７７８ｂｐ,编码１１个大于６ｋＤ的开放读码框架。对Ａｄ４ Ｅ３区ＯＲＦ分析结果表明,Ａｄ４ Ｅ３区编码的１９．３ｋ,１５ｋ,１０．４ｋ蛋白,分别与Ａｄ２ Ｅ３区的ｇｐ１９ｋ,１４．ｋ和１０     

3株肠道病毒71型毒株的全基因组序列分析

目的：对获得的3株肠道病毒71（EV71）型毒株进行全基因组序列测定,并对其进化特点及分型进行初步分析。方法：提取病毒RNA,反转录得到eDNA,PCR分段扩增覆盖病毒全长序列的6个重叠片段（不包括多聚腺苷酸尾）;用软件将3株EV71的备片段序列进行拼接、编辑和校正,随后进行氨基酸翻译及序列比较;用MEGA4.1软件构建系统进化树。结果：获得了3株EV71的全长序列：GDV103株基因组全长7404 nt,包括741bp的5’端非编码区（UTR）、6582bp的病毒基因组编码区（ORF）及81bp的3’UTR;安徽株（Anhui2007）基因组全长7405nt,包括742bp的5＇UTR、6582bp的ORF及81bp的3＇UTR;VR1432株基因组全长7408nt,包括743bp的5’UTR、6582bp的ORF及83bp的3’UTR长。经同源性比对和进化树分析,证实GDV103和安徽株EV71属于C基因型的C4基因亚型。而VR1432株则属于C基因型的C2基因亚型。结论：获得了3株EV71的全长基因组序列,并进一步探讨了其型别,为下一步的干扰素保护宴,哈重定了基础．     

家养野猪源猪圆环病毒2型安徽株的分离鉴定及其全基因组序列分析

目的对安徽省临诊疑似PMW S家养野猪病例进行猪圆环病毒2型(PCV2)分离鉴定,并对分离株的全基因组进行克隆与序列分析。方法应用PK-15细胞进行PCV2的分离与增殖,根据PCR、IFA、电镜技术进行PCV2分离株的鉴定,克隆分离株的全基因组,并对序列进行分析。结果获得1株来自安徽家养野猪源PCV2分离株,命名为YZ0901。该毒株全基因组长为1 767 bp,属于PCV2b基因型。与GenBank已发表的国内外参考毒株的同源性介于93.9%~99.2%,与安徽分离毒株彼此之间的同源性介于93.4%~99.5%。结论安徽省家养野猪中存在PCV2感染,分离毒株与家猪源病毒差异不大,PCV2核苷酸序列比较稳定,其进化不存在明显的地域相关性,家养野猪源PCV2的基因型与当地PCV2流行株的基因型密切相关。     

Draft genome sequence of ramie,Boehmeria nivea (L.) Gaudich

Ramie, Boehmeria nivea (L.) Gaudich, family Urticaceae, is a plant native to eastern Asia, and one of the world's oldest fibre crops. It is also used as animal feed and for the phytoremediation of heavy metal‐contaminated farmlands. Thus, the genome sequence of ramie was determined to explore the molecular basis of its fibre quality, protein content and phytoremediation. For further understanding ramie genome, different paired‐end and mate‐pair libraries were combined to generate 134.31 Gb of raw DNA sequences using the Illumina whole‐genome shotgun sequencing approach. The highly heterozygous B. nivea genome was assembled using the Platanus Genome Assembler, which is an effective tool for the assembly of highly heterozygous genome sequences. The final length of the draft genome of this species was approximately 341.9 Mb (contig N50 = 22.62 kb, scaffold N50 = 1,126.36 kb). Based on ramie genome annotations, 30,237 protein‐coding genes were predicted, and the repetitive element content was 46.3%. The completeness of the final assembly was evaluated by benchmarking universal single‐copy orthologous genes (BUSCO); 90.5% of the 1,440 expected embryophytic genes were identified as complete, and 4.9% were identified as fragmented. Phylogenetic analysis based on single‐copy gene families and one‐to‐one orthologous genes placed ramie with mulberry and cannabis, within the clade of urticalean rosids. Genome information of ramie will be a valuable resource for the conservation of endangered Boehmeria species and for future studies on the biogeography and characteristic evolution of members of Urticaceae.     

Complete genome sequence of an aerobic thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain7.

The complete genomic sequence of an aerobic thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain7 which optimally grows at 80 degrees C, at low pH, and under aerobic conditions, has been determined by the whole genome shotgun method with slight modifications. The genomic size was 2,694,756 bp long and the G + C content was 32.8%. The following RNA-coding genes were identified: a single 16S-23S rRNA cluster, one 5S rRNA gene and 46 tRNA genes (including 24 intron-containing tRNA genes). The repetitive sequences identified were SR-type repetitive sequences, long dispersed-type repetitive sequences and Tn-like repetitive elements. The genome contained 2826 potential protein-coding regions (open reading frames, ORFs). By similarity search against public databases, 911 (32.2%) ORFs were related to functional assigned genes, 921 (32.6%) were related to conserved ORFs of unknown function, 145 (5.1%) contained some motifs, and remaining 849 (30.0%) did not show any significant similarity to the registered sequences. The ORFs with functional assignments included the candidate genes involved in sulfide metabolism, the TCA cycle and the respiratory chain. Sequence comparison provided evidence suggesting the integration of plasmid, rearrangement of genomic structure, and duplication of genomic regions that may be responsible for the larger genomic size of the S. tokodaii strain7 genome. The genome contained eukaryote-type genes which were not identified in other archaea and lacked the CCA sequence in the tRNA genes. The result suggests that this strain is closer to eukaryotes among the archaea strains so far sequenced. The data presented in this paper are also available on the internet homepage (http://www.bio.nite.go.jp/E-home/genome_list-e.html/).     

De novo assembly of the Pseudomonas syringae pv. syringae B728a genome using Illumina/Solexa short sequence reads

Illumina's Genome Analyzer generates ultra-short sequence reads, typically 36 nucleotides in length, and is primarily intended for resequencing. We tested the potential of this technology for de novo sequence assembly on the 6 Mbp genome of Pseudomonas syringae pv. syringae B728a with several freely available assembly software packages. Using an unpaired data set, velvet assembled >96% of the genome into contigs with an N50 length of 8289 nucleotides and an error rate of 0.33%. edena generated smaller contigs (N50 was 4192 nucleotides) and comparable error rates. ssake and vcake yielded shorter contigs with very high error rates. Assembly of paired-end sequence data carrying 400 bp inserts produced longer contigs (N50 up to 15 628 nucleotides), but with increased error rates (0.5%). Contig length and error rate were very sensitive to the choice of parameter values. Noncoding RNA genes were poorly resolved in de novo assemblies, while >90% of the protein-coding genes were assembled with 100% accuracy over their full length. This study demonstrates that, in practice, de novo assembly of 36-nucleotide reads can generate reasonably accurate assemblies from about 40 × deep sequence data sets. These draft assemblies are useful for exploring an organism's proteomic potential, at a very economic low cost.     

背瘤丽蚌F型线粒体基因组全序列分析

部分双壳贝类的线粒体遗传方式是特殊的双重单亲遗传方式:F型存在于雌性体细胞组织和性腺中,M型仅存在于雄性个体的性腺中。通过LA-PCR扩增、SHOT-GUN测序、软件拼接获得背瘤丽蚌(Lamprotula leai)F型线粒体基因组全序列。线粒体基因组全长为16530 bp,包括13个蛋白质编码基因,22个tRNA其中包括2个tRNA^Ser和2个tRNA^Leu,2个SrRNA及27个长度不等的非编码区,最长的两个非编码区分别为969 bp、228 bp。比较分析已登录到GenBank中的淡水蚌类F型线粒体结构特征,结果显示背瘤丽蚌F型A+T含量为60.28%,表现出A+T偏好性,淡水蚌类线粒体基因组长度的差异主要表现为非编码区长度的差异。此外,背瘤丽蚌mtDNA的COⅡ-12S rRNA区域基因排列存在差异,是ND3、tRNA^His、tRNA^Ala、tRNA^Ser1、tRNA^Ser2、tRNA^Glu、ND2、tRNA^Met 8个基因发生重排造成。F型线粒体序列构建的系统进化树中,淡水蚌类和海水双壳贝类分别聚为一支。研究结果为进一步研究淡水珍珠蚌的DUI线粒体遗传方式和种质资源保护奠定基础,为双壳贝类mtDNA基因重排提供依据。     

Complete genome sequence of Streptococcus troglodytae TKU31 isolated from the oral cavity of a chimpanzee (Pan troglodytes)

Streptococcus troglodytae TKU31 was isolated from the oral cavity of a chimpanzee (Pan troglodytes) and was found to be the most closely related species of the mutans group streptococci to Streptococcus mutans. The complete sequence of TKU31 genome consists of a single circular chromosome that is 2,097,874 base pairs long and has a G + C content of 37.18%. It possesses 2082 coding sequences (CDSs), 65 tRNAs and five rRNA operons (15 rRNAs). Two clustered regularly interspaced short palindromic repeats, six insertion sequences and two predicted prophage elements were identified. The genome of TKU31 harbors some putative virulence associated genes, including gtfB, gtfC and gtfD genes encoding glucosyltransferase and gbpA, gbpB, gbpC and gbpD genes encoding glucan‐binding cell wall‐anchored protein. The deduced amino acid identity of the rhamnose‐glucose polysaccharide F gene (rgpF), which is one of the serotype determinants, is 91% identical with that of S. mutans LJ23 (serotype k) strain. However, two other virulence‐associated genes cnm and cbm, which encode the collagen‐binding proteins, were not found in the TKU31 genome. The complete genome sequence of S. troglodytae TKU31 has been deposited at DDBJ/European Nucleotide Archive/GenBank under the accession no. AP014612.     

Sequencing of Camelina neglecta,a diploid progenitor of the hexaploid oilseed Camelina sativa

Camelina neglecta is a diploid species from the genus Camelina, which includes the versatile oilseed Camelina sativa. These species are closely related to Arabidopsis thaliana and the economically important Brassica crop species, making this genus a useful platform to dissect traits of agronomic importance while providing a tool to study the evolution of polyploids. A highly contiguous chromosome-level genome sequence of C. neglecta with an N50 size of 29.1 Mb was generated utilizing Pacific Biosciences (PacBio, Menlo Park, CA) long-read sequencing followed by chromosome conformation phasing. Comparison of the genome with that of C. sativa shows remarkable coincidence with subgenome 1 of the hexaploid, with only one major chromosomal rearrangement separating the two. Synonymous substitution rate analysis of the predicted 34 061 genes suggested subgenome 1 of C. sativa directly descended from C. neglecta around 1.2 mya. Higher functional divergence of genes in the hexaploid as evidenced by the greater number of unique orthogroups, and differential composition of resistant gene analogs, might suggest an immediate adaptation strategy after genome merger. The absence of genome bias in gene fractionation among the subgenomes of C. sativa in comparison with C. neglecta, and the complete lack of fractionation of meiosis-specific genes attests to the neopolyploid status of C. sativa. The assembled genome will provide a tool to further study genome evolution processes in the Camelina genus and potentially allow for the identification and exploitation of novel variation for Camelina crop improvement.