mega操作过程-多序列比对、进化树、

1、基础生物信息学及应用,王兴平,多序列比对 分子进化分析系统发生树构建 核酸序列的预测与鉴定 酶切图谱制作 引物设计,内 容,多序列比对,内容： 多序列比对 多序列比对程序及应用,第一节、多序列比对 （Multiple sequence alignment）,概念 多序列比对的意义 多序列比对的打分函数 多序列比对的方法,1、概念,多序列比对（Multiple sequence alignment） align multiple related sequences to achieve optimal matching of the sequences. 为了便于描述，对多序列比对过程可以给出下

2、面的定义：把多序列比对看作一张二维表，表中每一行代表一个序列，每一列代表一个残基的位置。将序列依照下列规则填入表中： （a）一个序列所有残基的相对位置保持不变； （b）将不同序列间相同或相似的残基放入同一列，即尽可能将序列间相同或相似残基上下对齐（下表）。,表1 多序列比对的定义,表示五个短序列（I-V）的比对结果。通过插入空位，使5个序列中大多数相同或相似残基放入同一列，并保持每个序列残基顺序不变,2、多序列比对的意义,用于描述一组序列之间的相似性关系，以便了解一个分子家族的基本特征，寻找motif，保守区域等。 用于描述一组同源序列之间的亲缘关系的远近，应用到分子进化分析中。 序列同源性分

3、析：是将待研究序列加入到一组与之同源，但来自不同物种的序列中进行多序列同时比较，以确定该序列与其它序列间的同源性大小。 其他应用，如构建profile，打分矩阵等,手工比对 在运行经过测试并具有比较高的可信度的计算机程序（辅助编辑软件如bioedit，seaview，Genedoc等）基础上，结合实验结果或文献资料，对多序列比对结果进行手工修饰，应该说是非常必要的。 为了便于进行交互式手工比对，通常使用不同颜色表示具有不同特性的残基，以帮助判别序列之间的相似性。 计算机程序自动比对 通过特定的算法（如穷举法，启发式算法等），由计算机程序自动搜索最佳的多序列比对状态。,3、多序列比对的方法,穷举

4、法,穷举法（exhaustive alignment method） 将序列两两比对时的二维动态规划矩阵扩展到多维矩阵。即用矩阵的维数来反映比对的序列数目。这种方法的计算量很大，对于计算机系统的资源要求比较高，一般只有在进行少数的较短的序列的比对的时候才会用到这个方法 DCA (Divide-and-Conquer Alignment）：a web-based program that is semiexhaustive http:/bibiserv.techfak.uni-bielefeld.de/dca/,启发式算法,启发式算法（heuristic algorithms）： 大多数实用的多

5、序列比对程序采用启发式算法（heuristic algorithms），以降低运算复杂度。 随着序列数量的增加，算法复杂性也不断增加。用O（m1m2m3mn）表示对n个序列进行比对时的算法复杂性，其中mn是最后一条序列的长度。若序列长度相差不大，则可简化成O（mn），其中n表示序列的数目，m表示序列的长度。显然，随着序列数量的增加，序列比对的算法复杂性按指数规律增长。,第二节 多序列比对程序及应用,Progressive Alignment Method Iterative Alignment Block-Based Alignment DNASTAR DNAMAN,1、Progressive

6、 Alignment Method,Clustal: Clustal，是由Feng和Doolittle于1987年提出的。 Clustal程序有许多版本 ClustalW（Thompson等，1994）是目前使用最广泛的多序列比对程序 它的PC版本是ClustalX 作为程序的一部分，Clustal 可以输出用于构建进化树的数据。,ClustalW 程序：ClustalW 程序可以自由使用 在NCBI/EBI的FTP服务器上可以找到下载的软件包。ClustalW 程序用选项单逐步指导用户进行操作，用户可根据需要选择打分矩阵、设置空位罚分等。 ftp:/ftp.ebi.ac.uk/pub/sof

7、tware/ EBI的主页还提供了基于Web的ClustalW服务，用户可以把序列和各种要求通过表单提交到服务器上，服务器把计算的结果用Email返回用户（或在线交互使用）。 http:/www.ebi.ac.uk/clustalw/,Progressive Alignment Method,ClustalW 程序 ClustalW对输入序列的格式比较灵活，可以是FASTA格式，还可以是PIR、SWISS-PROT、GDE、Clustal、GCG/MSF、RSF等格式。 输出格式也可以选择，有ALN、GCG、PHYLIP和GDE等，用户可以根据自己的需要选择合适的输出格式。 用ClustalW

8、得到的多序列比对结果中，所有序列排列在一起，并以特定的符号代表各个位点上残基的保守性，“*”号表示保守性极高的残基位点；“.”号代表保守性略低的残基位点。,Progressive Alignment Method,Clustal W 使用 输入地址：http:/www.ebi.ac.uk/clustalw/ 设置选项 （next）,Progressive Alignment Method,Clustal W 使用 一些选项说明 PHYLOGENETIC TREE有三个选项 TREE TYPE：构建系统发育树的算法，有四个个选择none、nj（neighbour joining）、phylip、

9、dist CORRECT DIST：决定是否做距离修正。对于小的序列歧异（10），选择与否不会产生差异；对于大的序列歧异，需做出修正。因为观察到的距离要比真实的进化距离低。 IGNORE GAPS：选择on，序列中的任何空位将被忽视。 详细说明参见 http:/www.ebi.ac.uk/clustalw/clustalw_frame.html,Progressive Alignment Method,Clustal W 使用 输入5个16S RNA 基因序列 AF310602 AF308147 AF283499 AF012090 AF447394 点击“RUN”,Progressive Al

10、ignment Method,Progressive Alignment Method,T-Coffee (Tree-based Consistency Objective Function for alignment Evaluation）： Progressive alignment method /software/TCoffee.html In processing a query, T-Coffee performs both global and local pairwise alignment for all possible pairs inv

11、olved. A distance matrix is built to derive a guide tree, which is then used to direct a full multiple alignment using the progressive approach. Outperforms Clustal when aligning moderately divergent sequences Slower than Clustal,Progressive Alignment Method,PRALINE： web-based： http:/ibivu.cs.vu.nl/

12、programs/pralinewww/ First build profiles for each sequence using PSI-BLAST database searching. Each profile is then used for multiple alignment using the progressive approach. the closest neighbor to be joined to a larger alignment by comparing the profile scores does not use a guide tree Incorpora

13、te protein secondary structure information to modify the profile scores. Perhaps the most sophisticated and accurate alignment program available. Extremely slow computation.,Progressive Alignment Method,DbClustal: http:/igbmc.u-strasbg.fr:8080/DbClustal/dbclustal.html Poa (Partial order alignments):

14、 /poa/,2、Iterative Alignment,PRRN： web-based program http:/prrn.ims.u-tokyo.ac.jp/ Uses a double nested iterative strategy for multiple alignment. Based on the idea that an optimal solution can be found by repeatedly modifying existing suboptimal solutions,Block-Base

15、d Alignment,DIALIGN2： a web based program http:/bioweb.pasteur.fr/seqanal/interfaces/dialign2.html It places emphasis on block-to-block comparison rather than residue-to-residue comparison. The sequence regions between the blocks are left unaligned. The program has been shown to be especially suitab

16、le for aligning divergent sequences with only local similarity.,Block-Based Alignment,Match-Box： web-based server http:/www.fundp.ac.be/sciences/biologie/bms/matchbox_submit.shtml Aims to identify conserved blocks (or boxes) among sequences. The server requires the user to submit a set of sequences

17、in the FASTA format and the results are returned by e-mail.,DNASTAR DNAMAN,软件：,分子进化分析系统发生树构建,本章内容： 分子进化分析介绍 系统发生树构建方法 系统发生树构建实例,第一节 分子进化分析介绍,基本概念： 系统发生（phylogeny）是指生物形成或进化的历史 系统发生学(phylogenetics)研究物种之间的进化关系 系统发生树（phylogenetic tree）表示形式，描述物种之间进化关系,分子进化研究的目的 从物种的一些分子特性出发，从而了解物种之间的生物系统发生的关系。 蛋白和核酸序列 通过

18、序列同源性的比较进而了解基因的进化以及生物系统发生的内在规律,分子进化分析介绍,分子进化分析介绍,分子进化研究的基础 基本理论：在各种不同的发育谱系及足够大的进化时间尺度中，许多序列的进化速率几乎是恒定不变的。（分子钟理论， Molecular clock 1965 ）,实际情况：虽然很多时候仍然存在争议，但是分子进化确实能阐述一些生物系统发生的内在规律,分子进化分析介绍,直系同源与旁系同源 Orthologs(直系同源): Homologous sequences in different species that arose from a common ancestral gene dur

19、ing speciation; may or may not be responsible for a similar function. Paralogs(旁系同源): Homologous sequences within a single species that arose by gene duplication. 。 以上两个概念代表了两个不同的进化事件。用于分子进化分析中的序列必须是直系同源的，才能真实反映进化过程。,分子进化分析介绍,分子进化分析介绍,系统发生树（phylogenetic tree）: 又名进化树（evolutionary tree）已发展成为多学科交叉形成的一个

20、边缘领域。 包括生命科学中的进化论、遗传学、分类学、分子生物学、生物化学、生物物理学和生态学，又包括数学中的概率统计、图论、计算机科学和群论。 闻名国际生物学界的美国冷泉港定量生物学会议于1987年特辟出进化树专栏进行学术讨论，标志着该领域已成为现代生物学的前沿之一，迄今仍很活跃。,分子进化分析介绍,分子进化分析介绍,系统发生树结构 The lines in the tree are called branches(分支). At the tips of the branches are present-day species or sequences known as taxa (分类，th

21、e singular form is taxon) or operational taxonomic units（运筹分类单位）. The connecting point where two adjacent branches join is called a node（节点）, which represents an inferred ancestor of extant taxa. The bifurcating point at the very bottom of the tree is the root node（根节）, which represents the common a

22、ncestor of all members of the tree. A group of taxa descended from a single common ancestor is defined as a clade or monophyletic group (单源群). The branching pattern in a tree is called tree topology（拓扑结构）.,分子进化分析介绍,有根树与无根树 树根代表一组分类的共同祖先,分子进化分析介绍,如何确定树根 根据外围群：One is to use an outgroup（外围群）, which is

23、a sequence that is homologous to the sequences under consideration, but separated from those sequences at an early evolutionary time. 根据中点：In the absence of a good outgroup, a tree can be rooted using the midpoint rooting approach, in which the midpoint of the two most divergent groups judged by ove

24、rall branch lengths is assigned as the root.,Rooted by outgroup,分子进化分析介绍,分子进化分析介绍,树形 系统发生图（Phylograms）：有分支和支长信息 分支图（ Cladograms）只有分支信息，无支长信息,第二节 系统发生树构建方法,Molecular phylogenetic tree construction can be divided into five steps: (1) choosing molecular markers; (2) performing multiple sequence alignme

25、nt; (3) choosing a model of evolution; (4) determining a tree building method; (5) assessing tree reliability.,第三节 系统发生树构建实例,系统发生分析常用软件 (1) PHYLIP (2) PAUP (3) TREE-PUZZLE (4) MEGA (5) PAML (6) TreeView,(7) VOSTORG (8) Fitch programs (9) Phylo_win (10) ARB (11) DAMBE (12) PAL (13) Bionumerics,其它程序见：

26、 /phylip/software.html,系统发生树构建实例,Mega 3 下载地址,离散特征数据 (discrete character data)： 即所获得的是2个或更多的离散的值。如： DNA序列某一位置是或者不是剪切位点（二态特征）； 序列中某一位置，可能的碱基有A、T、G、C共4种（多态特征）； 相似性和距离数据 (similarity and distance data)： 是用彼此间的相似性或距离所表示出来的各分类单位间的相互关系。,核酸序列的预测和鉴定,内容： 序列概率信息的统计模型 核酸序列的

27、预测与鉴定,第一节、序列概率信息的统计模型,One of the applications of multiple sequence alignments in identifying related sequences in databases is by construction of some statistical models. Position-specific scoring matrices (PSSMs) Profiles Hidden Markov models (HMMs).,收集已知的功能序列和非功能序列实例 （这些序列之间是非相关的 ）,训练集 （training s

28、et）,测试集或控制集 （control set）,建立完成识别任务的模型,检验所建模型的正确性,对预测模型进行训练， 使之通过学习后具有 正确处理和辨别能力。,进行“功能”与“非功能”的 判断，根据判断结果计算 模识别的准确性。,识别“功能序列”和“非功能序列”的过程,多序列比对,相关序列选取,模型构建,模型训练,参数调整,应用,确立模型 Profile HMM,Hmmcalibrate,ClustalX,Hmmbuild,Hmmt,Hidden Markov Model,Hidden Markov Model,应用 HMMs has more predictive power than P

29、rofiles. HMM is able to differentiate between insertion and deletion states In profile calculation, a single gap penalty score that is often subjectively determined represents either an insertion or deletion.,Hidden Markov Model,应用 Once an HMM is established based on the training sequences, It can b

30、e used to determine how well an unknown sequence matches the model. It can be used for the construction of multiple alignment of related sequences. HMMs can be used for database searching to detect distant sequence homologs. HMMs are also used in Protein family classification through motif and patte

31、rn identification Advanced gene and promoter prediction, Transmembrane protein prediction, Protein fold recognition.,第二节 核酸序列的预测与鉴定,本节内容 核酸序列预测概念 基因预测 启动子和调控元件预测 酶切位点分析与引物设计,1、核酸序列预测概念,指利用一些计算方式（计算机程序）从基因组序列中发现基因及其表达调控元件的位置和结构的过程。包括： 基因预测（ Gene Prediction ） 基因表达调控元件预测（Promoter and Regulatory Element

32、 Prediction）,Structure of Eukaryotic Genes,AGCATCGAAGTTGCATGACGATGCATGACCTAGCAGCATCGAAGTTGCATGACGATGCATGACCTAGCAAGTTGCATGACGATGCATGACCTAGCAGCATCGAAGTTGCATGACGATGCATGACCTAGTGCATGACGATGCATGACCTAGCAGCATCGAAGTTGCATGACGATGCATGACCTAGCAAGTTGCATGACGATTGACCTAGTGCATGACGATGCATGACCTAGCAGCATCGAAGTTGCATGACGATGCAT

33、GACCTAGCAAGAAGTTGCATGACGATGCATGACCTAGTGCATGACGATGCATGACCTAGCAGCATCGAAGTTGCATGACGATGCATGACCTAGCAAGTTGCATGACGATTGACCTAGTGCATGACGATGCATGACCTAGCAGCATCGCGATGCATGACCTAGCAAGAAGTTGCATGACGATGCATGACCTAGTGCATGACGATGCATGACCTAGCAGCATCGAAGTTGCATGACGATGCATGACCTAGCAAGTTGCATGACGATTGACCTAGTGCATGACTGACCTAGCAGCATCGAAGT

34、TGCATGACGATGCATGACCTAGTGCATGACGATGCATGACCTAGCAGCATCGAAGTTGCATGACGATGCATGACCTAGCAAGTTGCATGACGATTGACCTAGTGCATGACGATGCATGACCTAGCAGCATCGAAGTTGCATGACGATGCATGACCTAGCAAGAAGTTGCATGACGATGCATGACCTAATGC,第二节 核酸序列的预测与鉴定,本节内容 核酸序列预测概念 基因预测 启动子和调控元件预测 酶切位点分析与引物设计,基因预测的概念及意义 原核基因识别 真核基因预测的困难性 真核基因预测的依据 真核基因预测的基本步骤及

35、策略 真核基因预测方法及其基本原理,2、基因预测,概念： Gene Prediction： Given an uncharacterized DNA sequence, find out: Where does the gene starts and ends? detection of the location of open reading frames (ORFs) Which regions code for a protein? delineation of the structures of introns as well as exons (eukaryotic),2.1 基因预

36、测的概念及意义,基因预测的概念及意义,意义： Computational Gene Finding (Gene Prediction) is one of the most challenging and interesting problems in bioinformatics at the moment. Computational Gene Finding is important because So many genomes have been being sequenced so rapidly. Pure biological means are time consuming

37、and costly. Finding genes in DNA sequences is the foundation for all further investigation (Knowledge of the protein-coding regions underpins functional genomics).,基因预测的概念及意义 原核基因识别 真核基因预测的困难性 真核基因预测的依据 真核基因预测的基本步骤及策略 真核基因预测方法及其基本原理,2、基因预测,2.2、原核基因识别,原核基因识别任务的重点是识别开放阅读框，或者说识别长的编码区域。 一个开放阅读框（ORF, ope

38、n reading frame）是一个没有终止编码的密码子序列。,原核基因预测工具介绍 ORF Finder HMM-based gene finding programs GeneMark Glimmer FGENESB RBSfinder,原核基因识别,ORF Finder (Open Reading Frame Finder) /gorf/gorf.html,原核基因识别,zinc-binding alcohol dehydrogenase, novicida(弗朗西丝菌 ),HMM-based gene finding program

39、s GeneMark: Trained on a number of complete microbial genomes /GeneMark/,原核基因识别,HMM-based gene finding programs Glimmer (Gene Locator and Interpolated Markov Modeler): A UNIX program /softlab/glimmer/glimmer.html,原核基因识别,HMM-based gene finding programs FGENESB

40、: Web-based program Trained for bacterial sequences ,原核基因识别,HMM-based gene finding programs RBSfinder: UNIX program Predicted start sites /pub/software/RBSfinder/,原核基因识别,基因预测的概念及意义 原核基因识别 真核基因预测的困难性 真核基因预测的依据 真核基因预测的基本步骤及策略 真核基因预测方法及其基本原理,2、基因预测,Why is Gene Prediction Challenging? C

41、oding density: as the coding/non-coding length ratio decreases, exon prediction becomes more complex. Some facts about human genome Coding regions comprise less than 3% of the genome There is a gene of 2400000 bps, only 14000 bps are CDS ( 0.5 are deemed reliable. This program is trained for sequenc

42、es from vertebrates, Arabidopsis, and maize. It has been used extensively in annotating the human genome.,真核基因预测方法及其基本原理,Ab InitioBased Programs GRAIL (Gene Recognition and Assembly Internet Link)： a web-based program： /public/tools/ based on a neural network algorithm. The pro

43、gram is trained on several statistical features such as splice junctions, start and stop codons, poly-A sites, promoters, and CpG islands. The program scans the query sequence with windows of variable lengths and scores for coding potentials and finally produces an output that is the result of exon

44、candidates. The program is currently trained for human, mouse, Arabidopsis, Drosophila, and Escherichia coli sequences.,真核基因预测方法及其基本原理,Ab InitioBased Programs FGENES (FindGenes) Web-based program: Uses LDA to determine whether a signal is an exon. In addition to FGENES, there are many variants of th

45、e program： FGENESH: make use of HMMs. FGENESH C: similarity based. FGENESH+: combine both ab initio and similarity-based approaches.,真核基因预测方法及其基本原理,Ab InitioBased Programs MZEF (Michael Zhangs Exon Finder) Web based： /genefinder/ Uses QDA for exon prediction. Has not been obvious

46、 in actual gene prediction.,真核基因预测方法及其基本原理,Ab InitioBased Programs HMMgene： Web based： www.cbs.dtu.dk/services/HMMgene HMM-based program. The unique feature of the program is that it uses a criterion called the conditional maximum likelihood to discriminate coding from noncoding features. If a seque

47、nce already has a subregion identified as coding region, which may be based on similarity with cDNAs or proteins in a database, these regions are locked as coding regions. An HMM prediction is subsequently made with a bias toward the locked region and is extended from the locked region to predict th

48、e rest of the gene coding regions and even neighboring genes. The program is in a way a hybrid algorithm that uses both ab initio-based and homology-based criteria.,真核基因预测方法及其基本原理,真核基因预测方法及其基本原理,Homology-Based Programs Homology-based programs are based on the fact that exon structures and exon seque

49、nces of related species are highly conserved. When potential coding frames in a query sequence are translated and used to align with closest protein homologs found in databases, near perfectly matched regions can be used to reveal the exon boundaries in the query. This approach assumes that the data

50、base sequences are correct. It is a reasonable assumption in light of the fact that many homologous sequences to be compared with are derived from cDNA or expressed sequence tags (ESTs) of the same species.,Homology-Based Programs： 优势：With the support of experimental evidence, this method becomes ra

51、ther efficient in finding genes in an unknown genomic DNA. 不足：The drawback of this approach is its reliance on the presence of homologs in databases. If the homologs are not available in the database, the method cannot be used. Novel genes in a new species cannot be discovered without matches in the

52、 database.,真核基因预测方法及其基本原理,Homology-Based Programs GenomeScan web-based server： /genomescan.html Combines GENSCAN prediction results with BLASTX similarity searches. The user provides genomic DNA and protein sequences from related species. The genomic DNA is translated in all six f

53、rames to cover all possible exons. The translated exons are then used to compare with the user-supplied protein sequences. Translated genomic regions having high similarity at the protein level receive higher scores. The same sequence is also predicted with a GENSCAN algorithm, which gives exons pro

54、bability scores. Final exons are assigned based on combined score information from both analyses.,真核基因预测方法及其基本原理,Homology-Based Programs EST2Genome： web-based program：http:/bioweb.pasteur.fr/seqanal/interfaces/est2genome.html To define intronexon boundaries. Purely based on the sequence alignment ap

55、proach The program compares an EST (or cDNA) sequence with a genomic DNA sequence containing the corresponding gene. The alignment is done using a dynamic programmingbased algorithm.,真核基因预测方法及其基本原理,Homology-Based Programs TwinScan / A similarity-based gene-finding server. Pre

56、dict exons How to works： it uses GenScan to predict all possible exons from the genomic sequence. The putative exons are used for BLAST searching to find closest homologs. The putative exons and homologs from BLAST searching are aligned to identify the best match. Only the closest match from a genom

57、e database is used as a template for refining the previous exon selection and exon boundaries.,真核基因预测方法及其基本原理,真核基因预测方法及其基本原理,Consensus-Based Programs These programs work by retaining common predictions agreed by most programs and removing inconsistent predictions. Such an integrated approach may imp

58、rove the specificity by correcting the false positives and the problem of over prediction. However, since this procedure punishes novel predictions, it may lead to lowered sensitivity and missed predictions. Two examples of consensus-based programs are given next.,Consensus-Based Programs GeneComber

59、： a web server： www.bioinformatics.ubc.ca/genecomber/index.php Combines HMMgene and GenScan prediction results. The consistency of both prediction methods is calculated. If the two predictions match, the exon score is reinforced. If not, exons are proposed based on separate threshold scores.,真核基因预测方法及其基本原理,Consensus-Based Programs DIGIT： web server：http:/digit.gsc.riken.go.jp/cgi-bin/index.cgi First, existing gene-finders （ FGENESH, GENSC