作者想表达自己的感激之情谁开发银河和DAVID，从而取得NGS科学界广泛接受的实验室，团体和个人。在我们的生物信息学的培训，在匹兹堡大学的同事提供的帮助和建议是公认的。这项工作是由一个埃里森医学基金会新学者在老龄化奖（AG-NS-0879-12）和美国国立卫生研究院（R01AG051659），以股份公司的资助。

<ol>
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作者什么都没有透露。

在现代生物学银河测序平台的意义银河项目已成为帮助生物学家没有生物信息学培训，以处理和快速，高效地分析高通量测序数据的工具。曾经被视为一个艰巨的任务，这个公开可用的平台，取得了运行复杂的生物信息学算法来分析NGS数据的简单，可靠，简单的过程。除了托管范围广泛的生物信息学工具，成功的关键银河也是它的用户界面的该鞋带复杂测序分析的各个方面一起以直观和无缝的方式的简单性。由于这些特征，银河管道已获得广泛应用之间的生物学家，包括线虫的研究人员。除了与RNA-Seq的分析管道熟悉的用户，银河也有助于奠定基础，基本生物学家把握数据分析的概念，并了解有关的工具。这方面的知识素数的用户可能进一步追求更复杂的生物信息学平台，如"R"和&#39;Python的。除了银河，其他工具和包可商业和开源解决方案，可用于RNA-Seq的分析。商业选项往往是独立的软件包，这是用户友好的，但也可以对个别研究者谁不经常使用NGS昂贵。可替代地，开源平台如BioWadrobe 34和35 ArrayExpressHTS要求命令行和运行脚本，这对非生物信息学家显著挑战的工作知识。因此，银河仍然是一个受欢迎的和不可缺少的资源。 在协议中的关键步骤银河和大卫费力优势尽管如此，一个成功的RNA-Seq的实验仍在从根本上依赖于精心设计和实验步骤执行。举例来说，关键是要确保遗传同质性的RNA-Seq的比较两个菌株之前，并确定是否有发育率的差异。从年龄匹配的菌株分离RNA是至关重要的为好。类似地，为了解释的相同菌株中的基因表达的变化，它运行每个菌株的两个或更多个"生物学重复"是重要的。这基本上意味着生长，并从菌株收获蠕虫在至少twoindependent实验被测序，虽然3个生物学重复是推荐的标准。星系结合来自多个生物重复的数据，使得菌株之间的报告基因表达的差异不是简单"样本内"的变异的结果。 一个关键的设计决策是对使用单端与双末端测序。同单末端测序，每个片段进行测序单向所以过程更快，更便宜，适合用于转录分析。在配对末端测序，一旦片段从一端测序，以其他的，第二轮测序是在相反方向上重新开始。它提供了更深入的数据和基因组的附加的定位信息，因此更适合于从头基因组组装，新SNP识别和用于识别表观遗传修饰，缺失，插入，倒位和。类似地，总读取次数和有足够的差异表达研究所需基因组覆盖度的程度是依赖于上下文的。对于小的基因组，如细菌和真菌，〜500万读取就足够了，而在蠕虫和苍蝇〜千万读取提供足够的覆盖范围。对于大基因组如小鼠和人类的生物，15-25万元是读取所需的范围。此外，在读人数和覆盖面，这也是小鬼ortant大多数NGS的读取对齐到参考基因组。的&lt;70％的读出对准指示差NGS或污染物的存在的。总体而言，对于线虫 RNA测序研究，三次生物学重复测序用50 bp的单向测序导致〜10-15百万读取并〜5到10倍的每个样品的基因组覆盖是一种理想的目标。 尽管易于使用银河，有几个点，以确保平稳，无毛刺数据分析方面的经验要记住。这是需要用户拥有的已使用的各种工具的用途和功能的一个基本的了解。每个星系工具需要的参数选择和理解该工具将帮助基于实验的需求的用户优化设置。银河帮助页面解释每一个参数，并建议用户仔细阅读这些细节上的测试变量决定。 基因名单所得对OST RNA测序分析仅仅是基因的列表，直到它被开采使用DAVID生物学相关的数据。这是一个至关重要的锻炼是基于个体的基因数据转换成基于生物处理结果。因此探索使用各种分析DAVID提供RNA测序的基因列表是该协议的一个不可或缺的重要组成部分。 修改后，故障排除和限制与NGS数据分析的常见故障是失败的，特别是在质量控制阶段的工作或测试。该FastQC在样品上运行测试，少数能想出为失败。然而，这并不必然意味着样品不符合FASTQ质量标准。该故障可能有一个应该仔细研究的另一种解释。 例如，如果" 每碱基序列内容 "测试失败（这表明之间有一个大于10％的差异在任何位置的碱基），检查该寡脱氧胸苷文库制备方法。以前的工作已经表明，Illumina的NGS库可以具有用于13 个碱基的倾向被测序，以具有用于使所述样品测试失败某些碱基的偏压。同样，"k聚体内容"试验失败有时可以归结为一个事实，即从随机引物衍生的文库将几乎总是显示在开始k聚偏由于随机引物，一个不完整的采样。因此，确定实验的命运之前，需要考虑这些和其他障碍在分析管道是非常重要的。 可能影响转录组测序数据分析的另一个重要特点是，在NGS方法和分析软件的出现和迅速进步指数。理想情况下，一个期望在两个管道或同一管道的两个版本分析样品NGS数据以产生相同的基因列表线。然而，虽然不断地改进算法降低像差RNA测序分析并产生更高的精度的基因列表，这经常会导致差异。例如，在分析使用较旧的更新与同一工具集的版本可能产生显著不同的基因列表的样品NGS数据。适度的变化预期，但用户需要知道大的差异可能是反射性的，实验的设计和性能的弱点。 总的来说，银河项目和DAVID分析工具已经改变了方式，NGS数据可被用来提取生物相关的信息。这已开通的独立性和调查的全新水平对科学界，包括线虫的研究人员。例如，测序加上更好更快的测序技术，不断降低成本以单一的蠕虫的水平迎来转录的时代，个别蠕虫组织甚至一些特定的蠕虫病毒细胞。这些努力包括产生在NGS数据急剧增加。与此工作流程的分析端紧跟将是一个挑战，但由于它的通用性，银河很可能是在增强从整个生物体转录到RNA测序在秀丽隐杆线虫单细胞水平的过渡工具。知识产生的进步有可能提供非凡的见解的生物学基础。

人类基因组的测序首先，执行使用弗雷德·桑格的双脱氧核苷酸测序方法，用了10年，耗资估计US $ 3十亿1，2。然而，自成立以来一点点过去十年，新一代测序（NGS）技术已经使人们有可能在两周内测序整个人类基因组和US $ 1,000。新NGS仪器的成本是允许日益增长的测序数据采集的速度令人难以置信的效率，与急剧下降一起，以难以想象的方式彻底改变现代生物学的基因组测序项目正在迅速成为家常便饭。此外，这些发展已镀锌在许多其他领域，例如通过RNA测序（RNA-SEQ），的基因组范围的表观遗传修饰的研究中，DNA-蛋白质相互作用的基因表达分析的进步，并筛选在人类宿主微生物多样性。 NGS-基于RNA的硒●在特定使得有可能鉴定和地图转录全面与准确度和灵敏度，并且已取代微阵列技术作为选择的一种绘制表达图谱的方法。虽然微阵列技术已经被广泛使用时，它通过其与已知基因组信息预先存在的阵列，以及其它的缺点的依赖的限制，诸如交叉杂交，并且能够可靠地计量的表达的变化限制范围。 RNA-SEQ，在另一方面，可用于检测已知和未知的转录而产生低的背景噪声，由于其明确的DNA映射性质。 RNA-Seq的，加上通过模式生物如酵母提供了众多的遗传工具，蝇，虫，鱼和小鼠，曾担任许多重要生物医学近期发现的基础。然而，显著挑战依然存在，使以更广泛的科学界NGS无法访问，包括存储的局限性，加工，最重要的是，男<html>大量的测序数据的eaningful生物信息学分析。 在测序技术和指数数据积累的快速发展创造了计算平台，将允许研究人员访问，分析和理解这些信息的巨大需求。早期的系统主要依赖于计算机编程知识，反之，基因组浏览器，比如NCBI，允许非程序员访问和可视化数据不允许复杂的分析。基于Web的，开放的接入平台，银河（ <a href="https://galaxyproject.org/" target="_blank">https://galaxyproject.org/</a> ），填补了这一空白，并证明是一个有价值的管道，使研究人员能够处理NGS数据并执行的频谱简单到复杂生物信息学分析。银河初步建立，并保持，由安东Nekrutenko（宾夕法尼亚州立大学）和詹姆斯·泰勒的实验室（约翰霍普金斯大学）F"&gt; 3。银河提供广泛的计算任务使其成为一个‘一站式’为无数生物信息学的需要，包括所有参与的RNA测序研究的步骤。Itallows用户无论是在其服务器或执行数据处理在当地自己的机器上，数据和工作流可以被复制和共享。在线教程，帮助部分和wiki页面 （ <a href="https://wiki.galaxyproject.org/Support" target="_blank">https://wiki.galaxyproject.org/Support</a> ）专用于银河项目提供持续的支持。但是，对于初次使用的用户，尤其是那些没有生物信息学培训，该管道可以出现艰巨，自主学习和熟悉的过程可能会非常耗时。此外，生物系统研究，实验和方法的具体使用，冲击在几个步骤的分析决策，而这些可能很难没有指令导航。 整体RN<html> A-SEQ银河工作流包括数据上传和质量检查，随后使用Tuxedo的套件4，5，6，7，8，9，这是用于RNA测序数据分析10的不同阶段所需的各种工具的集体分析， 11，12，13，14。典型的RNA测序实验由实验部分（样品制备，分离mRNA和cDNA文库制备）中，并NGS生物信息学分析数据。这些部分，以及涉及在银河管道中的步骤的概述，示于图1。 3fig1.jpg" /&gt; 图1:一个RNA测序工作流程的概述。参与的RNA测序实验，以比较两个蠕虫菌株（A和B，橙色和绿色线和箭头，分别地）的基因表达概况的实验和计算步骤图示。银河使用的不同的模块示于盒用在我们的协议以红色表示相应的步骤。各种操作的输出被写在灰色与以蓝色显示的文件格式。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55473/55473fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 在炫套房的第一个工具是一个名为" 高顶礼帽 "的比对程序。它分解的NGS输入读取成较小的片段，然后将它们映射到参考基因组。这种两步处理确保读取跨越内含子区，其比对可以以其他方式二srupted或错过入账和映射。这增加了覆盖范围并促进新的剪接点的鉴定。 顶帽输出被报告为两个文件，一个文件BED（具有约剪接点，其包括基因组位置信息）和一个文件BAM（具有每个读映射的详细信息）。接着，BAM文件针对参照基因组对齐来估计使用于所谓的" 袖扣" Tuxedo的套件随后工具每个样品内的单个转录物的丰度。通过扫描对准来报告全长转录物的片段或"transfrags"跨越所有在为每一个基因的输入数据可能的剪接变体袖扣功能。在此基础上，它会产生一个对于每个样品被测序（每个基因产生的每一个基因的所有转录的组件）"转录"。然后，这些袖扣组件崩溃或再一起合并在一起ference基因组以产生用于使用下一个工具，"Cuffmerge"下游差分分析的单个注释文件。最后，由每个样品的顶帽的输出进行比较，以最终Cuffmerge输出文件（ 图1） 的样品之间的"Cuffdiff"工具措施的差异基因表达。 袖扣使用FPKM / RPKM（片段/读取每百万映射成绩单的每千碱基读取）值报告成绩单丰度。这些值反映原始NGS数据的深度归一化和基因长度（基因具有不同的长度，所以计数必须被归一化了的基因，以比较电平的长度（平均数目从对齐到参考基因组中的样本读出）基因之间）。 FPKM和RPKM基本上是相同的与RPKM被用于单端RNA测序，每一个读出对应于一个单一片段，而，FPKM用于配对末端RNA测序，因为它占的事实，两次读取可以对应于相同的片段。最终，这些分析的结果是所测试的条件和/或株之间差异表达的基因的列表。 一旦成功银河运行完成并产生"基因名单"，下一个合乎逻辑的步骤需要更多的生物信息学分析，从数据集推断出有意义的知识。许多软件包应运而生，以满足这方面的需求，其中包括公开可用的基于网络的计算包，如DAVID 15（用于注释，可视化和集成发现数据库）。 DAVID利于通过比较基因上传列表，其集成的生物知识库和揭示与基因列表相关的生物注释分配生物学意义，从高通量研究大基因列表。其次是富集分析， 即测试IDEntify如果任何生物过程或基因类是在以统计学显著方式基因列表（一个或多个）过表达。因为一个广泛的，集成的知识基础和强大的分析算法，使研究人员能够检测富含内生物主题的组合，已成为一个流行的选择基因组衍生的"基因列表" 10，16。其它优点包括其处理的任何测序平台和高度友好的用户界面创建基因列表的能力。 所述线虫是一种遗传模型系统中，公知为它的许多优点，例如尺寸小，透明体，简单的身体计划，易于培养的和巨大的顺从的遗传和分子清扫。蠕虫有一个小的，简单的和注释良好的基因组，其包括高达40％保守的基因与已知的人类同系物17。事实上， 线虫是第一个后生动物，其基因组被完全测序18，并且其中RNA测序被用于映射生物体的转录19，20中的第一物种之一。早期的蠕虫的研究涉及的实验用不同的方法进行高通量RNA捕获，文库制备和测序及生物信息学管道是该技术21，22的发展作出了贡献。近年来，在蠕虫基于RNA-Seq的，实验已经司空见惯。但是，传统的蠕虫生物学家通过RNA-Seq的数据的计算分析所带来的挑战仍然是技术的更大和更好地利用的一个主要障碍。 在本文中，我们描述了使用Galaxy平台分析来自秀丽隐杆线虫中产生高通量的RNA测序数据的协议。对于许多第一次和小SCA勒用户来说，最具有成本效益的和直接的方式进行的RNA测序实验是在实验室中分离RNA，并利用商用（或内部）NGS设施用于制备测序的cDNA文库和NGS本身。因此，我们已首次详细涉及隔离的步骤，C.量化和质量评估线虫 RNA样品用于RNA测序。接下来，我们为使用Galaxy接口，用于NGS数据的分析中，有用于测序后质量控制检查，随后对准，装配，和基因表达的定量差试验开始提供一步一步的指示。此外，我们已包括方向审议银河使用DAVID生物富集研究产生的基因列表。由于工作流中的最后的步骤中，我们上传到公共服务器，如序列读数存档（SRA）在NCBI（RNA-SEQ数据提供指令<a href="http://www.ncbi.nlm.nih.gov/sra" target="_blank">的http://瓦特</a>ww.ncbi.nlm.nih.gov/sra），使其自由科学界访问。总体而言，我们预计，本文将蠕虫生物学家开展运行小样本首次RNA-Seq的实验以及频繁的用户提供全面的，充分的信息。

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transcriptomic analysis of c. elegans rna sequencing data through the tuxedo suite on the galaxy project

新一代测序（NGS）技术已经彻底改变了生物学研究的性质。在这些中，RNA测序（RNA-SEQ）已经成为用于基因表达分析和转录的映射的有力工具。然而，处理RNA-Seq的数据集需要复杂计算的专业知识和带来的生物学研究人员固有的挑战。这个瓶颈已经缓解由开放接入银河项目，使用户无需生物信息学技术分析RNA-Seq的数据，和注释，可视化和集成发现（DAVID）的数据库，基因本体论（GO）项的分析工具，帮助从大的数据集获得生物学意​​义。但是，对于初次使用者和生物信息学爱好者，自学习和熟悉这些平台可以是耗时且艰巨。我们描述一个简单的工作流程，这将有助于线虫研究人员分离出病毒RNA，进行了RNA-Seq的实验并分析利用银河DAVID平台的数据。这个协议提供了使用各种银河模块在每个步骤访问原始NGS数据，质量控制检查，对准和差异基因表达分析，指导与参数的用户生成可筛选的富集的基因列表逐步指令基因的类或使用DAVID生物过程。总体而言，我们预计，本文将提供信息，以秀丽隐杆线虫的研究人员正在进行运行的小样本首次RNA-Seq的实验以及频繁的用户。

在秀丽隐杆线虫 ，消去生殖系干细胞（GSC中）延长寿命，提高的应力复原力，和提升体脂肪24,28。 GSC中的损失，或者通过激光烧蚀或通过突变如GLP-1引起，导致寿命延长通过转录的网络因子29活化。一个这样的因素，TCER-1，编码人转录延伸和剪接因子，TCERG1 30的蜗杆同源物。下列代表性的结果示出了RNA-SEQ是如何用于鉴定基因的表达是通过TCER-1 / TCERG1调制以下在我们最近发表的研究31种系损失。的转录组年龄匹配，每日2名成人GLP-1和tCER-1的GLP-1突变体进行比较。对于每个菌株，的mRNA是从两个生物复制品分离TES（四个样品完全）使用部分1 RNA样品中描述的协议被运到该制备从四个样品的cDNA文库和进行50bp的单末端测序商业服务提供商。如在第2.1节中所描述的原始NGS数据被下载。 帖子测序数据分析 表1是测试结果的汇编，以评估质量的原始测序读取。 "FASTQ"质量检查分析强调没有"质量差"读序列的数目与48-49％GC含量和51 bp的恒定序列读长读取沿。此步骤还检查许多其他的功能，如k聚体含量的测序数据和共同地由从总计11次测试。在秀丽隐杆线虫基因组中是〜100 Mbp的。基于测序的数量从映射到基因组中各样品，克读取enome覆盖（最后一列）中的溶液使用兰德/沃特曼方程"C = LN / G"，其中，C代表覆盖估计，G为单倍体基因组的长度，L是读出长度，而N是读取次数。我们使用的所有步骤的默认参数并获得了48 - 所有样品中49％的GC含量。可以看出，基因组覆盖度是对样品中11倍之间的9倍。 TCER-1的鉴定/按银河基因差异表达分析TCERG-1调控的基因通过在部分2.2至2.4中详述的步骤，银河管道3被用来得到GLP-1和tCER-1之间差异表达的基因的列表; GLP-1突变体。星系使我们能够在NGS数据从两次重复每个菌株结合并进行差分析，以生成表格文件突出基因组范围表达PROFILE。使用在幅度和至少0.05的P值的至少一个倍的变化的阈值，产生31的835个基因的两种菌株之间差异表达该列表。基于是否所述基因的表达被下调于tCER-1列表被划分; GLP-1突变体（359个UP基因，其转录是可能通过增强TCER-1 / TCERG1）或上调（476个DOWN基因，其转录可能是由TCER-1 / TCERG1压抑）相比，GLP-1（ 图4）。 <img alt="图4" src="/files/ftp_upload/55473/55473fig4.jpg" /> 图4:在种系少C. TCER-1 / TCERG1调节的基因的鉴定使用线虫 RNA-SEQ突变体:星系（A）和DAVID（B）的分析结果。 （A）进行比较RNA测序数据的差异基因表达分析GLP-1和tCER-1的转录; GLP-1共835个基因，其中的359被确定为被TCER-1 / TCERG1（UP）上调和476作为由TCER-1下调产生/ TCERG1（DOWN）。鉴定为使用DAVID TCER-1 / TCERG1目标基因的" 功能注释聚类"分析的（B）的结果。 两者的上调（UP）和下调（DOWN）的TCER-1 / TCERG1目标类的生物过程百分比富集。这里所示的图形是通过绘制作为DAVID分析的输出中获得的富集的基因组（X轴）和它们各自的百分比富集（Y轴）中得到。图从阿姆里特等改性。 31和复制许可。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55473/55473fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 为了获得在TCER-1 / TCERG1目标富集的基因类的概述，我们进行了基因本体论（GO）术语分析使用DAVID。所述TCER-1 / TCERG1调节UP和DOWN基因名单被独立地上载到DAVID并且如在部分中所述3.很少有人知道有关通过TCER-1 / TCERG1先前30靶向的基因和细胞过程分析，所以我们发现，DAVID分析要特别揭示和帮助。向上的基因的功能注释分析显示5个注释集群具有&gt; 1.3的富集得分，最高的包括细胞色素P450酶编码基因和异生素应答基因，随后在脂质修饰有关的基因。这是由基因功能分类分析认为鉴定组归因类似molecula结果增强[R活动，显著富集分数。使用电子表格中，所识别的组分别作图各自的富集得分（ 图4）。我们以前的数据表明，TCER-1 / TCERG1运作与保守长寿转录因子DAF-16 / FOXO3A，促进GSC-成人少30的寿命。 DAF-16 / FOXO3A，反过来，已牵涉在最近的研究27，32，33调节脂质代谢。在此基础上的证据，和脂质代谢基因和途径在DAVID识别潜在TCER-1 / TCERG1目标分析，我们专注于在详细机理研究的转录组测序研究发现脂肪代谢的基因。在此之后铅，并通过随后的分子遗传，生物化学和功能试验，我们表明，TCER-1 DAF-16 / FOXO3A沿/ TCERG1协调enhan土木工程署响应于种系损失31两者的脂质分解代谢和合成代谢过程。类似地，DOWN的功能注释聚类 TCER-1 / TCERG1鉴定富集细胞骨架功能，生长，繁殖和老化的正调控注释集群目标（ 图4）。这些观察结果，我们的支撑实验证据，表明在种系损失，TCER-1 / TCERG1也阻遏在体细胞的生长和生殖生理学以及抗长寿基因31的表达。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 样品 </td><td> 共有序列 </td><td> 长度 </td><td> ％GC </td><td> 总读取（银河） </td><td> 短片段（银河） </td><td> 基因组覆盖 </s仲&gt; </td></tr><tr><td> GLP-1 </td><td> 4000000 </td><td> 51 </td><td> 49 </td><td> 20700539 </td><td> 〜16000000 </td><td> 11X </td></tr><tr><td> GLP-1;于tCER-1 </td><td> 4000000 </td><td> 51 </td><td> 49 </td><td> 18055444 </td><td> 〜13000000 </td><td> 9X </td></tr><tr><td> GLP-1 </td><td> 4000000 </td><td> 51 </td><td> 48 </td><td> 18947463 </td><td> 〜14000000 </td><td> 10倍</td></tr><tr><td> GLP-1;于tCER-1 </td><td> 4000000 </td><td> 51 </td><td> 48 </td><td> 13829643 </td><td> 〜10,000,000 </td><td> 7X </td></tr></tbody></table> 表1:RNA-Seq的样品详细信息。原始数据的属性编制评估后测序，确认测序运行成功。从代表性实验测序数据由两个生物的条件下，对照菌株（GLP-1 </em&gt;）和突变菌株（ 于tCER-1; GLP-1）与测序对于每两个生物学重复。 "FastQC"质量检查分析强调没有"质量差"读，读48的序列的数量- 49％GC含量和51bp的恒定序列读长。改性，并与来自阿姆里特等人许可再现。 31。 补充文件:在短暂的命令链在工具上银河管道的RNA-Seq的数据分析运行。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55473/Amrit_Ghazi_JoVE_Suppl.docx">请点击这里下载此文件。</a>

Watch this Scientific Journal Video about Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project at JoVE.com

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

银河和大卫已经成为流行的工具，让没有生物信息学培训调查分析和解释RNA-Seq的数据。我们描述了一种协议，用于秀丽隐杆线虫的研究人员进行RNA测序实验，访问和使用银河处理数据集，并获得从使用DAVID的基因列表有意义的生物信息。

的转录分析<em&gt;ç</em&gt;。<em&gt;线虫</em&gt; RNA测序数据通过炫套房的银河项目

Next generation sequencing (NGS) technologies have revolutionized the nature of biological investigation. Of these, RNA Sequencing (RNA-Seq) has emerged ...

transcriptomic-analysis-c-elegans-rna-sequencing-data-through-tuxedo

Nanyang Technological University

Research

JoVE Journal

Genetics

Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

17.2K 次观看.  University of Pittsburgh School of Medicine, Children's Hospital of Pittsburgh. 银河和大卫已经成为流行的工具，让没有生物信息学培训调查分析和解释RNA-Seq的数据。我们描述了一种协议，用于秀丽隐杆线虫的研究人员进行RNA测序实验，访问和使用银河处理数据集，并获得从使用DAVID的基因列表有意义的生物信息。 

视频: Transcriptomic Analysis of C. elegans RNA Sequencing Data Through the Tuxedo Suite on the Galaxy Project

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

接合交配测定在接合介入转移蛋白的序列特异性分析

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

科研

遗传学

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

单细胞测序拷贝数改变的检测

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

通过诱导瞬时转染可控离子通道表达

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation&#160;of the DNA-protein interaction, followed by&#160;immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-&#946; (TGF-&#946;)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF).

DNA-protein interactions are essential for multiple biological processes. During the evaluation of cellular functions, the analysis of DNA-protein interactions is indispensable for understanding gene regulation. Chromatin immunoprecipitation (ChIP) is a powerful tool to analyze such interactions in vivo.

使用染色质免疫沉淀分析c-KIT配体启动子

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

Manipulation of Ploidy in Caenorhabditis elegans

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has been associated with both the progression of cancer and the development of drug resistance. Until now, it has not been feasible to easily manipulate the ploidy of a multicellular animal without generating mostly sterile progeny. Presented here is a simple and rapid protocol for generating tetraploid Caenorhabditis elegans animals from any diploid strain. This method allows the user to create a bias in chromosome segregation during meiosis, ultimately increasing ploidy in C. elegans. This strategy relies on the transient reduction of expression of the rec-8 gene to generate diploid gametes. A rec-8 mutant produces diploid gametes that can potentially produce tetraploids upon fertilization. This tractable scheme has been used to generate tetraploid strains carrying mutations and chromosome rearrangements to gain insight into chromosomal dynamics and interactions during pairing and synapsis in meiosis. This method is efficient for generating stable tetraploid strains without genetic markers, can be applied to any diploid strain, and can be used to derive triploid C. elegans. This straightforward method is useful for investigating other fundamental biological questions relevant to genome instability, gene dosage, biological scaling, extracellular signaling, adaptation to stress, development of resistance to drugs, and mechanisms of speciation.

Manipulation of Ploidy in Caenorhabditis elegans

This method allows for the generation of tetraploid and triploid Caenorhabditis nematodes from any diploid strain. Polyploid strains generated by this method have been used to study chromosome interactions in meiotic prophase, and this method is useful for examining important basic questions in cell, developmental, evolutionary, and cancer biology.

在秀丽线虫中对倍体的操控

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has ...

Informatic Analysis of Sequence Data from Batch Yeast 2-Hybrid Screens

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing high-throughput short-read DNA sequencing. The resulting sequence datasets can not only track what genes in a population that are enriched during selection for positive yeast 2-hybrid interactions, but also give detailed information about the relevant subdomains of proteins sufficient for interaction. Here, we describe a full suite of stand-alone software programs that allow non-experts to perform all the bioinformatics and statistical steps to process and analyze DNA sequence fastq files from a batch yeast 2-hybrid assay. The processing steps covered by these software include: 1) mapping and counting sequence reads corresponding to each candidate protein encoded within a yeast 2-hybrid prey library; 2) a statistical analysis program that evaluates the enrichment profiles; and 3) tools to examine the translational frame and position within the coding region of each enriched plasmid that encodes the interacting proteins of interest.

Deep sequencing of yeast populations selected for positive yeast 2-hybrid interactions potentially yields a wealth of information about interacting partner proteins. Here, we describe the operation of specific bioinformatics tools and customized updated software to analyze sequence data from such screens.

间歇酵母 2-混合筛序列数据的信息化分析

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing ...

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to normal and pathological conditions. The goal of this protocol is to generate high-quality, large-scale transcriptome data of each endocrine cell type with the use of a droplet-based microfluidic single-cell RNA sequencing technology. Such data can be utilized to build the gene expression profile of each endocrine cell type in normal or specific conditions. The process requires careful handling, accurate measurement, and rigorous quality control. In this protocol, we describe detailed steps for human pancreatic islets dissociation, sequencing, and data analysis. The representative results of about 20,000 human single islet cells demonstrate the successful application of the protocol.

Here, we present a protocol to generate high-quality, large-scale transcriptome data of single cells from isolated human pancreatic islets using a droplet-based microfluidic single-cell RNA sequencing technology.

人类胰腺胰岛的单细胞RNA测序与分析

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to ...

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT employs two purification steps. First, immunoprecipitation of a tagged RNP subunit is followed by mild RNase digestion and subsequent non-denaturing affinity elution. A second immunoprecipitation of another RNP subunit allows for enrichment of a defined complex. Following a denaturing elution of RNAs and proteins, the RNA footprints are converted into high-throughput DNA sequencing libraries. Unlike the more popular ultraviolet (UV) crosslinking followed by immunoprecipitation (CLIP) approach to enrich RBP binding sites, RIPiT is UV-crosslinking independent. Hence RIPiT can be applied to numerous proteins present in the RNA interactome and beyond that are essential to RNA regulation but do not directly contact the RNA or UV-crosslink poorly to RNA. The two purification steps in RIPiT provide an additional advantage of identifying binding sites where a protein of interest acts in partnership with another cofactor. The double purification strategy also serves to enhance signal by limiting background. Here, we provide a step-wise procedure to perform RIPiT and to generate high-throughput sequencing libraries from isolated RNA footprints. We also outline RIPiT&#39;s advantages and applications and discuss some of its limitations.

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing RIPiT-Seq

Here, we present a protocol to enrich endogenous RNA binding sites or &#34;footprints&#34;&#160;of RNA:protein (RNP) complexes from mammalian cells. This approach involves two immunoprecipitations of RNP subunits and is therefore dubbed RNA immunoprecipitation in tandem (RIPiT).

RNA足迹的识别:通过RNA免疫沉淀在串联中的蛋白质复合物,然后测序(RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT ...

Identification of Circular RNAs using RNA Sequencing

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein interactions, and regulation of parental gene transcription. In classical next generation RNA sequencing (RNA-seq), circRNAs are typically overlooked as a result of poly-A selection during construction of mRNA libraries, or are found at very low abundance, and are therefore difficult to isolate and detect. Here, a circRNA library construction protocol was optimized by comparing library preparation kits, pre-treatment options and various total RNA input amounts. Two commercially available whole transcriptome library preparation kits, with and without RNase R pre-treatment, and using variable amounts of total RNA input (1 to 4 &#181;g), were tested. Lastly, multiple tissue types; including liver, lung, lymph node, and pancreas; as well as multiple brain regions; including the cerebellum, inferior parietal lobe, middle temporal gyrus, occipital cortex, and superior frontal gyrus; were compared to evaluate circRNA abundance across tissue types. Analysis of the generated RNA-seq data using six different circRNA detection tools (find_circ, CIRI, Mapsplice, KNIFE, DCC, and CIRCexplorer) revealed that a stranded total RNA library preparation kit with RNase R pre-treatment and 4 &#181;g RNA input is the optimal method for identifying the highest relative number of circRNAs. Consistent with previous findings, the highest enrichment of circRNAs was observed in brain tissues compared to other tissue types.

Circular RNAs (circRNAs) are non-coding RNAs that may have roles in transcriptional regulation and mediating interactions between proteins. Following assessment of different parameters for construction of circRNA sequencing libraries, a protocol was compiled utilizing stranded total RNA library preparation with RNase R pre-treatment and is presented here.

使用RNA测序识别循环RNA

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein ...

Characterization of In Vitro Differentiation of Human Primary Keratinocytes by RNA-Seq Analysis

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported for in vitro differentiation of keratinocytes cultured in two-dimensional (2D) submerged manners using various induction conditions. Described here is a procedure for 2D in vitro keratinocyte differentiation method by contact inhibition and subsequent molecular characterization by RNA-seq. In brief, keratinocytes are grown in defined keratinocyte medium supplemented with growth factors until they are fully confluent. Differentiation is induced by close contacts between the keratinocytes and further stimulated by excluding growth factors in the medium. Using RNA-seq analyses, it is shown that both 1) differentiated keratinocytes exhibit distinct molecular signatures during differentiation and 2) the dynamic gene expression pattern largely resembles cells during epidermal stratification. As for comparison to normal keratinocyte differentiation, keratinocytes carrying mutations of the transcription factor p63 exhibit altered morphology and molecular signatures, consistent with their differentiation defects. In conclusion, this protocol details the steps for 2D in vitro keratinocyte differentiation and its molecular characterization, with an emphasis on bioinformatic analysis of RNA-seq data. Because RNA extraction and RNA-seq procedures have been well-documented, it is not the focus of this protocol. The experimental procedure of in vitro keratinocyte differentiation and bioinformatic analysis pipeline can be used to study molecular events during epidermal differentiation in healthy and diseased keratinocytes.

Presented here is a stepwise procedure for in vitro differentiation of human primary keratinocytes by contact inhibition followed by characterization at the molecular level by RNA-seq analysis.

通过RNA-Seq分析，人类原主角细胞的体外分化特征

Human primary keratinocytes are often used as in vitro models for studies on epidermal differentiation and related diseases. Methods have been reported ...