基于RefSeq的人类基因荧光定量PCR引物库的构建

利用NCBI提供的RefSeq序列,通过BLAT、Sim4和自主开发的剪接比对程序Ealter1.0对人类RefSeq转录本进行外显子预测,根据预测的外显子信息,采用自主开发SYBR Green I Real Time PCR引物设计程序E-qPCR-Design1.0高通量设计21,118对SYBR Green I Real Time PCR引物,同时选取5000条基因进行SYBR Green I Real Time PCR引物验证,95.92%的基因引物取得良好效果,1.64%的基因引物产生引物二聚体,1.08%的基因引物有非特异性扩增,通过生物信息技术分析与实验验证,建立了基于RefSeq的人类基因荧光定量PCR引物库。     

常用肿瘤基因分析方法及基于TCGA数据库的分析应用

随着二代测序技术的快速发展,数据量不断累积,肿瘤学家的目光逐渐由多物种测序转移至高通量测序数据的分析和比对。基因数据分析方法层出不穷,高通量的组学分析手段不断优化和创新,基因数据的挖掘和分析工作正处于飞速发展的时期。以肿瘤病人样本为核心的数据库The Cancer Genome Atlas (TCGA)由此应运而生,该数据库全方位记录了从临床肿瘤病人样本得到的基因数据如DNA序列、转录本信息、表观遗传学修饰等。本文主要从数据分析方法、TCGA数据库及其应用实例等3个方面详细介绍了肿瘤相关基因数据的深入挖掘和生物信息学分析方法的最新研究进展,以期为研究人员利用大数据发现肿瘤防治相关的新靶点提供借鉴和参考。     

真核基因起始与终止密码子旁侧序列特征分析

真核基因起始与终止密码子旁侧序列的特征对于确定cDNA开放阅读框架 (ORF)和预测基因组序列中的编码区 (CDS)非常重要。基于高质量RefSeq数据库 ,在较大数据规模下统计分析了起始密码子旁侧序列所具有的“Kozak规则” ,发现不同物种之间存在差别。同时分析了不同终止密码子旁侧序列的统计学特征 ,给出了相应的正则表达式。由于发现多种基因中存在同相位起始、终止密码子串联使用的情况 ,亦对此进行了讨论。     

朝仓花椒幼芽转录组测序数据组装及基因功能注释

采用2代Illumina Hi-Seq测序技术对朝仓花椒雌株结果期幼芽的转录组进行测序,构建了转录组数据库,获得49 672 080条Clean Reads数据,包含总长度为4 967 208 000 nt序列数据信息;经拼接组装,获得转录组基因信息长达55 902 243 nt的176 407个Contig片段,再通过进一步拼接,共获得平均长度为878 nt的96 475个Unigene片段。与Nt、Nr、Swiss-Prot、COG、GO、KEGG等数据库进行BLAST信息比对(E-value艽10-5),共获得68 315个注释基因,以及62 150个表达序列标签(EST)。与NR数据库比对,发现花椒转录组基因序列与同科柑橘属的甜橙(Citrus sinensis)和克莱门柚(Citrus clementina)具有较高的同源性,分别为43.96%及41.86%,与其他物种的同源性较低,均不足10%;可将花椒转录组的Unigene的功能通过与COG数据库进行注释比对划分为25类;根据GO数据库的注释,可将其分为生物过程、细胞组分和分子功能3大类共55分支,根据与KEGG数据库的比较分析发现朝仓花椒的转录组数据中含有代谢通路相关基因128类,其中包括多种化合物的代谢途径和次生代谢产物的生物合成途径,其中有较多的次生物质代谢途径,如萜类化合物生物合成途径、黄酮类生物合成代谢途径以及花青素的生物合成途径等。     

基于Cytb基因的NCBI数据库序列可靠性分析

本研究以常用于动物种属鉴定的Cytb基因位点为研究对象,利用所测得的30种盗猎案件中常见的野生动物Cytb基因部分片段序列及NCBI数据库中下载的该物种序列及其近缘物种序列,构建系统进化树。根据进化树的聚类情况,判断NCBI数据库中的相关基因序列或物种名称的正确性,并对其中错误序列的登陆号进行标记,以防对后续涉案动物的准确鉴定造成影响。分别从30种常见涉案野生动物(共35份样本)中提取线粒体DNA,并利用通用引物扩增线粒体DNA上的Cytb基因部分片段并进行测序分析。通过NCBI数据库的Blast比对功能,筛选出与本研究物种同源性由高到低的物种,并从NCBI基因数据库中下载此类近缘物种的Cytb基因序列共482条,利用MEGA软件构建该物种的系统进化树。比对发现NCBI中登录号为DQ246798、KP202269、AY286434等3个序列所对应物种拉丁名错误。登录号为AY509634、AJ131639、AM072747等5个序列所对应物种拉丁名可能存在疑问。NCBI数据库数据可靠性有待进一步验证,只能作为涉案物种鉴定的参考数据之一,可借助构建系统进化树等方法来确认其结果的准确性。     

基于Web的水稻芯片数据注释和分析平台

国际水稻基因组测序计划(IEGSP)顺利完成, 水稻基因的研究也进入了后基因组研究阶段. 水稻基因芯片数据注释分析是一项重要的功能基因组学研究内容, 它为理解水稻基因的生物学意义提供了帮助. 本研究开发了一个基于Web的水稻基因芯片数据注释和分析平台(RiceChip), 它比同类的注释数据库更加全面快捷. 本平台共由5个功能模块组成: BioChip模块为水稻基因表达数据提供快速检索和高级检索, 可依次按照Probe Set ID, Locus ID, Analysis Name等字段进行检索; BioAnno模块整合多个生物学数据库, 为水稻基因提供基因功能、蛋白质结构、生物代谢途径以及转录调控等方面的注释信息; BioSeq模块则收集水稻基因组的序列信息, 支持对水稻基因与芯片探针的序列查询; BioView模块是系统图形可视化的核心模块, 提供友好的访问界面与结果输出, 方便研究人员使用; BioAnaly模块结合R/Bioconductor统计分析工具提供高通量芯片数据的在线分析. 本系统从不同的方面依次提供了数据检索、基因注释、序列分析、数据可视化和数据分析等功能, 其数据收集的全面性与功能分析的强大性在同类水稻基因芯片数据注释和分析平台中都较突出.     

日本七鳃鳗(Lampetra japonica)肝脏ESTs 分析与比较转录组研究

以日本七鳃鳗(Lampetra japonica)肝脏为材料构建cDNA文库, 在文库中随机挑选克隆子进行测序共得到10077条有效ESTs(expressed sequence tags)序列. ESTs序列分析显示, 8515条ESTs拼成648条片段重叠群, 共得到2210条转录本, 其中47.06%的转录本预测为全长序列; 利用BLAST程序在GenBank数据库中进行同源性搜索发现2053条转录本有同源序列匹配, 占总转录本的92.9%. 更进一步对这些基因产物进行Gene Ontology注释, 结果发现, 在日本七鳃鳗肝脏中与有颌类免疫、凝血和代谢相关的基因大量表达, 并预测了8个新基因. 通过对日本七鳃鳗与底鳉(Fundulus heteroclitus)、鼠(Mus musculus)、牛(Bos taurus)和人(Homo sapiens)肝脏转录组的比较分析, 发现日本七鳃鳗肝脏中比其他物种优势表达的是甲壳质酶和多糖代谢等相关的基因, 这些基因可能在日本七鳃鳗免疫中发挥重要作用. 此外, 也利用TargetScan软件对日本七鳃鳗肝脏转录组中3′UTR区进行microRNA靶标识别, 结果发现了与人类癌症基因调控同源的microRNA靶标, 这为研究人类癌症提供了有益的线索. 上述结果将为七鳃鳗功能基因和蛋白组学的研究以及脊椎动物的基因组进化提供重要的理论基础.     

新疆天山雪莲转录组注解知识库

新疆天山雪莲(Sasussured involucrata)具有较高的极端低温耐受特性,为低温耐受机制研究提供了一种非常好的模式植物。新疆天山雪莲转录组注解知识库(http://www.shengtingbiology.com/Saussurea KBase/index.jsp)是基于网络数据资源的综合性数据库,由html、Perl、Perl CGI/DBD/DBI、Java和Java Script编程所设计的前端界面和用于数据存取、注释及管理的后端数据库管理系统Postgrel SQL构成。知识库包含基因组数据、转录组原始数据、质量控制数据、GC含量、功能基因序列及注释、功能基因代谢通路、功能基因的注释统计、雪莲与其它物种的转录组或基因组比较分析数据和生物分析软件包等资源。该数据库不仅有利于低温功能基因组学及低温耐受机制研究,而且为冷耐受性状物种的分子育种提供基因资源平台和理论依据。     

基于多传感器的声景大数据在线采集与分析系统研究

声景包含重要的生态信息,具有实时性强、信息密度高的特点,有重要研究价值。现有的声景研究中,音频及相关环境参数采集和分析仍需要大量的人工作业,耗时耗力。基于多传感集成、边缘计算和深度学习技术,建立了一套声景大数据在线采集与分析系统,包括边缘计算节点和中心计算服务器。并通过3个实验站点,进行了近1年的技术验证,实现了声景大数据的自动化在线采集、传输和分析。该系统能适应户外恶劣的自然环境,能根据任务需求持续不断地进行声景大数据在线采集和分析,稳定性好。声学指数可以反映声景变化,但因指数侧重点不同,不同的声学指数之间变化特征差异较大,需要组合使用。通过声纹特征图能直观地识别出不同发声源,对物种的快速识别、声源的分类等具有较强的借鉴意义。系统借助VGGish网络提取的高维声景特征图能很好地识别不同站点和不同时间的声景变化,在不同站点和昼夜上具有较高的区分精度,有快速和直观地反映不同生态系统的类型特征、生态系统动态变化的潜力。丰富声纹特征库、优化声景特征分析神经网络、建设声景长期监测共享网络,有助于扩展系统在物种识别、生物多样性快速分析、生物与环境相互作用机制方面的应用。研究为声景大数据的在线采集...     

Automation of a primer design and evaluation pipeline for subsequent sequencing of the coding regions of all human Refseq genes

Screening for mutations in human disease-causing genes in a molecular diagnostic environment demands simplicity with a view to allowing high throughput approaches. In order to advance these requirements, we have developed and applied a primer design program, termed BatchPD, to achieve the PCR amplification of coding exons of all known human Refseq genes. Primer design, in silico PCR checks and formatted primer information for subsequent web-based interrogation are queried from existing online tools. BatchPD acts as an intermediate to automate queries and results processing and provides exon-specific information that is summarised in a spreadsheet format.     

hsa-miR-192-3p的靶基因预测及功能分析

通过生物信息学方法预测hsa-miR-192-3p的靶基因及其靶基因的可能功能。首先通过miRbase在线数据库对hsamiR-192-3p的碱基序列及序列在各物种间的保守性进行分析,再通过miRGator v3. 0在线数据库查看hsa-miR-192-3p在各个组织器官中的表达丰富度情况;其次,应用Target Scan和miRanda在线数据库预测hsa-miR-192-3p的靶基因;最后,将预测得到的两个数据库的靶基因交集用DAVID在线数据库进行功能富集分析和信号转导通路富集分析。结果表明:hsa-miR-192-3p在人、家鼠、猕猴等生物中存在高度保守性; hsa-miR-192-3p在胃肠道、肾脏、肝胆系统、干细胞、鼻、脾、胸腺中表达丰富度较高;通过两个靶基因预测软件预测的靶基因取交集后共有190个;功能富集分析发现hsa-miR-192-3p靶基因富集在细胞质、细胞核、质膜、高尔基体等15个细胞组件(p0. 05),参与蛋白结合、GTP酶活性、锌离子跨膜转运蛋白活性等7个分子功能(p0. 05),涉及金属离子运输、RNA聚合酶II启动子的转录阳性调控、基因表达调节、钙离子跨膜运输、胚胎发育等18个生物过程(p0. 05);预测靶基因集合显著富集于癌症通路与催乳素信号通路中(p0. 05)。得出结论:hsa-miR-192-3p预测的靶基因集合富集于多个生物过程,与肿瘤密切相关,生物信息预测为今后的研究奠定了一定的理论基础,为后续实验验证提供了研究方向。     

ArrayExpress--a public repository for microarray gene expression data at the EBI

ArrayExpress is a new public database of microarray gene expression data at the EBI, which is a generic gene expression database designed to hold data from all microarray platforms. ArrayExpress uses the annotation standard Minimum Information About a Microarray Experiment (MIAME) and the associated XML data exchange format Microarray Gene Expression Markup Language (MAGE-ML) and it is designed to store well annotated data in a structured way. The ArrayExpress infrastructure consists of the database itself, data submissions in MAGE-ML format or via an online submission tool MIAMExpress, online database query interface, and the Expression Profiler online analysis tool. ArrayExpress accepts three types of submission, arrays, experiments and protocols, each of these is assigned an accession number. Help on data submission and annotation is provided by the curation team. The database can be queried on parameters such as author, laboratory, organism, experiment or array types. With an increasing number of organisations adopting MAGE-ML standard, the volume of submissions to ArrayExpress is increasing rapidly. The database can be accessed at http://www.ebi.ac.uk/arrayexpress.     

MOTUs, Morphology, and Biodiversity Estimation: A Case Study Using Nematodes of the Suborder Criconematina and a Conserved 18S DNA Barcode

DNA barcodes are increasingly used to provide an estimate of biodiversity for small, cryptic organisms like nematodes. Nucleotide sequences generated by the barcoding process are often grouped, based on similarity, into molecular operational taxonomic units (MOTUs). In order to get a better understanding of the taxonomic resolution of a 3' 592-bp 18S rDNA barcode, we have analyzed 100 MOTUs generated from 214 specimens in the nematode suborder Criconematina. Previous research has demonstrated that the primer set for this barcode reliably amplifies all nematodes in the Phylum Nematoda. Included among the Criconematina specimens were 25 morphologically described species representing 12 genera. Using the most stringent definition of MOTU membership, where a single nucleotide difference is sufficient for the creation of a new MOTU, it was found that an MOTU can represent a subgroup of a species (e.g. Discocriconemella limitanea), a single species (Bakernema inaequale), or a species complex (MOTU 76). A maximum likelihood phylogenetic analysis of the MOTU dataset generated four major clades that were further analyzed by character-based barcode analysis. Fourteen of the 25 morphologically identified species had at least one putative diagnostic nucleotide identified by this character-based approach. These diagnostic nucleotides could be useful in biodiversity assessments when ambiguous results are encountered in database searches that use a distance-based metric for nucleotide sequence comparisons. Information and images regarding specimens examined during this study are available online.     

Genome-wide identification, characterization and phylogenetic analysis of the rice LRR-kinases

LRR-kinases constitute the largest subfamily of receptor-like kinases in plants and regulate a wide variety of processes related to development and defense. Through a reiterative process of sequence analysis and re-annotation, we identified 309 LRR-kinase genes in the rice genome (Nipponbare). Among them, 127 genes in the Rice Annotation Project Database and 85 in Refseq of NCBI were amended (in addition, 62 LRR-kinase genes were not annotated in Refseq). The complete set of LRR-kinases was characterized. These LRR-kinases were classified into five groups according to phylogenetic analysis, and the genes in groups 1, 2, 3 and 4 usually have fewer introns than those in group 5. The introns in the LRR domain, which are highly conserved in regards to their positions and configurations, split the first Leu or other amino residues at this position of the 'xxLxLxx' motif with phase 2 and usually separate one or more LRR repeats exactly. Tandemly repeated LRR motifs have evolved from exon duplication, mutation and exon shuffling. The extensive distribution and diversity of the LRR-kinase genes have been mainly generated by tandem duplication and mutation after whole genome duplication. Positive selection has made a limited contribution to the sequence diversity after duplication, but positively selected sites located in the LRR domain are thought to involve in the protein-protein interaction.     

Mixed messages from multiple information sources on invasive species: a case of too much of a good thing?

Aim The increasing number and availability of online databases of alien species beg a question of their comparability given most do not adopt standard criteria in the definition of species status or taxonomic treatment and vary in their comprehensiveness. In this study, we compare the consistency of two major European databases for the regions they have in common. We assess whether they use consistent terminology to classify species status, provide similar taxonomic classification and coverage, deliver comparable estimates of alien richness per country and identify comparable correlates of alien richness. Location Northern Europe. Methods Data on the total number of alien species as well as the number of established alien species were extracted from the online databases DAISIE and NOBANIS for 13 European countries and classified into comparable taxonomic groups. Analyses across countries examined trends in alien species richness, correlations among taxonomic groups and the explanatory power of population density, country area and per capita GDP on alien species richness. Results Alien species richness, intertaxon correlations and the significance of individual drivers of invasion were all strongly database dependent. Differences were more marked for total numbers of aliens than established aliens. Over all taxonomic groups, DAISIE had lower species richness and fewer significant intertaxon correlations but presented a greater number of significant explanatory models of alien species richness. Trends in species richness were not generally correlated between the two databases with human population density being a more important driver in DAISIE while country area had greater explanatory power in NOBANIS. Main conclusions Considerable caution should be applied when collating data from different databases because often their underlying structure and content may differ markedly. For Europe, the analysis indicates that having two contrasting databases is not an ideal basis for implementing invasive species policy and moves should be made soon to establish a central pan‐European database.     

WebACT--an online companion for the Artemis Comparison Tool

SUMMARY: WebACT is an online resource which enables the rapid provision of simultaneous BLAST comparisons between up to five genomic sequences in a format amenable for visualization with the well-known Artemis Comparison Tool (ACT). Comparisons can be generated on-the-fly using sequences directly retrieved via EMBL database queries, or by entering or uploading user sequences. Furthermore, pre-computed comparisons are available between all publicly available, completed prokaryotic genomes and plasmids currently contained within the Genome Reviews database (372 sequences, representing 175 different species). The system is designed to minimize the volume of downloaded data and maximize performance. Genome sequences, annotation and pre-computed comparisons are stored in a relational database allowing flexible querying based on user-defined sequence regions, from whole genome to a defined region flanking a specified gene. Comparison and sequence files, whether computed online or retrieved from the database of pre-computed genome comparisons, can be viewed online using ACT and are available for download. AVAILABILITY: Freely accessible at http://www.webact.org. SUPPLEMENTARY INFORMATION: User guide and worked examples are available at http://www.webact.org/WebACT/docs.     

基因结构分析软件对实时定量PCR引物进行设计和筛选

目的：以人丝裂原活化蛋白激酶3(mitogen-activated protein kinase 3, ) 基因结构为例,利用不同生物相关软件分析、 设计和筛选合适的定量PCR 引物。方法：利用NCBI 的Gene 数据库查找人基因的参考序列、UniGene 数据库查找标准 参考序列;并用在线软件如Spidey, UCSC, Ensembl 等分析基因结构;利用Primer3,Oligo6,IDT 等软件进行引物设计;用MFOLD 程序分析基因二级结构后,选择引物可定位的外显子位置;利用电子PCR进行引物扩增特异性的检验;最后通过实验检验引物的 扩增效果。结果：从程序软件推荐的引物列表中筛选出一对能特异扩增人基因的引物。结论：基因结构分析软件有助于定 量PCR 引物的设计。