我们感谢洛伦实验室的成员，对稿件的意见。我们感谢乔治华盛顿大学系生物科学与艺术和科学的哥伦比亚学院拨款，D.洛伦。

1。基本局部比对搜索工具（BLAST）：在线界面<ol><li>点击该链接访问BLAST 1 web服务器在国家生物技术信息中心（NCBI）。 - <a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi">http://blast.ncbi.nlm.nih.gov/Blast.cgi</a> （ 图1）。 </li><li>输入一个FASTA格式的文本序列（ 见图2为例）在查询框。 </li><li>单击相应的BLAST程序和相关数据库或感兴趣的个别品种在搜索中使用，然后单击“BLAST”。 注：FASTA格式的序列开始与一个“&gt;”符号表示的描述一致。描述必须与“&gt;”符号，序列（ 即 ，核苷酸或氨基酸）跟随在下一行的描述后，立即跟进。从BLAST搜索的输出被看作是HTML，纯文本，XML或打TA统计局（文本或CSV）的默认设置为HTML（ 图3）。 </li></ol> 2。基本局部比对搜索工具（BLAST）：本地可执行文件<ol><li>从这个链接下载最新的BLAST命令行BLAST可执行文件： <a href="ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/">ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/</a> - </li><li> EM&gt; 对于PC用户：双击最新疾风win32.exe文件并接受许可协议，然后单击安装。 注意：默认安装目录为C： NCBI-BLAST-2.2.27 +。 </li><li>配置PC环境变量，如下所示： <ol><li>点击电脑的“开始”按钮，然后用鼠标右键单击“计算机”， </li><li>点击“属性”，在弹出点击“高级”选项卡</li><li>点击“环境变量按钮”，并在新弹出的点击下个“新”按钮e“的用户变量用户”一节</li><li>在弹出的添加变量名“路径”和变量值为“C： NCBI-BLAST-2.2.27 + bin中。 注：在bin目录包 ​​含可执行文件（ 即 BLASTP 等 ）。 </li></ol></li><li> EM&gt; 对于Mac用户：打开终端应用程序（要做到这一点只需打开“搜索”，然后搜索“终端”，这将显示“终端”图标）。到终端窗口输入： &gt; FTP <a href="ftp://ftp.ncbi.nih.gov">ftp.ncbi.nih.gov</a> 注：也可以输入在这个例子中为PC上面使用的URL </li><li>要访问NCBI的ftp站点类型为“无名氏”的名称和密码，然后键入： &gt; CD爆炸/可执行文件/最新</li><li>通过键入以下列出的可执行文件： &gt; LS </li><li>获取最新版本通过键入以下内容（或任何最新的版本是目前）： 2，获得NCBI-BLAST-2.2.7-macosx.tar.gz </li><li>通过键入“exit”退出NCBI的ftp服务器站点。 </li><li>通过键入以下命令解压下载的文件： &gt;焦油XZF NCBI-BLAST-2.2.7-macosx.tar.gz </li><li>添加二进制文件的爆炸可执行文件的位置到您的路径，以便shell可以通过输入查找命令时，通过此目录搜索： &gt; PATH = $ PATH：new_folder_location </li><li>检查这个附加的位置到您的路径，键入： &gt;回声$ PATH </li><li>点击这里下载一个预先格式化的BLAST数据库（这是每日更新）： <a href="ftp://ftp.ncbi.nlm.nih.gov/blast/db/">ftp://ftp.ncbi.nlm.nih.gov/blast/db/</a> </li><li>将数据库置于“DB”文件夹中。 </li><li> EM&gt; 在PC上：打开一个MS-DOS提示符（为此，请单击“开始”，在搜索栏输入“cmd”），并通过键入更改目录到NCBI-BLAST的文件夹： C：用户&gt; CD .. [移动上一级文件夹] C：&gt; CD NCBI-BLAST-2.2.27 + 这将目录更改为： C： NCBI-BLAST-2.2.27 +&gt; </li><li>创建使用下面的“makedb”命令数据库： &gt; makedb - 在DB / briggsae.fasta-DBTYPE普罗特出DB / briggsae 注意：在下面的例子中（ 图4）中的数据库被命名为“briggsae”，并从生物体线虫briggsae由一个连接基团的。 </li><li>创建一个通过插入一个FASTA格式的文本蛋白质序列进“DB”文件夹，名为“测试”查询蛋白质序列。 </li><li>通过BLASTP搜索通过键入以下命令审问数据库： &gt; BLASTP查询DB / test.txt的-分贝分贝/ briggsae出的text.txt </li><li> EM&gt; 在Mac上：通过访问NCBI的ftp网站按照上面的操作步骤（步骤2.4），并下载本地BLAST搜索数据库中的n型： &gt; LCD .. /数据库/ </li><li>通过键入下载感兴趣的基因或序列： &gt;得到NC_ [加入＃]。FNA 注：“FNA”指的是FASTA格式的核苷酸序列和“FAA”指的是FASTA格式的氨基酸序列。 </li><li>输入“quit”退出ftp站点。 </li><li>使数据库键入： &gt; makeblastdb - 在DB / mouse.faa出鼠标DBTYPE普罗特</li><li>插入一张格式化的快速查询序列到“bin”文件夹，并用以下命令审问数据库： &gt; BLASTP查询“你query.fasta”-DB“数据库”出results.txt </li></ol> 3。产生多序列比对<ol><li>点击这些链接可以访问常用的多序列比对（MSA）计划： ClustalW比6 <a href="http://www.clustal.org/">http://www.clustal.org/</a> 卡利GN 7 <a href="http://msa.sbc.su.se/cgi-bin/msa.cgi">http://msa.sbc.su.se/cgi-bin/msa.cgi</a> MAFFT 8,9 <a href="http://mafft.cbrc.jp/alignment/software/">http://mafft.cbrc.jp/alignment/software/</a> 肌肉10 <a href="http://www.drive5.com/muscle/">http://www.drive5.com/muscle/</a> T型咖啡11 <a href="http://www.tcoffee.org/Projects/tcoffee/">http://www.tcoffee.org/Projects/tcoffee/</a> PROBCONS 12 <a href="http://toolkit.tuebingen.mpg.de/probcons">http://toolkit.tuebingen.mpg.de/probcons</a> </li><li>点击此链接- <a href="http://tcoffee.crg.cat/apps/tcoffee/do:regular">http://tcoffee.crg.cat/apps/tcoffee/do:regular</a> -和输入FASTA格式的序列数据，在查询框 注：从T型咖啡机的输出示例可以在图5中可以看出，类似残渣的颜色编码。 </li><li>下载CLUSTAL MSA作为一个命令行版本（ClustalW比）或图形v版为（CLUSTALX）通过点击这个链接： <a href="http://www.clustal.org/clustal2/">http://www.clustal.org/clustal2/</a> -然后点击相应的可执行文件（ 即赢，在Linux，Mac OS X）。 </li><li>上传数据为FASTA格式的序列文本和调整（ 图6）。 </li></ol> 4。确定进化的最佳拟合模型<ol><li>点击这里下载ProtTest 13个方案： <a href="http://darwin.uvigo.es/our-software/">http://darwin.uvigo.es/our-software/</a> </li><li>一旦ProtTest被下载，就ProtTest.jar双击文件</li><li>一旦ProtTest推出后，点击“选择文件”和加载序列数据（ 图7）。 </li><li>然后点击“开始”，程序将开始（ 图8）。 注：运行（ 图8）完成后，程序会显示基于标准的最佳模式，例如<html> “根据AIC最佳模式：WAG + I + G” </li></ol> 5。通过最大似然或贝叶斯推理推断基于序列的系统发育<ol><li>下载PHYML 4在这里： <a href="https://code.google.com/p/phyml/">https://code.google.com/p/phyml/</a> </li><li>通过双击相应的应用程序（ 即 PHYML的Windows，Linux的PHYML 等 ），并在界面窗口就会弹出（ 图9）启动的可执行文件。 </li><li>通过键入以下命令加载输入序列作为PHYLIP格式的序列： &gt;“文件名”。PHY 注意： 要序列格式之间进行转换，使用可用的“Readseq”网络计划在- <a href="http://iubio.bio.indiana.edu/cgi-bin/readseq.cgi">http://iubio.bio.indiana.edu/cgi-bin/readseq.cgi</a> 。 </li><li>通过键入“Y”启动该程序。 </li><li>下载MrBayes 5的位置： rceforge.net / download.php“&gt; http://mrbayes.sourceforge.net/download.php </li><li>要启动该程序，点击可执行文件并读取NEXUS格式的序列数据到程序中键入： &gt;执行“文件名”。NEX </li><li>将进化模型。 </li><li>选择世代数通过键入运行： &gt; mcmcp NGEN = 1000000 [这台世代数1000000] &gt;油底壳燃尽= 10000 [此设置燃尽10000] </li><li>保存结果文件的分支长度，键入： &gt; mcmcp savebrlens =是</li><li>运行由打字分析： &gt; MCMC </li><li>使用“SUMT”命令总结的树木。 </li></ol> 6。可视化系统发育<ol><li>查看树查看器程序列表在这里： <a href="http://www.treedyn.org/overview/editors.html">http://www.treedyn.org/overview/editors.html</a> </li><li>下载的TreeView 14 PROGR在这里： <a href="http://taxonomy.zoology.gla.ac.uk/rod/treeview.html">http://taxonomy.zoology.gla.ac.uk/rod/treeview.html</a> </li></ol>

<ol>
	<li>Altschul, S. F., Carroll, R. J., Lipman, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Weights+for+data+related+by+a+tree.">Weights for data related by a tree.</a> J. Mol. Biol. 207 (4), 647-653 (1989).</li><li>Akaike, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+look+at+the+statistical+model+identification.">A new look at the statistical model identification.</a> IEEE Trans. Automat. Contr. 19 (6), 706-723 (1974).</li><li>Schwarz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+the+dimension+of+a+model.">Estimating the dimension of a model.</a> Ann. Stat. 6 (2), 461-464 (1978).</li><li>Guindon, S., Gascuel, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple,+fast,+and+accurate+algorithm+to+estimate+large+phylogenies+by+maximum+likelihood.">A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood.</a> Syst. Biol. 52 (5), 696-704 (2003).</li><li>Huelsenbeck, J. P., Ronquist, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MRBAYES:+Bayesian+inference+of+phylogenetic+trees.">MRBAYES: Bayesian inference of phylogenetic trees.</a> Bioinformatics. 17 (8), 754-755 (2001).</li><li>Thompson, J. D., Higgins, D. G., Gibson, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CLUSTAL+W:+Improving+the+sensitivity+of+progressive+multiple+sequence+alignment+through+sequence+weighting,+position-specific+gap+penalties+and+weight+matrix+choice.">CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.</a> Nucleic Acids Res. 22 (22), 4673-4680 (1994).</li><li>Lassmann, T., Sonnhammer, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kalign--an+accurate+and+fast+multiple+sequence+alignment+algorithm.">Kalign--an accurate and fast multiple sequence alignment algorithm.</a> BMC Bioinformatics. 6, 298 (2005).</li><li>Katoh, K., Kuma, K., Toh, H., Miyata, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAFFT+version+5:+Improvement+in+accuracy+of+multiple+sequence+alignment.">MAFFT version 5: Improvement in accuracy of multiple sequence alignment.</a> Nucleic Acids Res. 33 (2), 511-518 (2005).</li><li>Katoh, K., Misawa, K., Kuma, K., Miyata, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAFFT:+A+novel+method+for+rapid+multiple+sequence+alignment+based+on+fast+fourier+transform.">MAFFT: A novel method for rapid multiple sequence alignment based on fast fourier transform.</a> Nucleic Acids Res. 30 (14), 3059-3066 (2002).</li><li>Edgar, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MUSCLE:+Multiple+sequence+alignment+with+high+accuracy+and+high+throughput.">MUSCLE: Multiple sequence alignment with high accuracy and high throughput.</a> Nucleic Acids Res. 32 (5), 1792-1797 (2004).</li><li>Notredame, C., Higgins, D. G., Heringa, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T-coffee:+A+novel+method+for+fast+and+accurate+multiple+sequence+alignment.">T-coffee: A novel method for fast and accurate multiple sequence alignment.</a> J. Mol. Biol. 302 (1), 205-217 (2000).</li><li>Do, C. B., Mahabhashyam, M. S., Brudno, M., Batzoglou, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ProbCons:+Probabilistic+consistency-based+multiple+sequence+alignment.">ProbCons: Probabilistic consistency-based multiple sequence alignment.</a> Genome Res. 15 (2), 330-340 (2005).</li><li>Darriba, D., Taboada, G. L., Doallo, R., Posada, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ProtTest+3:+Fast+selection+of+best-fit+models+of+protein+evolution.">ProtTest 3: Fast selection of best-fit models of protein evolution.</a> Bioinformatics. 27 (8), 1164-1165 (2011).</li><li>Page, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TreeView:+An+application+to+display+phylogenetic+trees+on+personal+computers.">TreeView: An application to display phylogenetic trees on personal computers.</a> Comput. Appl. Biosci. 12 (4), 357-358 (1996).</li><li>Darriba, D., Taboada, G. L., Doallo, R., Posada, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=jModelTest+2:+More+models,+new+heuristics+and+parallel+computing.">jModelTest 2: More models, new heuristics and parallel computing.</a> Nat. Methods. 9 (8), 772 (2012).</li><li>Chevenet, F., Brun, C., Banuls, A. L., Jacq, B., Christen, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TreeDyn:+Towards+dynamic+graphics+and+annotations+for+analyses+of+trees.">TreeDyn: Towards dynamic graphics and annotations for analyses of trees.</a> BMC Bioinformatics. 7, 439 (2006).</li></ol>

我们什么都没有透露。

我们希望这篇文章是它将作为一个出发点，引导研究人员或学生，是新的系统发育。基因组测序计划已经变得不那么昂贵，在过去的几年里，因而这种技术，用户需求在不断增加，现在生产的大型数据集的顺序是司空见惯的小型实验室。这些数据集通常为研究人员提供的基因组需要一个进化的框架开始了解它们的功能。此外，由于系统发育是找到一个家在越来越多的研究实验室，我们还打算对这?...

为了了解两个（或更多）的物种是如何进化，首先需要从每个样品获得的序列或形态数据，这些数据代表的数量，我们可以通过使用进化空间来衡量他们的关系。只是测量直线距离时，其可用（ 例如，英里，英寸，微米）更多的数据将等同于一个更精确的测量喜欢。测功，与研究者可以推断进化距离的精度在很大程度上受到可用来测量关系信息的数据量的影响。此外，因为不同样品演变以不同的速率和通过不同的机制，我们用来测量两个类群之间的关系的方法也直接影响进化测量的精度。因此，由于进化的关系并不直接观察，而是从序列或形态学数据，推断进化的问题外推关系变成统计中的一个。系统发生学是生物学关注的运用统计模型的进化模式，以优化重构类群间的进化史的分支。分类单元之间的这种重建被称为类群的系统发育关系 。 为了帮助弥合分子生物学家和进化生物学家我们在这里介绍一步步管道从一组序列​​的系统发育推断之间在专业知识的差距。首先，我们详细地使用基本局部比对搜索工具（BLAST 1）的算法，通过基于web的接口，并通过使用本地可执行涉及数据库询问的步骤，这是经常在获得相似序列的列表，以一个身份不明的第一步查询，虽然一些研究人员还可能有兴趣在收集数据的单个组通过网络接口，如Phylota（http://www.phylota.net/）。 BLAST是一个算法Comparing对序列数据库的一级氨基酸或核苷酸序列数据来搜索“命中”，类似的查询序列。 BLAST程序是由斯蒂芬Altschul 等设计。在美国国立卫生研究院（NIH）1。爆炸服务器由许多不同的程序，这里是一些最常见的BLAST程序的列表： I） 核苷酸-核苷酸BLAST（BLASTN）：此程序需要的DNA序列输入和从DNA数据库返回最相似的DNA序列，用户指定（ 如特定的生物）。 ⅱ） 蛋白质-蛋白质BLAST（BLASTP）：在这里，用户输入的蛋白序列和程序从用户指定的蛋白质数据库返回最相似的蛋白序列。 三） 特定位置的迭代BLAST（PSI-BLAST）（blastpgp）：用户输入是一个保护制服在序列，其返回一组密切相关的蛋白质，并从这个数据集在一个保守的更新生成的。接下来一个新的查询只使用其中用于询问蛋白质数据库中的这些保守的“图案”产生，这会返回一个更大的组蛋白的这一套新的保守的“图案”被提取，然后用询问的蛋白质数据库中，直到一个更大的组蛋白被重新调谐，并产生另一个配置文件并重复该过程。由包括相关蛋白质进入每个步骤中的查询这个程序允许用户确定比较发散序列。 ⅳ） 核苷酸6帧翻译蛋白质（BLASTX）：这里的用户提供了转换成六格概念翻译产物（ 即核苷酸序列输入两条链）与蛋白质序列数据库。 V） 核苷酸6帧翻译核苷酸6帧转换（TBLASTX）：这个程序需要一个DNA核苷酸序列的输入和转换输入到它比较反对的核苷酸序列数据库的六框翻译全部六个框架概念翻译产物。 六） 蛋白质-核苷酸6帧转换（TBLASTN）：这个程序使用了蛋白质序列输入要与之比较的核苷酸序列数据库的所有六个阅读框。 接下来，我们将介绍常用的方案，以便从序列数据集生成多序列比对（MSA），这之后是一个用户指南，以确定进化的最佳拟合模型序列数据集节目。系统发育重建是一个统计问题，并且因为这一点，系统发育分析方法需要将一个统计框架。这个统计框架成为它采用了数据集中的顺序变化的演化模型。这种进化莫德尔是由一组有关的核苷酸或氨基酸的替换过程的假设的，并为特定的数据集的最佳拟合模型可以通过统计测试来选择。将适合不同型号的数据可以通过似然比检验（LRTS）或信息标准，在一组可能的人选择最合适的模型进行比较。两种常见的信息标准是赤池信息量准则（AIC）2和贝叶斯信息准则（BIC）3。一旦一个最佳比对产生时，有许多不同的方法来创建自对准的数据的系统发育关系。还有推断的进化关系的多种方法;广泛地说，它们可分为两类：基于距离的方法和基于序列的方法。基于距离的计算方法从序列成对距离，然后用这些距离来获得树。基于序列的方法，直接使用序列比对，通常搜索吨采用最优标准REE空间。我们概述2基于序列的方法来重建系统发育关系：这些都是PHYML 4，它实现了最大似然框架，MrBayes 5它采用贝叶斯马尔可夫链蒙特卡罗推论。似然和贝叶斯方法提供了系统发育重建一个统计框架。通过提供对常用树构建工具的用户信息，我们将为读者介绍来推断亲缘关系所需的必要数据。

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		<tr height="21">
			<td height="21" style="height:21px;">BLAST webpage&#160;</td>
			<td></td>
			<td></td>
			<td><a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi">http://blast.ncbi.nlm.nih.gov/Blast.cgi</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BLAST executables&#160;</td>
			<td></td>
			<td></td>
			<td><a href="ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/">ftp://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Preformatted BLAST databases</td>
			<td></td>
			<td></td>
			<td><a href="ftp://ftp.ncbi.nlm.nih.gov/blast/db/">ftp://ftp.ncbi.nlm.nih.gov/blast/db/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Clustal</td>
			<td></td>
			<td></td>
			<td><a href="http://www.clustal.org/">http://www.clustal.org/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kalign</td>
			<td></td>
			<td></td>
			<td><a href="http://msa.sbc.su.se/cgi-bin/msa.cgi">http://msa.sbc.su.se/cgi-bin/msa.cgi</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MAFFT</td>
			<td></td>
			<td></td>
			<td><a href="http://mafft.cbrc.jp/alignment/software/">http://mafft.cbrc.jp/alignment/software/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MUSCLE</td>
			<td></td>
			<td></td>
			<td><a href="http://www.drive5.com/muscle/">http://www.drive5.com/muscle/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T-Coffee</td>
			<td></td>
			<td></td>
			<td><a href="http://www.tcoffee.org/Projects/tcoffee/">http://www.tcoffee.org/Projects/tcoffee/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PROBCONS</td>
			<td></td>
			<td></td>
			<td><a href="http://toolkit.tuebingen.mpg.de/probcons">http://toolkit.tuebingen.mpg.de/probcons&#160;</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Se-Al&#160;</td>
			<td></td>
			<td></td>
			<td><a href="http://tree.bio.ed.ac.uk/software/seal/">http://tree.bio.ed.ac.uk/software/seal/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSEdit&#160;</td>
			<td></td>
			<td></td>
			<td><a href="http://www.bsedit.org/">http://www.bsedit.org/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">JalView</td>
			<td></td>
			<td></td>
			<td><a href="http://www.jalview.org/">http://www.jalview.org/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SeaView</td>
			<td></td>
			<td></td>
			<td><a href="http://pbil.univ-lyon1.fr/software/seaview.html">http://pbil.univ-lyon1.fr/software/seaview.html</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ProtTest&#160;</td>
			<td></td>
			<td></td>
			<td style="width:447px;"><a href="https://code.google.com/p/prottest3/">https://code.google.com/p/prottest3/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Java Runtime&#160;</td>
			<td></td>
			<td></td>
			<td><a href="http://www.java.com/en/download/chrome.jsp">http://www.java.com/en/download/chrome.jsp</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Readseq</td>
			<td></td>
			<td></td>
			<td><a href="http://iubio.bio.indiana.edu/cgi-bin/readseq.cgi">http://iubio.bio.indiana.edu/cgi-bin/readseq.cgi</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">jModelTest</td>
			<td></td>
			<td></td>
			<td style="width:447px;"><a href="https://code.google.com/p/jmodeltest2/">https://code.google.com/p/jmodeltest2/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PhyML</td>
			<td></td>
			<td></td>
			<td><a href="https://code.google.com/p/phyml/">https://code.google.com/p/phyml/</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MrBayes</td>
			<td></td>
			<td></td>
			<td><a href="http://mrbayes.sourceforge.net/download.php">http://mrbayes.sourceforge.net/download.php</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TreeView</td>
			<td></td>
			<td></td>
			<td><a href="http://taxonomy.zoology.gla.ac.uk/rod/treeview.html">http://taxonomy.zoology.gla.ac.uk/rod/treeview.html</a></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TreeDyn</td>
			<td></td>
			<td></td>
			<td style="width:447px;"><a href="http://www.treedyn.org/">http://www.treedyn.org/</a></td>
		</tr>
	</tbody>
</table>

a practical guide to phylogenetics for nonexperts

许多研究者，跨越令人难以置信的多样化灶，正在申请系统学他们的研究问题（次）。然而，许多研究人员是新的这个话题，所以它呈现固有的问题。在这里，我们编译一个实用介绍系统发育的非专家。我们概述了一步一步的方式，管道用于从基因序列数据集可靠的系统发育。我们从一个用户指南的相似性搜索工具通过在线接口以及本地可执行文件。接下来，我们将探讨产生多序列比对后，使用软件来确定进化的最佳拟合模型协议的程序。然后，我们通过最大似然法和贝叶斯准则概述协议进行重建亲缘关系，最后描述了工具的可视化系统进化树。虽然这是不以任何手段的进化方法的详尽描述，它为读者提供实用的开始了信息离子由phylogeneticists普遍使用的关键软件应用程序。该愿景这篇文章将是它可以作为一个实用的培训工具，研究人员着手进行系统发育研究，也作为可能被纳入到教室或教学实验室的教育资源。

寻找相似的查询可以让研究人员归咎于一个潜在的身份，以新的序列，并推断序列之间的关系。高炉1文件输入类型是FASTA格式的文本序列或GenBank登录号。 FASTA格式的序列开始与一个“&gt;”符号（ 图2）所示的描述一致。描述必须与“&gt;”符号，序列（ 即 ，核苷酸或氨基酸）跟随在下一行的描述后，立即跟进。在保存和编辑序列文件，最好使用文本编辑器如PC或TextWrang...

Watch this Scientific Journal Video about A Practical Guide to Phylogenetics for Nonexperts at JoVE.com

A Practical Guide to Phylogenetics for Nonexperts

这里，我们描述了一步一步管道，用于从核苷酸或氨基酸序列数据集可靠的系统发育。本指南的目的是服务于研究人员或学生对新的系统发育分析。

实用指南系统发育的非专家

Many researchers, across incredibly diverse foci, are applying phylogenetics to their research question(s). However, many researchers are new to this ...

a-practical-guide-to-phylogenetics-for-nonexperts

Research

JoVE Journal

Biology

35.2K 次观看.  The George Washington University. 这里，我们描述了一步一步管道，用于从核苷酸或氨基酸序列数据集可靠的系统发育。本指南的目的是服务于研究人员或学生对新的系统发育分析。 

视频: A Practical Guide to Phylogenetics for Nonexperts

The Ladder Rung Walking Task: A Scoring System and its Practical Application.

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to elaborate distinct aspects of motor function and to determine even subtle loss of movement capacity. To enhance efficacy and resolution of testing, tests should permit qualitative and quantitative measures of motor function and be sensitive to changes in performance during recovery periods. The present study describes a new task to assess skilled walking in the rat to measure both forelimb and hindlimb function at the same time. Animals are required to walk along a horizontal ladder on which the spacing of the rungs is variable and is periodically changed. Changes in rung spacing prevent animals from learning the absolute and relative location of the rungs and so minimize the ability of the animals to compensate for impairments through learning. In addition, changing the spacing between the rungs allows the test to be used repeatedly in long-term studies. Methods are described for both quantitative and qualitative description of both fore- and hindlimb performance, including limb placing, stepping, co-ordination. Furthermore, use of compensatory strategies is indicated by missteps or compensatory steps in response to another limb&#x2019;s misplacement. 

The ladder rung walking task is a new test to assess skilled walking and measure both forelimb and hindlimb placing, stepping, and inter-limb co-ordination.

梯级步行任务：一个评分系统，其实际应用。

Progress in the development of animal models for/stroke, spinal cord injury, and other neurodegenerative disease requires tests of high sensitivity to ...

科研

生物学

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing and adult tissue. An advance in this concept is Genetic Inducible Fate Mapping (GIFM), linking gene expression, cell fate, and cell behaviors in vivo, to create fate maps based on genetic lineage.
GIFM exploits X-CreER lines where X is a gene or set of gene regulatory elements that confers spatial expression of a modified bacteriophage protein, Cre recombinase (CreERT). CreERT contains a modified estrogen receptor ligand binding domain which renders CreERT sequestered in the cytoplasm in the absence of the drug tamoxifen. The binding of tamoxifen releases CreERT, which translocates to the nucleus and mediates recombination between DNA sequences flanked by loxP sites. In GIFM, recombination typically occurs between a loxP flanked Stop cassette preceding a reporter gene such as GFP.
Mice are bred to contain either a region- or cell type-specific CreER and a conditional reporter allele. Untreated mice will not have marking because the Stop cassette in the reporter prevents further transcription of the reporter gene. We administer tamoxifen by oral gavage to timed-pregnant females, which provides temporal control of CreERT release and subsequent translocation to the nucleus removing the Stop cassette from the reporter. Following recombination, the reporter allele is constitutively and heritably expressed. This series of events marks cells such that their genetic history is indelibly recorded. The recombined reporter thus serves as a high fidelity genetic lineage tracer that, once on, is uncoupled from the gene expression initially used to drive CreERT.
We apply GIFM in mouse to study normal development and ascertain the contribution of genetic lineages to adult cell types and tissues. We also use GIFM to follow cells on mutant genetic backgrounds to better understand complex phenotypes that mimic salient features of human genetic disorders.
This video article guides researchers through experimental methods to successfully apply GIFM. We demonstrate the method using our well characterized Wnt1-CreERT;mGFP mice by administering tamoxifen at embryonic day (E)8.5 via oral gavage followed by dissection at E12.5 and analysis by epifluorescence stereomicroscopy. We also demonstrate how to micro-dissect fate mapped domains for explant preparation or FACS analysis and dissect adult fate-mapped brains for whole mount fluorescent imaging. Collectively, these procedures allow researchers to address critical questions in developmental biology and disease models.

A Practical Approach to Genetic Inducible Fate Mapping: A Visual Guide to Mark and Track Cells In Vivo

Genetic Inducible Fate Mapping (GIFM) marks and tracks cells with fine spatial and temporal control in vivo and elucidates how cells from a specific genetic lineage contribute to developing and adult tissues. Demonstrated here are the techniques required to fate map E12.5 mouse embryos for epifluorescent and explant analysis.

一个实用的方法遗传诱导的命运映射：一个视觉引导标记和跟踪细胞在体内</em

Fate maps are generated by marking and tracking cells in vivo to determine how progenitors contribute to specific structures and cell types in developing ...

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

Laboratory tests can be done on the cellular or fluid portions of the blood. The use of different blood collection tubes determines the portion of the blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.
The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes1, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.
We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.

We are describing a new method of isolating genomic DNA from whole blood collected for plasma/serology. After plasma collection, the compacted blood is usually discarded. Our novel method represents a significant improvement over existing methods and makes DNA and plasma available from a single collection, without requesting additional blood.

一种实用的新的方法来提取基因组DNA保存血浆蛋白从血液采集套件

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a &#34;black box&#34; because an experimentalist does not know if it has been performed successfully until the last of several steps. Moreover, the quality of western blot data is sometimes challenged due to a lack of effective quality control tools in place throughout the western blotting process. Here we describe the V3 western workflow, which applies stain-free technology to address the major concerns associated with the traditional western blot protocol. This workflow allows researchers: 1) to run a gel in about 20-30 min; 2) to visualize sample separation quality within 5 min after the gel run; 3) to transfer proteins in 3-10 min; 4) to verify transfer efficiency quantitatively; and most importantly 5) to validate changes in the level of the protein of interest using total protein loading control. This novel approach eliminates the need of stripping and reprobing the blot for housekeeping proteins such as &#946;-actin, &#946;-tubulin, GAPDH, etc. The V3 stain-free workflow makes the western blot process faster, transparent, more quantitative&#160;and reliable.

V3 workflow is a western blot procedure using stain-free gels. The stain-free technology allows researchers to visualize protein separation quality, to verify the transfer efficiency, and most importantly, to validate the change in the protein of interest using total protein quantification as a reliable loading control.

V3 无污渍工作流，适用于西式印迹中实用、方便和可靠的总蛋白质加载控制

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a &#34;black ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

指南现代荧光定量免疫印迹与故障排除策略

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

Endotracheal Intubation of Rabbits Using a Polypropylene Guide Catheter

Endotracheal intubation in rabbits can be challenging due to their unusual anatomy. Achieving a patent airway during anesthesia is critical for the avoidance of airway obstruction, prevention of gastric tympany, and to allow ventilatory support. Due to the difficulty of intubation, alternative methods such as the use of laryngeal mask airways or laryngeal tubes have been explored. However, these methods do not result in direct access to the trachea and thus may present a risk for development of complications. In addition, lack of direct intubation of the trachea can result in personnel exposure to waste anesthetic gases. Numerous methods for endotracheal intubation have been described, including blind placement, use of a fiberoptic laryngoscope or endoscope, and cricoid placement. Despite these numerous publications, many still struggle to achieve success. Here we provide a detailed description of an intubation technique that can be taught with minimal training with a short time to proficiency. Briefly, after administration of injectable anesthesia and proper positioning of the rabbit, a polypropylene catheter is placed into the trachea by direct visualization using a laryngoscope. The catheter is then used as a guide to direct the endotracheal tube into the trachea. This method allows for intubation without the need for expensive equipment and can be performed by a single individual without the need for an assistant. In conclusion, this technique can be easily taught and performed at very little cost in any clinical or research setting.

Endotracheal intubation in rabbits is challenging due to their unusual anatomy. Here we present a technique for direct intubation of the trachea using a polypropylene catheter as a guide. This method utilizes relatively inexpensive supplies, requires minimal training, and can be easily performed in any clinical setting.

用聚丙烯导管引导家兔气管插管

Endotracheal intubation in rabbits can be challenging due to their unusual anatomy. Achieving a patent airway during anesthesia is critical for the ...

Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool operating by disruption of essential gene expression. Although different methods, such as injection, feeding, and soaking, have been reported for successful delivery of double-stranded RNA (dsRNA), the efficiency of RNAi through oral delivery of dsRNA is highly variable among different insect groups. The German cockroach, Blattella germanica, is highly sensitive to the injection of dsRNA, as shown by many studies published previously. The present study describes a method to demonstrate that the dsRNA encapsulated with liposome carriers is sufficient to retard the degradation of dsRNA by midgut juice. Notably, the continuous feeding of dsRNA encapsulated by liposomes significantly reduces the tubulin expression in the midgut, and led to the death of cockroaches. In conclusion, the formulation and utilization of dsRNA lipoplexes, which protect dsRNA against nucleases, could be a practical use of RNAi for insect pest control in the future.

This manuscript demonstrates the depletion of gene expression in the midgut of the German cockroach through oral ingestion of double-stranded RNA encapsulated in liposomes.

rna 干扰的实际应用: 口服双链 rna 在脂质体载体为蟑螂

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool ...

A Sectioning, Coring, and Image Processing Guide for High-Throughput Cortical Bone Sample Procurement and Analysis for Synchrotron Micro-CT

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been studied substantially using traditional two-dimensional techniques. Recent advancements in X-ray imaging technology via desktop micro-computed tomography (&#181;CT) and synchrotron radiation micro-computed tomography (SR&#181;CT) have allowed for the acquisition of high-resolution three-dimensional (3D) scans of a larger field of view (FOV) than other 3D imaging techniques (e.g., SEM) providing a more complete picture of microscopic structures within human cortical bone. The specimen should be accurately centered within the FOV, however, to limit the appearance of streak artifacts known to impact data analysis. Previous studies have reported procurement of irregularly shaped rectilinear bone blocks that result in imaging artifacts due to uneven edges or image truncation. We have applied a geological sampling protocol (coring) to procure consistently sized cortical bone core specimens for SR&#181;CT experiments from the anterior aspect of human femora. This coring method is efficient and minimally destructive to tissue. It creates uniform cylindrical samples that decrease imaging artifacts by nature of being isometric during rotation and providing a uniform path length for X-ray beams throughout scanning. Image processing of X-ray tomographic data of cored and irregularly shaped samples confirms the potential of the technique to improve visualization and analysis of cortical bone microarchitecture. A goal of this protocol is to deliver a reliable and repeatable method for the extraction of cortical bone cores that is adaptable for various types of high-resolution bone imaging experiments. An overarching goal of the work is to create a standardized cortical bone procurement for SR&#181;CT that is affordable, consistent, and straightforward. This procedure may further be adapted by researchers in related fields who commonly evaluate hard composite materials such as in biological anthropology, geosciences, or material sciences.

We employed a geological (coring) sampling protocol to procure cortical bone specimens of uniform size for SR&#181;CT experiments from the anterior aspect of human femora. This method is minimally destructive, efficient, results in cylindrical specimens that minimize imaging artifacts from irregular sample shapes and improves microarchitectural visualization and analysis.

同步加速器微CT高通量皮质骨样品采购和分析的剖面、科林和图像处理指南

Bone is a dynamic and mechanically active tissue that changes in structure over the human lifespan. The products of the bone remodeling process have been ...

A Step-by-Step Guide to Mosquito Electroantennography

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when acquiring a blood-meal. To locate a host to feed on, mosquitoes rely on a wide range of sensory cues, including visual, mechanical, thermal, and olfactory. The study details a technique, electroantennography (EAG), that allows researchers to assess whether the mosquitoes can detect individual chemicals and blends of chemicals in a concentration-dependent manner. When coupled with gas-chromatography (GC-EAG), this technique allows to expose the antennae to a full headspace/complex mixture and determines which chemicals present in the sample of interest, the mosquito can detect. This is applicable to host body odors as well as plant floral bouquets or other ecologically relevant odors (e.g., oviposition sites odorants). Here, we described a protocol that permits long durations of preparation responsiveness time and is applicable to both female and male mosquitoes from multiple genera, including Aedes, Culex, Anopheles, and Toxorhynchites mosquitoes. As olfaction plays a major part in mosquito-host interactions and mosquito biology in general, EAGs and GC-EAG can reveal compounds of interest for the development of new disease vector control strategies (e.g., baits). Complemented with behavioral assays, the valence (e.g., attractant, repellent) of each chemical can be determined.

The present article details a step-by-step protocol for successful and low noise electroantennograms in several genera of mosquitoes, including both females and males.

蚊子电图分步指南

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when ...

开发 FSH 水平升高的卵巢切除小鼠模型

Exploring Independent Effects of Follicle-Stimulating Hormone In Vivo in a Mouse Model

During the transition from a reproductive to a nonreproductive phase (menopause), many women experience significant physiological and pathological changes, including decreased bone mass, increased blood lipids, and increased visceral adiposity. Levels of follicle-stimulating hormone (FSH) rise during the menopausal transition. Many studies have shown that FSH in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. Thus, building an animal model that can help study the independent effects of FSH in vivo is particularly important. In this study, C57BL/6 female mice were ovariectomized and supplemented with estradiol valerate (OVX + E2) to eliminate the effect of the hypothalamic-pituitary-gonadal axis. The OVX + E2 mice received solvent (N.S.) or different doses of recombinant FSH via intraperitoneal injection to create a mouse model (OVF) characterized by relatively stable estrogen and rising FSH levels. Thus, we successfully generated an experimental mouse model to mimic the early stage of menopause transition, characterized by elevated serum FSH levels. The OVF model has the advantages of being stable, low cost, and easy to operate, which is suitable for studies to explore the extragonadal actions of FSH. Here, we describe detailed protocols for the mouse OVF model.

作者聚焦：研究围绝经期女性 FSH 与病理生理变化之间的关系 - 来自小鼠模型的见解 

Follicle-stimulating hormone (FSH) in various extragonadal tissues and organs is associated with the pathogenesis of multiple diseases. The ovariectomized and FSH-treated mouse model (OVF) can be used to explore the extragonadal actions of FSH.