Name: laboratory protocol for genetic gut content analyses of aquatic macroinvertebrates using group-specific rdna primers
Uploaded: 2017-10-05T12:55:00.000+00:00
Duration: 10 min 17 s
Description: Watch this Scientific Journal Video about Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers at JoVE.com

我们感谢 Silke 克拉森的生态系统分析和评估研究所 (创), RWTH 亚琛为她的帮助建立了该协议中使用的特定于组的入门集。我们还要感谢基尔大学的彼得马丁和劳拉 Schynawa 的合作, 在水螨的实验。我们还感谢技术实验室助理保持实验室运行顺畅, 并准备注册的公司和目录编号。这项工作得到了德国研究基金会 (DFG; 项目 GE 2219/3-1) 的支持。

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作者没有什么可透露的。

在这里, 我们提出了一个简单和经济高效的方法, pcr 确定的捕食-猎物的相互作用, 从田间样品通过组特定引物的 rDNA 地区, 可以结合其他 non-molecular 分析相同的标本。但是, 该协议中有几个关键步骤。首先, 保证所用底漆的特殊性是必不可少的。其次, 在胃肠道的制备和随后的 DNA 提取过程中, 避免肠道内容物质的污染和丢失是至关重要的。虽然 cross-sample 污染可能导致对猎物 DNA 的假阳性检测, 但肠道内容物质的丢失可能导致很少被消耗的猎物物种。核查属于各自亚单位的 rDNA 的存在和用于分析的模板的功能效率 (《议定书》2节) 对于收集关于所消耗的猎物的代表性信息是必不可少的。这是特别重要的, 如果消费者的猎物群的脱氧核糖核酸不应该哺养可以被检测25,27。此外, 当准备 PCRs (步骤 3.1, 5.1.1 和 5.1.2), 这是至关重要的混合物和协议, 使用完全符合标准, 其中各自的底漆被指定给某一组的分类群。否则, PCR 反应中的 dna 放大可能会完全失效, 或者没有靶向的类群 dna 也可能被放大。因此, 必须在每个 pcr 运行中使用正负控制, 以确保 pcr 反应有效, 不发生污染。此外, 有必要为每个 PCR 运行的样品的数组是准确的文件, 以使正确分配的猎物消费到各自的消费者标本。对于这项任务, 需要特别谨慎, 同时结合 PCR 产品, 以并行检测多个猎物群通过自动片段分析 (步骤 5.2.3)。
对于使用琼脂糖凝胶电泳 (步骤3.2 和 3.3) 检测扩增的碎片, 琼脂糖凝胶应尽可能薄, 在图像的目视检查期间必须小心, 以避免视觉分辨率低的缺失碎片。对于结果和一个适当的结论关于猎物消耗量由消费者, 它是关键的甚而很少被消耗的猎物分类群被认出。浓琼脂糖凝胶降低了潜在的低数量的猎物 DNA 的能见度, 从而减少了错过很少消耗的猎物的可能性, 并引入了分析得出的结论的不确定性。此外, 从健康的角度来看, 使用比溴溴化物更少或无毒的替代品来染色凝胶可能是可取的。通过使用聚丙烯酰胺凝胶进行 SSCP 分析后进一步分析扩增碎片的议定书包括一些应特别注意的步骤。重要的是, 凝胶盒组装是防漏 (步骤 4.1.3) 和聚丙烯酰胺溶液有足够的时间聚合完全 (步骤 4.1.6)。过早打开卡带会导致液体丙烯酰胺溶液泄漏, 尽管准备工作必须从头开始, 但也需要进行大量的清理工作。此外, 将聚丙烯酰胺凝胶转移到染色浴 (步骤 4.7), 并从那里到 UV 表 (步骤 4.8) 需要非常小心, 因为这种凝胶的不稳定性。否则, 凝胶可能撕裂和碎片可能被打乱, 这导致必要重复的程序。
成功处理从自动片段分析中获得的数据的一个主要要求是, 样本的内部尺寸标准与制造商 (步骤 5.3.3) 提供的大小标准模式紧密匹配, 从而能够确定适当的片段长度。这是特别重要的, 因为否则, 不同的片段, 标签与相同的荧光染色染料, 可能被分配到错误的猎物分类, 因此导致一个完整的误判的捕食者-猎物的相互作用。此外, electropherograms 经常显示背景噪音。为了建立对解释的信心, 重要的是, 内部尺寸标准的峰值明显高于样品的背景噪声。因此, 每个标记/底漆的峰值只有当它们明显高于背景噪声时才应计算。
这一协议可以修改, 以检测多个群体的利益, 在不同的消费者感兴趣的猎物群。诚然, 对于每一个潜在的猎物群来说, 特定的引物可能已经没有了。然而, 组特定的引物不仅适用于几个淡水动物类群28, 而且也适用于其他种类繁多的分类群 (例如,几个海洋动物分类群16,30和飞行昆虫的常见分类群31)。因此, 选择适合于放大特定分类的所有捕食物种的 dna 的基团特异性引物集, 但不成功地从不属于这个分类的物种的 dna 放大, 是必不可少的32。现在已有许多特定的群特异性引物被指定用于给定范围的分类群 (例如, 在河莱茵系统28中共有的淡水无脊椎动物的130类群)。因此, 为了避免假阴性或假阳性结果, 此类引物的特异性应检查在其应用于目标分类群之前, 在其所关注的生态系中常见的分类群的范围内, 而不包括在分类群的范围内的消费者该底漆已建立。此外, 如果这些分子分析不能与其他完全相同个体的分析相结合, 胃肠道解剖可能会被忽略。
然而, 解剖更大的标本的胃肠道也防止 dna 提取失败, 因为太多的材料, 并减少在样本提取物的消费者 dna 的存在。然而, 研究表明, 使用特定的阻断引物, 它附着在消费者的 DNA, 从而阻止它, 为 PCR 反应可以有效地提高在混合样品的稀有序列的放大33。因此, 即使在处理较大的消费者标本时, 也可以通过使用特定的阻断底漆来避免胃肠道剥离。对于通过自动片段分析从多组特定的底漆中获得的增的并行检测, 应注意染色染料作为标签的选择。因此, 底漆的放大片段, 大致相同的长度应该被贴上明确的可区分荧光染料。此外, 有一系列的毛细管电泳系统可从几个制造商。这些系统中的一些甚至可以通过不染料来测定多个碎片。这里提出的协议可以很容易地被调整, 以检测放大的片段与任何这些系统。除此之外, 减少 t 所需的时间他分析, 这将是可能的优化单复用 PCR 方法, 以扩大一个片段分析批次的猎物群的 DNA 同时 (例如, 同时放大12捕食生物的甲虫34或多达六飞行昆虫分类群31)。
肠道内容分析的一个潜在的缺点一般, 因此也描述的分析在这个协议, 是低时间分辨率。有了这些方法, 只可能发现最近摄入的有机体, 这可能导致不确定的重要性单一的猎物生物体2。然而, 我们证明, 根据这个协议的遗传分析可以很容易地结合分析稳定的同位素 (δ15N 和δ13C) 的确切相同的消费者标本25,27。作为捕食者-猎物相互作用的时间自然示踪剂, 稳定同位素分析经常被用来描述食物 web 结构1, 但不同捕食物种的潜在重叠同位素值往往会阻碍精确捕食者与猎物的相互作用的定义2。因此, 利用这一协议进行遗传分析, 结合稳定同位素分析, 克服每种方法的弊端, 可以进一步了解食物网及其与养分或能量通量的关系。

捕食-食饵的相互作用, 构成了大多数的营养相互作用和食物网络动力学, 是关键的方面, 以表征物质和能量的通量在整个食物链内和之间的生态系统, 这是生态学的主要目标之一1. 碳和养分的来源和流动的确定是进一步了解生态系统之间的连通性2。然而, 生态系统, 如河流及其汇水区, 不仅与有机物和养分的通量有关, 而且还由生物体的运动3。因此, 生境的改变中断了与这些系统挂钩的资源流动, 这不仅直接而且间接地改变了捕食者-猎物群落的组成, 从而极大地改变了这两个生态系的食物网。例如, 食物网的变化已经被证明与单一捕食物种 (例如, 虹鳟鱼)4的移动有关。这种变化可能威胁生物多样性和水生生态系统的运作。因此, 分析这一领域中的捕食者与猎物的相互作用, 对于确定人类引起的环境变化 (如水管理做法) 对水生生态系统当地生物多样性的影响至关重要。
由于在复杂的系统中跟踪营养关联是困难的, 因此已经建立了几种方法来评估在5字段中的喂养交互。传统上, 在田间觅食相互作用的研究是基于对猎物遗骸的视觉识别在解剖内脏和需要广泛的知识关于形态学猎物多样性。视觉肠内容分析提供了一些消费者群体的资源使用的洞察力 (例如,龙虾的饮食的季节性变化6和鱼7,8或喂养偏好的桡足类)。然而, 消化的物理过程使视觉肠内容分析变得困难, 通常会漏掉软体的猎物-有机体9。对于以液体摄取为食的物种或某些无脊椎动物的消费者, 在进食前密集粉碎他们的食物, 如片, 在肠道内容中对猎物种类的视觉识别是不可能的10。由于这些限制, 分子分析提供了一个有希望的选择。
分子分析现在已经成为一种常见的工具, 允许快速和精确的猎物检测肠道内容。这种技术的范围多种多样: 基于单克隆抗体或聚合酶链反应 (PCR) 的策略经常被使用, 因为它们具有高特异性和敏感性11。开发新的单克隆抗体是时间和成本密集型, 因此, 当抗体不存在11时, 其他分子技术的应用就更有用了。另一种常见的方法是放大脱氧核糖核酸 (脱氧核糖核酸) 的区域, 象核糖体核糖核酸 (RNA) 基因存在于多数种类, 使用普遍引物设置12,13。在使用这种技术时, 通常不可能在混合来源的 DNA14中确定整个捕食生物体的范围。有效的方法, 以避免这种缺点是使用组特定的入门集的遗传肠道内容分析。设计为只放大特定目标群的 DNA 区域并排除所有其他物种15,16, 组特定的引物可在指定组的分类级别上识别捕食生物体, 而无需时间和成本密集型的二次分析。然而, 像所有的肠道内容分析, 这种分析只提供了一个喂养行为的快照。因此, 结合分子肠含量分析与时间自然示踪剂 (例如,稳定同位素) 的分析被认为是有益的1,2。
在这里, 我们描述了一个详细的方法, pcr 调查的捕食-猎物的相互作用使用组特定的引物的核核糖体 DNA (rDNA) 地区与稳定同位素分析的同一标本。我们描述了通过琼脂糖凝胶电泳检测单个猎物群的 DNA。此外, 我们还提供了一个机会, 进一步下游分析 PCR 产品的这种特定的群体特异性底漆适用于任何时候更高的分类分辨率比底漆的特异性是必需的。由于单股 DNA (ssDNA) 片段形成了由其主序17确定的三级构象, 因此由这种基团特异的引物放大的片段的微小变化会导致构象变化。这种变化可以检测到单链构象多态性 (SSCP) 分析与聚丙烯酰胺凝胶17,18, 使更精确的识别猎物生物体 (下至物种水平)。
虽然琼脂糖凝胶电泳是一个常见的和廉价的工具, 可视化 dna 片段和确定其近似长度19, 分辨率取决于 dna 的数量和染色染料使用20。通常, 在使用纯 DNA 样本时, 可视化是直接的, 但是在消费者的肠道中潜在的少量的猎物 dna 可能会使琼脂糖凝胶电泳结果的评分复杂化。尽管如此, 这种检测方法是可行的, 以筛选少量的消费样本从外地的一个或几个猎物群, 但复杂的评分, 使筛选的大量样本的多个猎物群非常时间密集, 从而不切实际.更灵敏的检测方法是通过毛细管电泳自动分析碎片, 另外还允许确定片段的确切长度21。一些微卫星研究表明, 通过使用不同的荧光染料作为标签, 可以通过自动片段分析22,23来检测和确定可比较长度的不同片段. 24。因此, 我们还提出了一个详细的协议, 并行检测 DNA 从多个猎物群使用 PCR 与标记组特定的底漆集和检测通过自动片段分析与自动排序器。此外, 我们提出的案例研究的结果表明, 通过自动片段分析检测猎物的 DNA 是一种方法, 也使相对量化的摄入猎物。

<table><tbody><tr><td>liquid nitrogen</td><td>Any</td><td>NA</td><td>For transport of field samples of macroinvertebrates to the laboratory.</td></tr><tr><td>1.5 ml reaction tubes - Safelock</td><td>Any</td><td>NA</td><td>For collection of consumer specimens in the field and subsequent conservation in liquid nitrogen reaction tubes do not necessarily need to be PCR clean. To avoid exploding of the tubes, the lid of the tubes should be punctured with a needle.</td></tr><tr><td>Stereomicroscope</td><td>Any</td><td>NA</td><td>Make sure magnification is suitable to prepare the gastro-intestinaltract of the size of consumer of interest.</td></tr><tr><td>Petri dishes</td><td>Any</td><td>NA</td><td>To dissect the gastro-intestinal tract of consumer specimens under the stereomikroscope.</td></tr><tr><td>Stainless steel foceps: Dumont forceps No. 7</td><td>Bioform</td><td>B40d</td><td>This curved stainless steel forceps can sometimes be very helful to hold specimen fixed inbetween the curved part without puncturing the gastro-intestinal tract.</td></tr><tr><td>Stainless steel foceps: Dumont forceps Electronic No. 5</td><td>Bioform</td><td>B40b</td><td>This fine stainless steel forceps is ideally suited for the preparation of the gastro-intestinal tract of consumer specimens.</td></tr><tr><td>DNA-ExitusPlus</td><td>AppliChem</td><td>A7409,1000RF</td><td>Decontamination solution for the removal of DNA and RNA contaminations</td></tr><tr><td>fuzz-free paper towel</td><td>Any</td><td>NA</td><td></td></tr><tr><td>Lab scale</td><td>Any</td><td>NA</td><td>Precision needs to be at least 0.01 g.</td></tr><tr><td>Microlitre pipettes (0.5-10/10-100/50-200/100-1000 &amp;mu;L)</td><td>Any</td><td>NA</td><td></td></tr><tr><td>Safe-Lock Tubes 2.0 ml</td><td>VWR</td><td>211-2165</td><td>It is crucial that these raction tubes are free of DNA, DNase, RNase and PCR inhibitors (PCR clean)!</td></tr><tr><td>Safe-Lock Tubes 1.5 ml</td><td>VWR</td><td>211-2164</td><td>It is crucial that these raction tubes are free of DNA, DNase, RNase and PCR inhibitors (PCR clean)!</td></tr><tr><td>Sodium chloride &amp;ge;99.5%, p.a., ACS, ISO</td><td>carlroth</td><td>3957.3</td><td>For salt extraction buffer (SEB) and 5M NaCl-solution required for extraction of DNA.</td></tr><tr><td>Tris(hydroxymethyl)-aminomethane Tris</td><td>carlroth</td><td>4855.2</td><td>For salt extraction buffer (SEB) and TBE buffer</td></tr><tr><td>Microlitre pipettes (0.5-10/10-100/50-200/100-1000 &amp;mu;L)</td><td>carlroth</td><td>8043.1</td><td>For salt extraction buffer (SEB) and TBE buffer</td></tr><tr><td>Boric acid &amp;ge;99.8 %, p.a., ACS, ISO</td><td>carlroth</td><td>6943.2</td><td>For TBE buffer. Not needed if commercialy available TBE buffer is obtained.</td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>carlroth</td><td>4360.1</td><td>For extraction of DNA</td></tr><tr><td>Proteinase K lyophil.</td><td>carlroth</td><td>7528.1</td><td>Prepare a working solution containing 10 mg/ml.</td></tr><tr><td>TissueLyser II</td><td>Quiagen</td><td>85300</td><td>Bead mill for homogenization of the samples.</td></tr><tr><td>Stainless Steel Beads, 5 mm</td><td>Quiagen</td><td>69989</td><td>For homogenization of samples.</td></tr><tr><td>Heating Thermoshaker</td><td>Any</td><td>NA</td><td></td></tr><tr><td>Vortex Mixer</td><td>Any</td><td>NA</td><td></td></tr><tr><td>Refrigerated centrifuge: himac CT 15 RE</td><td>Hitachi</td><td>NA</td><td>Refrigerated centrifuge used for centrifugation during the extraction of DNA (Protocol 1).</td></tr><tr><td>Isopropanol</td><td>carlroth</td><td>6752.5</td><td></td></tr><tr><td>Ethanol 99.8 %, p.a.</td><td>Any</td><td>NA</td><td></td></tr><tr><td>Water Molecular biology grade (ddH&lt;sub&gt;2&lt;/sub&gt;O)</td><td>AppliChem</td><td>A7398,1000</td><td>nuclease-free (stream sterilized and DEPC treated) water used for re-suspension of DNA and whenever refered to ddH&lt;sub&gt;2&lt;/sub&gt;O within the manuscript</td></tr><tr><td>Unmodified Olegonucleotides</td><td>Eurofins MWG Operon</td><td>NA</td><td>Primers unlabeled</td></tr><tr><td>Modified olegonucleotides</td><td>Metabion International AG</td><td>NA</td><td>Primers labelled</td></tr><tr><td>peqGOLD Taq all inclusive</td><td>VWR Peqlab</td><td>01-1000</td><td>Taq all inclusive for PCR reaction mixtures (reaction buffer Y and S, dNTPs and MgCl2)</td></tr><tr><td>Primus 96 advanced gradient thermocycler or peqStar96x Universal Gradient</td><td>Peqlab</td><td>NA</td><td>Primer sets were originally established on a Primus 96 advanced gradient thermocycler. As this cycler was quite old, we than checked if our primer sets are efficient and specific under the same conditions using the peqSta96x (which also could simulate the older model). However, tests showed that our primers work well and are still specific (see Koester et al. 2013) without using the simulation of the old machine.</td></tr><tr><td>Standard 96 Well PCR Plates</td><td>VWR Peqlab</td><td>732-2879</td><td>Used for PCR reactions.</td></tr><tr><td>Low Profile Tube Stripes 0.15 ml with Flat Cap Stripes</td><td>VWR Peqlab</td><td>732-3223</td><td>Cap stripes used to close the PCR plates.</td></tr><tr><td>Agarose</td><td>Peqlab</td><td>35-1020</td><td>Used for gel electrophoresis.</td></tr><tr><td>Microwave</td><td>Any</td><td>NA</td><td></td></tr><tr><td>Conical flask</td><td>Any</td><td>NA</td><td></td></tr><tr><td>Measuring cylinder</td><td>Any</td><td>NA</td><td></td></tr><tr><td>Horizontal electrophoresis unit and respective combs with teeth</td><td>Any</td><td>NA</td><td>For agarose gel electrophoresis.</td></tr><tr><td>Electrophoresis Power Supply PS3002</td><td>Life Technologies</td><td>NA</td><td></td></tr><tr><td>Ethidium bromide solution 1 %</td><td>carlroth</td><td>2218.1</td><td>Used to prepare staining bath for agarose gels.</td></tr><tr><td>Formamide</td><td>carlroth</td><td>6749.1</td><td>Needded for the denaturation buffer for SSCP analyses.</td></tr><tr><td>Bromphenol blue</td><td>AppliChem</td><td>A3640.0010</td><td>Needed for the loading dye for agarose gel electrophoresis.</td></tr><tr><td>Sucrose</td><td>carlroth</td><td>4621.1</td><td>Needed for the loading dye for agarose gel electrophoresis.</td></tr><tr><td>100 bp-DNA-Ladder extended</td><td>carlroth</td><td>T835.1</td><td>As size standard for agarose gel electrophoresis.</td></tr><tr><td>UV-Documentation system</td><td>Any</td><td>NA</td><td>To visualize gel electrophoresis results.</td></tr><tr><td>Rotiphorese NF-Acrylamide/Bis-solution 40 % (29:1)</td><td>carlroth</td><td>A121.1</td><td>For the preparation of polyacrylamid gels.</td></tr><tr><td>Ammonium peroxydisulphate</td><td>carlroth</td><td>9592.2</td><td>For the preparation of polyacrylamid gels.</td></tr><tr><td>foldback clips (32 mm)</td><td>Any</td><td>NA</td><td>For the preparation of polyacrylamid gels.</td></tr><tr><td>square-shaped plastic box (21 &amp;times; 6.5 &amp;times; 5 cm)</td><td>Any</td><td>NA</td><td>For the preparation of polyacrylamid gels.</td></tr><tr><td>Tetramethylethylenediamine (TEMED)</td><td>carlroth</td><td>23.67.1</td><td>For the preparation of polyacrylamid gels.</td></tr><tr><td>Beaker</td><td>Any</td><td>NA</td><td></td></tr><tr><td>RC 10 Digital chiller</td><td>VWR</td><td>462-0138</td><td>For SSCP analyses using polyacrylamide gel electrophoresis.</td></tr><tr><td>Vertical electrophoresis unit P10DS (OWL Separation Systems)</td><td>VWR</td><td>700-2361</td><td>For SSCP analyses using polyacrylamide gel electrophoresis.</td></tr><tr><td>Notched glass plate</td><td>VWR</td><td>730-0261</td><td>For SSCP analyses using polyacrylamide gel electrophoresis.</td></tr><tr><td>Blank glass plate</td><td>VWR</td><td>730-0260</td><td>For SSCP analyses using polyacrylamide gel electrophoresis.</td></tr><tr><td>Spacers</td><td>VWR</td><td>730-0262</td><td>For SSCP analyses using polyacrylamide gel electrophoresis.</td></tr><tr><td>Comb with 25 teeth</td><td>VWR</td><td>730-0294</td><td>For SSCP analyses using polyacrylamide gel electrophoresis.</td></tr><tr><td>Shaking platform</td><td>Any</td><td>NA</td><td>For constant shaking of the staining bath.</td></tr><tr><td>GelStar Nucleic Acid Stain (Lonza Rockland, Inc.)</td><td>Biozym</td><td>850535 (50535)</td><td>For staining of polyacrylamide gels.</td></tr><tr><td>GenomeLab DNA Size Standard Kit 400bp</td><td>AB Sciex</td><td>608098</td><td>For automated fragment analyses. Size stanard for Batch B.</td></tr><tr><td>GenomeLab DNA Size Standard Kit 600bp</td><td>AB Sciex</td><td>608095</td><td>For automated fragment analyses. Size stanard for Batch A.</td></tr><tr><td>Sample Loading Solution (SLS)</td><td>AB Sciex</td><td>608082</td><td>For automated fragment analyses.</td></tr><tr><td>Sequenzing plate - 96 Well Polypropylene PCR Microplate</td><td>Costar</td><td>6551</td><td>For automated fragment analyses.</td></tr><tr><td>Plate centrifuge, PerfectSpin P</td><td>VWR Peqlab</td><td>NA</td><td>mini plate centrifuge used to spin down PCR products.</td></tr><tr><td>Buffer plate - 96 Well EIA/RIA Clear Flat Bottom Polystyrene Not Treated Microplate</td><td>Corning</td><td>9017</td><td>For automated fragment analyses.</td></tr><tr><td>GenomeLab Seperation buffer</td><td>AB Sciex</td><td>608012</td><td>For automated fragment analyses.</td></tr><tr><td>Separation Gel - LPAI</td><td>AB Sciex</td><td>608010</td><td>For automated fragment analyses.</td></tr><tr><td>Sequenzer - CEQ8000 Genetic Analysis System</td><td>Beckman Coulter</td><td>NA</td><td>For automated fragment analyses.</td></tr><tr><td>GeneMarker V1.95</td><td>Softgenetics</td><td>NA</td><td>To analyze results of automated fragment analyses.</td></tr></tbody></table>

laboratory protocol for genetic gut content analyses of aquatic macroinvertebrates using group-specific rdna primers

分析食物网对于更好地了解生态系统是至关重要的。例如, 食物网络的相互作用可能会因非土著物种的入侵而发生严重的变化。然而, 在许多情况下, 对场捕食者-食饵相互作用的精确识别是困难的。这些分析通常是基于对肠道内容的视觉评估或稳定同位素比率的分析 (δ15N 和δ13C)。这些方法需要对个别捕食生物体的形态多样性或同位素特征进行全面的了解, 从而导致对猎物生物体的准确识别的障碍。视觉肠道内容分析, 特别是低估了软体的猎物有机体, 因为浸泡, 摄食和消化的猎物有机体, 使鉴定的特定物种很难。因此, 聚合酶链反应 (PCR) 为基础的策略, 例如使用组特定的入门集, 提供了一个强大的工具, 调查食物网络互动。在这里, 我们描述了详细的协议, 以调查的肠道内容的动物消费者从现场使用的组特定的入门套核核糖体脱氧核糖核酸 (rDNA)。DNA 可以从整个标本中提取 (在小分类群的情况下), 或者从野外采集的标本的肠道内容。dna 模板的存在和功能的效率需要直接从被测试的个体中得到确认, 使用针对各自 dna 亚基的通用引物集。我们还证明, 被消耗的猎物可以进一步确定的物种水平通过 PCR 与未修改的组特异性引物结合随后单链构象多态性 (SSCP) 分析使用聚丙烯酰胺凝胶。此外, 我们还表明, 使用不同的荧光染料作为标签, 可以通过自动片段分析从多个肠道含量样本中对不同猎物群的 DNA 片段进行平行筛选。

我们成功地使用了本协议中描述的步骤进行不同研究中的饮食分析。在本节中, 我们将介绍该协议不同部分的适用性的示例。
以 SSCP 分析为例, 演示了作者28建立的组特定引物集的有效性以及对 PCR 产物进行进一步下游分析的潜力, 并进行了饲喂试验。个体的Dikerogammarus villosus作为捕食者和Caenis的猎物被捕获的河流莱茵河和死水附近的卡尔斯鲁厄 (德国)。在实验室中, 捕食者和捕食类群分别在20° c 的过滤河水中被单独保存, 并被饿死36小时。在实验中, 片标本分别放置在烧杯过滤流水 (100 毫升), 喂食两到三Caenis幼虫30分钟, 并随后冷冻在液氮中。对每个villosus的胃肠道进行解剖, 并按照协议1中的描述提取 DNA。DNA 提取物的检测与 Caep28S 底漆集适合探测Caenis , 使用适当的 PCR 协议28 , 如协议3所述。为了验证不同Caenis物种的放大片段之间的差异, 采用了三个Caenis参考物种的肠道含量样本和纯 DNA 样本进行 PCR。琼脂糖凝胶电泳导致单波段双绞股 DNA, 不能区分不同的Caenis物种与这种类型的电泳 (图 2A)。相比之下, 通过 SSCP 凝胶电泳 (如协议4所述), 通过将肠道含量样本与参考样本 (图 2B) 进行比较, 可以将猎物生物体识别为物种级别。三参考类群的 SSCP 片段模式Caenis beskidensis (图 2B、lane 1)、 Caenis horaria (图 2B、lane 2)、和Caenis luctuosa (图 2B, 车道 3)由四带可微的模式组成。两个肠道内容样本 (图 2B, 车道4和 5) 的片段模式等于C. luctuosa (图 2B, 3 车道)。第三种肠道内容样本 (图 2B, lane 6) 的片段模式与C. horaria (图 2B, lane 2) 相同。两种肠道含量样品的序列分析被确定为luctuosa和c. horaria (图 2B, 车道4和 6) 和各自的参考种类证实了这个结果 (参见电子补充对准 fasta 文件)。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56132/56132fig2.jpg"> 
图 2:原始图像 (A) 琼脂糖凝胶电泳和 (B) SSCP 分析 PCR 片段的肠道内容和参考样品放大的 Caep28S 底漆集。车道1-3 显示的参考样本的物种Caenis beskidensis (1), Caenis horaria (2) 和Caenis luctuosa (3)。在4-6 车道, 介绍了片Dikerogammarus villosus的不同肠道含量样本的片段模式。7车道显示了 100 bp DNA 阶梯的片段模式。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56132/56132fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
此外, 还进行了一项实验室试验, 其中的水螨属于该物种的Hygrobates fluviatilis , 以摇幼虫为食, 不仅可以确定在水螨中是否能检测到摇个体的 DNA,还要测试检测到的 DNA 数量是否取决于从消耗猎物的时间29。在饥饿螨大约一个星期以后, 每螨被提供了一摇幼虫作为食物。每3-4 个水螨个体被固定在乙醇 (绝对) 的遗传分析使用 Chiro18S 底漆集特定的 dipteran 家族摇28在 1, 2, 3, 7, 9, 24, 32 和 50 h 后, 被喂食29。作为一个消耗量由水螨个体, 各自的猎物个体被分类入以下五哺养的类别: 1 = 完全地被吮, 珠完全地倒空 (和 #62; 90%), 2 = 几乎完全地被吮 (和 #62;75-90%), 3 = 半吮 (和 #62; 50-75%), 4 = 只部份地吮了 (和 #62; 5-50%), 5 = 没有明显的变化在猎物的身体结构 (5%)29。DNA 是从水螨中提取的, 如协议 1 (不含步骤1.2.1 和 1.2.2), 并且在提取物中存在 18S rDNA 和模板的功能效率, 如协议2所述。根据协议4的规定, 使用荧光染料 (见表 2) 和 DNA 尺寸标准试剂盒-400 的 Chiro18S 底漆, 对猎物 dna 进行检测。为了允许在自动排序器的不同运行中分析的样本进行比较, 计算了每个样本的峰值高度与最接近的内部标准的峰值高度之间的比值 (在本例中为即, 360 bp), 并将其用作用于检测到的 DNA 数量的代理。结果表明, 在摇幼虫29上, 几乎所有的Hygrobates fluviatilis的个体都能检测到摇 DNA。从最短的时间间隔 (1 小时后喂养) 到喂养后最长的时间 (50 h), 检测到的猎物 DNA 的相对数量明显减少 (图 3A)29。此外, 还可以确定摄食后的时间与猎物的分类之间的关系 (图 3B)29。在进食后增加的时间内发现的猎物 dna 的减少最有可能完全反映在 dna 的降解和猎物的消化, 因为排的猎物 dna 是非常难以置信的, 由于缺乏肛门在水螨29。这些结果证实了这种方法对摄食猎物的定量的适用性。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56132/56132fig3.jpg"> 
图 3:国家 ln 高度-比率 (样本-峰值高度/标准360峰值高度) 和 (A) 在螨喂养后的时间和 (B) 喂养类别 (n = 23) 之间的关系。"实验和应用 Acarology, 第一次检测猎物 DNA 在Hygrobates fluviatilis (Hydrachnidia, 蜱螨亚纲): 一个新的方法来确定在水螨的捕食者-猎物关系, 67, 376, p. 马丁, m. Koester, l. Schynawa, r. Gergs, (©国际出版瑞士和 2015) "允许的斯普林斯。<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56132/56132fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
为了研究侵入性片在野外的重要作用, 在villosus的作用下, 强稳定同位素分析δ13C 和δ15N 的villosus和潜在的食物资源得到了进一步的分子检测最近摄入的猎物在肠道内的内容完全相同的个人。总共收集了来自不同地点的 206 villosus个体, 解剖了每个个体的胃肠道, 并根据协议1提取了 DNA。如协议2所述, 所使用模板的存在和功能效率得到保证。最近的捕食由villosus个人对多个动物的捕食类群进行了测试, 如协议4所述。总的来说, 只有16% 的D. villosus肠道内容对属于16种目标的动物猎物群的 DNA 进行了阳性检测, 因此支持稳定同位素分析结果, 表明低捕食摄食行为侵入性片在字段27。在遗传分析中, 在至少1的肠道含量样本中发现了12种被测猎物群的 dna, 但从未检测到其他4个猎物分类群的 dna (图 4)27。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56132/56132fig4.jpg"> 
图 4:直方图, 用于说明villosus的各自数量, 来自所有十站点的个人, 其肠道内容检测为动物的猎物 DNA 阳性.用16组特定的引物进行 PCR。"Hydrobiologia, 是Dikerogammarus villosus (甲壳, Gammaridae) 在莱茵河系统中的 ' 杀手虾 '？, 768, 2016, 310, Koester 美, 拜耳巴斯蒂安, Gergs ren é, (©斯普林国际出版瑞士 2015)" 允许springer.<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56132/56132fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
补充文件Sequence_alignement_feeding_trial. fasta<a href="//ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/56132/Sequence_alignement_feeding_trial.fasta" target="_blank">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers at JoVE.com

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

无脊椎动物最常见的肠道成分分析是可视的。由于需要对猎物生物体的形态多样性有强烈的了解, 他们会想念身体柔软的猎物, 由于对猎物的强烈粉碎, 对包括片在内的一些生物体来说几乎是不可能的。我们提供了详细的, 新的遗传方法, 动物猎物识别在饮食中的片。

用基团特异 rDNA 引物对水生无脊椎动物进行遗传肠道含量分析的实验室规程

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

laboratory-protocol-for-genetic-gut-content-analyses-aquatic

Universidad San Sebastian

Research

JoVE Journal

Biology

8.8K 次观看.  University of Koblenz-Landau. 无脊椎动物最常见的肠道成分分析是可视的。由于需要对猎物生物体的形态多样性有强烈的了解, 他们会想念身体柔软的猎物, 由于对猎物的强烈粉碎, 对包括片在内的一些生物体来说几乎是不可能的。我们提供了详细的, 新的遗传方法, 动物猎物识别在饮食中的片。

视频: Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time PCR (RT-qPCR). It provides accurate, sensitive, reliable, and reproducible results. Several factors can affect the sensitivity and specificity of RT-qPCR. Residual genomic DNA (gDNA) contaminating RNA samples is one of them. In gene expression analysis, non-specific amplification due to gDNA contamination will overestimate the abundance of transcript levels and can affect the RT-qPCR results. Generally, gDNA is detected by qRT-PCR using primer pairs annealing to intergenic regions or an intron of the gene of interest. Unfortunately, intron/exon annotations are not yet known for all genes from vertebrate, bacteria, protist, fungi, plant, and invertebrate metazoan species.
Here we present a protocol for detection of gDNA contamination in RNA samples by using ribosomal DNA (rDNA)-based primers. The method is based on the unique features of rDNA: their multigene nature, highly conserved sequences, and high frequency in the genome. Also as a case study, a unique set of primers were designed based on the conserved region of ribosomal DNA (rDNA) in the Poaceae family. The universality of these primer pairs was tested by melt curve analysis and agarose gel electrophoresis. Although our method explains how rDNA-based primers can be applied for the gDNA contamination assay in the Poaceae family,&#160;it could be easily used to other prokaryote and eukaryote species

Here, we present a protocol for tracing genomic DNA (gDNA) contamination in RNA samples. The presented method utilizes primers specific for the internal transcribed spacer region (ITS) of ribosomal DNA (rDNA) genes. The method is suited for reliable and sensitive detection of DNA contamination in most eukaryotes and prokaryotes.

基于核糖体 dna 的 RNA 样本 dna 污染评价

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time ...

科研

遗传学

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of taxonomic and functional groups in environmental samples. Used in combination with a reverse transcriptase reaction (RT-q-PCR), it can also be employed to quantify gene transcripts. q-PCR makes use of highly sensitive fluorescent detection chemistries that allow quantification of PCR amplicons during the exponential phase of the reaction. Therefore, the biases associated with &#39;end-point&#39; PCR detected in the plateau phase of the PCR reaction are avoided. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. First, a method for the co-extraction of DNA and RNA from coastal sediments, including the additional steps required for the preparation of DNA-free RNA, is outlined. Second, a step-by-step guide for the quantification of 16S rRNA genes and transcripts from the extracted nucleic acids via q-PCR and RT-q-PCR is outlined. This includes details for the construction of DNA and RNA standard curves. Key considerations for the use of RT-q-PCR assays in microbial ecology are included.

A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. The methodology includes the co-extraction of DNA and RNA; preparation of DNA-free RNA; and 16S rRNA gene and transcript quantification via RT-q-PCR, including standard curve construction.

从海岸沉积物和16S量化的rRNA基因和转录同时DNA-RNA提取采用Real-time PCR检测

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of ...

环境科学

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

从土壤、根际和根 Endosphere 提取细菌群落数据的根菌群探析

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

Development and Testing of Species-specific Quantitative PCR Assays for Environmental DNA Applications

New, non-invasive methods for detecting and monitoring species presence are being developed to aid in fisheries and wildlife conservation management. The use of environmental DNA (eDNA) samples for detecting macrobiota is one such group of methods that is rapidly becoming popular and being implemented in national management programs. Here we focus on the development of species-specific targeted assays for probe-based quantitative PCR (qPCR) applications. Using probe-based qPCR offers greater specificity than is possible with primers alone. Furthermore, the ability to quantify the amount of DNA in a sample can be useful in our understanding of the ecology of eDNA and the interpretation of eDNA detection patterns in the field. Careful consideration is needed in the development and testing of these assays to ensure the sensitivity and specificity of detecting the target species from an environmental sample. In this protocol we will delineate the steps needed to design and test probe-based assays for the detection of a target species; including creation of sequence databases, assay design, assay selection and optimization, testing assay performance, and field validation. Following these steps will help achieve an efficient, sensitive, and specific assay that can be used with confidence. We demonstrate this process with our assay designed for populations of the mucket (Actinonaias ligamentina), a freshwater mussel species found in the Clinch River, USA.

Environmental DNA assays require rigorous design, testing, optimization and validation before the collection of field data can begin. Here, we present a protocol to take users through each step of designing a species-specific, probe-based qPCR assay for the detection and quantification of a target species DNA from environmental samples.

开发和测试针对物种的定量PCR检测环境DNA应用

New, non-invasive methods for detecting and monitoring species presence are being developed to aid in fisheries and wildlife conservation management. The ...

Visualizing Methane-Cycling Microbial Dynamics in Coastal Wetlands

Coastal wetlands are the largest biotic source of methane, where methanogens convert organic matter into methane and methanotrophs oxidize methane, thus playing a critical role in regulating the methane cycle. The wetlands in South Texas, which are subject to frequent weather events, fluctuating salinity levels, and anthropogenic activities due to climate change, influence methane cycling. Despite the ecological importance of these processes, methane cycling in South Texas coastal wetlands remains insufficiently explored. To address this gap, we developed and optimized a method for detecting genes related to methanogens and methanotrophs, including mcrA as a biomarker for methanogens and pmoA1, pmoA2, and mmoX as biomarkers for methanotrophs. Additionally, this study aimed to visualize the spatial and temporal distribution patterns of methanogen and methanotroph abundance utilizing the geographic information system (GIS) software ArcGIS Pro. The integration of these molecular techniques with advanced geospatial visualization provided critical insights into the spatial and temporal distribution of methanogen and methanotroph communities across South Texas wetlands. Thus, the methodology established in this study offers a robust framework for mapping microbial dynamics in wetlands, enhancing our understanding of methane cycling under varying environmental conditions, and supporting broader ecological and environmental change studies.

The protocol detects key methane-cycling genes in South Texas coastal wetlands and visualizes their spatial distribution to enhance understanding of methane regulation and its environmental impacts in these dynamic ecosystems.

可视化沿海湿地中的甲烷循环微生物动力学

Coastal wetlands are the largest biotic source of methane, where methanogens convert organic matter into methane and methanotrophs oxidize methane, thus ...

DNA-based Fish Species Identification Protocol

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. This versatile method employs PCR amplification of genomic DNA extracted from fish samples, followed by restriction fragment length polymorphism (RFLP) analysis to generate fragment patterns that can be resolved on the Agilent 2100 Bioanalyzer and matched to the correct species using RFLP pattern matching software.
The fish identification method uses a simple, reliable, spin column- based protocol to isolate DNA from fish samples. The samples are treated with proteinase K to release the nucleic acids into solution. DNA is then isolated by suspending the sample in binding buffer and loading onto a micro- spin cup containing a silica- based fiber matrix. The nucleic acids in the sample bind to the fiber matrix. The immobilized nucleic acids are washed to remove contaminants, and total DNA is recovered in a final volume of 100 &mu;l. The isolated DNA is ready for PCR amplification with the provided primers that bind to sequences found in all fish genomes. The PCR products are then digested with three different restriction enzymes and resolved on the Agilent 2100 Bioanalyzer. The fragment lengths produced in the digestion reactions can be used to determine the species of fish from which the DNA sample was prepared, using the RFLP pattern matching software containing a database of experimentally- derived RFLP patterns from commercially relevant fish species.

This publication describes how to use the Agilent Fish Species Identification System to identify the species of a fish by extracting DNA and performing PCR and RFLP analysis.

以DNA为基础的鱼类物种鉴定协议

We have developed a fast, simple, and accurate DNA-based screening method to identify the fish species present in fresh and processed seafood samples. ...

生物学

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to identify commensal or mutualistic bacteria species. The latter ones, have been observed to serve multiple functions to the insect, i.e. helping in insect reproduction1, boosting the immune response2, pheromone production3, as well as nutrition, including the synthesis of essential amino acids4, among others. &#160; &#160;
Due to the importance of these associations, many efforts have been made to characterize the communities down to the individual members. However, most of these efforts were either based on cultivation methods or relied on the generation of 16S rRNA gene fragments which were sequenced for final identification. Unfortunately, these approaches only identified the bacterial species present in the gut and provided no information on the metabolic activity of the microorganisms.
To characterize the metabolically active bacterial species in the gut of an insect, we used stable isotope probing (SIP) in vivo employing 13C-glucose as a universal substrate. This is a promising culture-free technique that allows the linkage of microbial phylogenies to their particular metabolic activity. This is possible by tracking stable, isotope labeled atoms from substrates into microbial biomarkers, such as DNA and RNA5. The incorporation of 13C isotopes into DNA increases the density of the labeled DNA compared to the unlabeled (12C) one. In the end, the 13C-labeled DNA or RNA is separated by density-gradient ultracentrifugation from the 12C-unlabeled similar one6. Subsequent molecular analysis of the separated nucleic acid isotopomers provides the connection between metabolic activity and identity of the species.
Here, we present the protocol used to characterize the metabolically active bacteria in the gut of a generalist insect (our model system), Spodoptera littoralis (Lepidoptera, Noctuidae). The phylogenetic analysis of the DNA was done using pyrosequencing, which allowed high resolution and precision in the identification of insect gut bacterial community. As main substrate, 13C-labeled glucose was used in the experiments. The substrate was fed to the insects using an artificial diet.

Identification of Metabolically Active Bacteria in the Gut of the Generalist Spodoptera littoralis via DNA Stable Isotope Probing Using 13C-Glucose

The active bacterial community associated with the gut of Spodoptera littoralis, was determined by stable-isotope-probing (SIP) coupled to pyrosequencing. Using this methodology, identification of the metabolically active bacteria species within the community was done with high resolution and precision.

的代谢活性细菌在通才的肠道鉴定<em&gt;海灰翅夜蛾</em&gt;通过DNA的稳定同位素探测使用<sup&gt; 13</sup&gt; C-葡萄糖

Guts of most insects are inhabited by complex communities of symbiotic nonpathogenic bacteria. Within such microbial communities it is possible to ...

Rapid and Efficient Zebrafish Genotyping Using PCR with High-resolution Melt Analysis

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic tools have been introduced, including multiple strategies for inducing mutations and generating transgenic lines. However, large-scale screening is limited by traditional genotyping methods, which are time-consuming and labor-intensive. Here we describe a technique to analyze zebrafish genotypes by PCR combined with high-resolution melting analysis (HRMA).&#160;This approach is rapid, sensitive, and inexpensive, with lower risk of contamination artifacts.&#160;Genotyping by PCR with HRMA can be used for embryos or adult fish, including in high-throughput screening protocols.

PCR combined with high-resolution melt analysis (HRMA) is demonstrated as a rapid and efficient method to genotype zebrafish.

快速，高效的斑马鱼基因分型采用PCR与高分辨率熔体分析

Zebrafish is a powerful vertebrate model system for studying development, modeling disease, and performing drug screening. Recently a variety of genetic ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

We present a protocol for the functional assessment of comprehensive single-site saturation mutagenesis libraries of proteins utilizing high-throughput sequencing. Importantly, this approach uses orthogonal primer pairs to multiplex library construction and sequencing. Representative results using TEM-1 &#946;-lactamase selected at a clinically relevant dosage of ampicillin are provided.

经过一蛋白饱和诱变图书馆的功能评价规范利用高通量测序

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...

Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. Deviations from what is considered a healthy microbiota composition (dysbiosis) may impair vital functions leading to pathologic conditions. Recent and ongoing research efforts have been directed toward the characterization of associations between microbial composition and human health and disease.
Advances in high-throughput sequencing technologies enable characterization of the gut microbial composition. These methods include 16S rRNA-amplicon sequencing and shotgun sequencing. 16S rRNA-amplicon sequencing is used to profile taxonomical composition, while shotgun sequencing provides additional information about gene predictions and functional annotation. An advantage in using a targeted sequencing method of the 16S rRNA gene variable region is its substantially lower cost compared to shotgun sequencing. Sequence differences in the 16S rRNA gene are used as a microbial fingerprint to identify and quantify different taxa within an individual sample.
Major international efforts have enlisted standards for 16S rRNA-amplicon sequencing. However, several studies report a common source of variation caused by batch effect. To minimize this effect, uniformed protocols for sample collection, processing, and sequencing must be implemented. This protocol proposes the integration of broadly used protocols starting from fecal sample collection to data analyses. This protocol includes a column-free, direct-PCR approach that enables simultaneous handling and DNA extraction of large numbers of fecal samples, along with PCR amplification of the V4 region. In addition, the protocol describes the analysis pipeline and provides a script using the latest version of QIIME (QIIME 2 version 2017.7.0 and DADA2). This step-by-step protocol is aimed to guide those interested in initiating the use of 16S rRNA-amplicon sequencing in a robust, reproductive, easy to use, detailed way.

This manuscript describes a detailed standardized protocol of high-throughput 16S rRNA-amplicon sequencing. The protocol introduces an integrated, uniformed, feasible, and inexpensive protocol starting from fecal sample collection through data analyses. This protocol enables analysis of large numbers of samples with rigorous standards and several controls.

16S rRNA-扩增子测序法对粪便微生物特性的指导协议

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. ...