这个项目是支持部分由约翰斯·霍普金斯大学，教务长办公室通过网关科学倡议和QIAGEN公司

简介:一个单一的菌落，应使用适当的孵育过夜培养肉汤接种。甲细菌沉淀，然后处理使用市售的基因组提取试剂盒纯化的基因组DNA。纯化的基因组DNA应该有一个260/280（nm）的比率&gt; 1.8，以确保样品是无蛋白污染。生成焦磷酸测序模板所需的基因组DNA的量是10-20纳克。 A.模板的扩增 （PyroMark PCR手册; <a href="http://www.qiagen.com/handbooks" target="_blank">www.qiagen.com /手册</a> 1） 设置PCR反应 注:一个引物必须在其5&#39;端生物素化，它的建议，是为模板制备高效液相色谱纯化。我们建议PyroMark PCR和SE检测设计软件2.0优化短读测序的序列设计引物，然而，底漆，的BLAST（ <a href="http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome" target="_blank">http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome</a> ）也可以从NCBI用于引物设计。 <ol><li>解冻所有所需的解决方案（ 表1中列出），并混合每一个彻彻底底。根据表1的反应。所有的工作都可以在室温下进行。 </li></ol> 注意:PCR扩增预混试剂含有MgCl 2的终浓度为1.5mM的，在大多数情况下提供令人满意的结果。如果需要更高的Mg 2 +浓度是必需的，可以被添加到高达3.5微升的25mM的MgCl 2反应。 <ol start="2"><li>充分混合反应混合物轻轻吹打向上和向下，然后分配适当的卷PCR管。 </li><li>添加templat"每个PCR管êDNA。我们建议10 ng的基因组DNA。 </li><li>方案的热循环仪，按照表2中。最后一个保持在4℃，建议方便。如果使用热循环仪没有热盖，用100μl矿物油覆盖反应。 </li><li> PCR管放置在PCR仪中，并开始循环程序。 </li></ol> B.真空工作站协议固定的生物素标记的PCR产物<ol><li>根据图4，使主结构。 </li></ol> 注:移液之前，链霉亲和素涂层琼脂糖珠轻轻摇晃瓶子，以确保均匀的悬浮液。 <ol start="2"><li>根据使用的PCR产物的体积，取60-75微升的PCR板的各孔中，以使每孔的总体积为80微升混合液到。 </li><li>向每孔中加入5-20μl的生物素化的PCR产物。 </li><li>密封条带长约井ps和PCR板在1400 rpm搅拌5-10分钟，在室温下（15-25℃），使用轨道摇床。 </li></ol>变性的DNA和测序引物的添加<ol><li>用退火缓冲液稀释的测序引物，以0.3μM和分配到一个反应板的各孔中25微升。定位板在工作站上。 </li><li>根据图5的图，填充工作站槽。 </li><li>开启真空泵，确保真空工具的开关设置（ 图6）。冲洗过滤器探针槽5的高纯度水（Milli-Q系统18.2兆瓦为x cm或同等学历）（ 图5）。在步骤12中使用新鲜的高纯度水笔芯的低谷。 </li><li>工作迅速，定位的PCR板珠在工作站上。确保两板在同一方向时，将样品加载。 </li><li>随着真空开关ON，降低真空工具的PCR板入井，持续20秒或直到所有音量都已经被吸。通过真空工具（ 图7），将被捕获的有孔玻璃珠。 </li><li>随着真空，冲洗工具，用70％乙醇（槽1），持续20秒或直到所有的音量已经吸气。 </li><li>真空开，冲洗工具变性溶液（槽2），持续20秒或直到所有卷已经被吸。 </li></ol> 注意:该变性溶液含有氢氧化钠，这是眼睛和皮肤的刺激性。总是穿白大褂，手套和护目镜。 <ol start="8"><li>真空开，用洗涤缓冲液（槽3）冲洗工具，持续20秒或直到所有音量都已经被吸。 </li><li>真空开，真空工具提高到超过90°垂直，持续5秒，让所有的液体排出真空工具。关闭循环泵停止，并对齐与反应板的真空工具。降低进入真空工具井，轻轻摇晃从一边到另一边释放珠入井含有测序引物。 </li><li>为了清洁真空工具，搅拌过滤器探头在高纯度的水（4槽），持续10秒。 </li><li>真空切换和刷新工具用高纯度的水（5槽），持续20秒或直到所有音量都已经被吸。 </li><li>真空工具提高到超过90°C垂直范围为5秒，然后真空开关关闭，并存储在"停车"位置。 </li></ol>测序引物退火到DNA链<ol><li>在预热的板固定器，将反应板。 </li><li>加热板在80℃下加热块上，持续2分钟。 </li><li>拆下该板从固定器在柜台上，在室温下冷却5分钟。 </li></ol> Pyromark Q24操作 注:以上的电源线使用电源开关位于仪器上的开关。明星锤头可能需要长达1分钟。 <ol><li>填充墨盒PyroMark黄金Q24试剂手册中提供的指示。要确定需要，设置了仪器软件运行文件的卷，并在"工具"菜单下，选择预运行信息</li><li>如果仪器已经开启，确保它不执行运行，然后打开盖子。将发出可听见的警告信号，如果盖子被打开时，这样做是不安全的。 </li><li>打开墨盒门和插入墨盒标签朝前（ 图8）。确保墨盒已正确插入（一条线在前面应该是可见的），并关闭大门。 </li><li>打开板保持框和定位仪器内部的样品板。 </li><li>关闭平板保持框和仪器的盖子（ 图9）。 </li></ol>启动运行<ol><li>插入USB记忆棒运行文件Ş仪器软件创建到USB端口在前面的仪表。 </li><li>在主菜单中选择"运行"，然后按"确定"。 </li><li>使用滚动箭头来选择所需的运行文件，按"选择"。 </li></ol> 注:该仪器将开始分配时达到所有预设的压力和温度水平（这可能需要几分钟）的试剂。在运行过程中，仪​​器屏幕上显示的实时选择的热解图。使用滚动箭头来查看其他井的热解图。 完成一个运行<ol><li>当仪器确认，程序运行结束并运行文件已被保存到U盘，按"关闭"。 </li><li>取下USB记忆棒。如果它被删除完成运行前，插入USB记忆棒。选择"管理"，然后"复制未保存的运行"。 </li><li>现在运行的文件可以被打开和使用仪器软件分析和比较的库已知微生物使用Identifire软件的。 </li></ol>

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生产和自由地进入本文由Qiagen公司赞助。 作者艾哈迈德·迪罗谢，杰森和瓦迪Qiagen公司产生在这篇文章中所使用的试剂和仪器的公司员工。

几个新的下一代测序方法已经商品化。三，最广泛使用的方法是离子半导体测序，单分子实时测序，并通过合成测序（SBS）。3-11每一种方法都依赖于通过合成测序，但结合的核苷酸的检测，采用新颖的平台。在离子半导体测序，测序链作为模板的DNA单链供应12聚合酶和核苷酸依次加入焦磷酸测序。当一个核苷酸加入到生长的DNA链中，氢离子被释放。的场效应晶体管基于传感器检测到的氢离子。 单分子实时测序取决于零模式波导（中贸网）13中贸网是一个小的结构，在一个单一的的聚合酶分子被连接到井底。在这样一种方式，一个花神照明气味分子可以被检测到。每个核苷酸用荧光分子标记。作为荧光标记核苷酸注册成立，探测器识别的核苷酸。当添加的下一个核苷酸，荧光分子被裂解14 通过合成测序（SBS）使用一种独特的方法扩增靶DNA等独特的序列的簇生成15要生成并合成互补链的单链模板。每个核苷酸用荧光分子标记和碱加成后，每个记录的添加的碱的荧光。

焦磷酸测序法是一种快速，准确的方法，以"合成测序"的原则是根据序列核酸。 "通过合成的序列"涉及到使用一个单链DNA模板合成互补链的一个基站在一个时间，和检测法团的碱（A，T，G或C）在每个步骤通过检测的化学发光信号。焦磷酸测序反应包括模板DNA的酶，dNTPs（三磷酸的dATP，dTTP的，dCTP标记），DNA聚合酶，ATP硫酸化酶，萤光素，萤光素酶，三磷酸腺苷双磷酸酶。过程是由一个另外的四种dNTP和DNA聚合酶的生物素化的单链模板DNA的合成反应。 DNA聚合酶结合到模板上的互补的dNTP，与随后释放的焦磷酸盐（ 图1）。 ATP硫酸按比例转换成的焦ATP。 ATP作为氧化萤光素，荧光素的荧光素酶介导的转化用催化剂产生光（ 图2）。掺入 ​​的核苷酸的数量成比例的光的强度，确定是否一个或多个特定的dNTP（的dATP，dTTP的，三 ​​磷酸，或dCTP标记）是模板链顺序（ 图3）上存在的。任何非法人的dNTP降解三磷酸腺苷双磷酸酶之前，加入下一个dNTP的合成反应的延续。这种"合成测序"反应与另外四种dNTP各重复，直到确定的DNA序列的单链DNA模板。 要查看焦磷酸测序反应的动画， <a href="http://www.jove.com/files/ftp_upload/50405/50405movie1.mov" target="_blank">点击这里观看电影</a> 。

<table><tbody><tr><td>PyroMark PCR Master Mix, 2x</td><td>Qiagen</td><td>978703</td><td></td></tr><tr><td>CoralLoad Concentrate, 10x</td><td>Qiagen</td><td>978703, 978705</td><td></td></tr><tr><td>Q-Solution, 5x</td><td>Qiagen</td><td>978703, 978705</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;, 25 mM</td><td>Qiagen</td><td>978703, 978705</td><td></td></tr><tr><td>RNase-Free Water</td><td>Qiagen</td><td>129112</td><td></td></tr><tr><td>Primers</td><td>Qiagen</td><td>978776 or 978777</td><td>Primers should be purchased from an established oligonucleotide manufacturer (&lt;em&gt;i.e.&lt;/em&gt; QIAGEN Pyromark Custom Assays, IDT, etc). Lyophilized primers should be dissolved in TE to provide a stock solution of 100 &amp;mu;M.</td></tr><tr><td>Pyromark Gold Q24 Reagents</td><td>Qiagen</td><td>970802</td><td></td></tr><tr><td>High-purity water (Milli-Q 18.2 M&amp;Omega; x cm or equivalent)</td><td></td><td></td><td></td></tr><tr><td>Streptavidin Sepharose High Performance Beads</td><td>GE Healthcare</td><td>17-5113-07</td><td></td></tr><tr><td>PyroMark Annealing Buffer</td><td>Qiagen</td><td>979009</td><td></td></tr><tr><td>PyroMark Denaturation Solution</td><td>Qiagen</td><td>979007</td><td></td></tr><tr><td>PyroMark Wash Buffer</td><td>Qiagen</td><td>979008</td><td></td></tr><tr><td>PyroMark Q24</td><td>Qiagen</td><td>9001514</td><td></td></tr><tr><td>PyroMark Q24 Cartridge</td><td>Qiagen</td><td>979202</td><td></td></tr><tr><td>PyroMark Q96 HS Tip Holder Box</td><td>Qiagen</td><td>979105</td><td></td></tr><tr><td>PyroMark Q24 Vacuum Workstation</td><td>Qiagen</td><td>9001516</td><td></td></tr><tr><td>PyroMark Q24 Plate Holder</td><td>Qiagen</td><td>9022273</td><td></td></tr><tr><td>Orbital shaker</td><td>Fisher</td><td>11-675-297</td><td></td></tr><tr><td>Heat block</td><td>Fisher</td><td>11-718Q</td><td></td></tr></tbody></table>

pyrosequencing for microbial identification and characterization

焦磷酸测序法是一种通用的技术，可用于确定细菌种类，歧视细菌菌株，并检测抗微生物药物抗性的基因突变，有利于微生物的基因组测序。焦磷酸测序微生物应用程序的优点包括快速和可靠的微生物和微生物基因组突变的高通量筛选和准确的鉴定。焦磷酸测序法测序的DNA合成的互补链在一个时间一个单独的基，而确定的特定核苷酸的合成反应过程中被纳入。该反应发生在固定的单链模板DNA的四种脱氧核糖核苷酸（dNTP的）中依次加入下一个dNTP的合成反应，然后加入酶降解，并非法团的dNTPs。被由一代chemilum监测检测模板纳入特定的碱基信号inescent。的dNTPs，产生化学发光信号的顺序决定的模板的DNA序列。焦磷酸测序技术使实时测序能力快速微生物鉴定，在一个单一的检测。此外，焦磷酸测序仪，分析抗微生物药物耐药性的，包括分型的SNP位点，点突变，插入，缺失，以及定量分析中可能发生的一些抗微生物性的多基因拷贝的完整遗传多样性图案。

使用该软件的一个典型的焦磷酸测序运行的结果显示的位置的正向和反向引物，用于扩增子的产生，和保守的16S核糖体序列（ 图10）内的焦磷酸测序反应所用的测序引物，引物之间的序列的高变网站允许了大量的细菌种类，使用保守的测序引物（5&#39;-TACATGCAAGTCGA）进行细菌鉴定。作为一个内部的质量控制中，可变区的DNA序列包围可以被检查，以确保了正确的DNA区域已被分析。焦磷酸测序软件允许到内部数据库中进行细菌鉴定细菌核糖体序列生成的序列的比较和调整。此外，序列可以被分析以确定赋予抗生素耐药性的突变。例如，突变分析的23S核糖体基因Helicob字符幽门螺杆菌演示了两种抗生素抗性的突变模式（GAA或AGA）（ 图11）。分析多株来自同一病人可同时检测跟踪耐药性的出现，随着时间的推移，以帮助微生物耐药性爆发的流行病学调查。 <img alt="图1" src="/files/ftp_upload/50405/50405fig1.jpg" /> 图1。使用生物素化的PCR产物作为模板，将导致生成的焦磷酸（PPi）的DNA聚合酶，dNTPs浓度。 <img alt="图2" src="/files/ftp_upload/50405/50405fig2.jpg" /> 图2。 ATP硫酸按比例转换成焦磷酸，三磷酸腺苷，三磷酸腺苷的催化剂作为荧光素的荧光素酶介导的转化率氧化萤光素，产生光，是亲正比于ATP的量。记录光热解图跟踪峰值，并表示核苷酸成立。非法人的dNTPs先下一加入的dNTP继续合成三磷酸腺苷双磷酸酶降解。 <img alt="图3" src="/files/ftp_upload/50405/50405fig3.jpg" /> 图3。如果一个或多个特定的dNTP（的dATP，dTTP的，三 ​​磷酸，或dCTP标记）被合并到模板链上的顺序所产生的光的强度。 <img alt="图4" src="/files/ftp_upload/50405/50405fig4.jpg" /> 图4。预混固定生物素化的PCR产物编制流程图。 <img alt="图5" src="/files/ftp_upload/50405/50405fig5.jpg" /> GE ="总是"&gt; 图5。 PyroMark工作站，PyroMark板，PCR板，和波谷的位置。 <img alt="图6" src="/files/ftp_upload/50405/50405fig6.jpg" /> 图6。真空开关ON和OFF位置的位置。 <img alt="图7" src="/files/ftp_upload/50405/50405fig7.jpg" /> 图7。真空工具。正确处理真空工具。 <img alt="图8" src="/files/ftp_upload/50405/50405fig8.jpg" /> 图8。门打开墨盒与墨盒到位。 <img alt="图9" src="/files/ftp_upload/50405/50405fig9.jpg" /> 图9所示。正确插入墨盒门关闭。 "FO:保持together.within"="总是"&gt; <img alt="图10" src="/files/ftp_upload/50405/50405fig10.jpg" /> 图10。基于焦磷酸测序的细菌鉴定结果，设计PCR引物保守区域的DNA模板和测序引物的位置的紧上游，其特征在于良好的吞吐量高变的DNA序列内的扩增子（显示为蓝色）。 <img alt="图11" src="/files/ftp_upload/50405/50405fig11.jpg"/> 图11。使用焦磷酸测序幽门螺杆菌抗生素耐药检测 23S含有GAA AGA序列的幽门螺杆菌基因，赋予抗菌性的突变分析。 <a href="https://www.jove.com/files/ftp_upload/50405/50405fig11large.jpg" target="_blank">点击这里查看大图</a> 。 <table border="1"><tbody><tr> &lt;TD&gt; 组件 </td><td> 每个反应体积 </td><td> 终浓度 </td></tr><tr><td>火焰兵PCR预混，2 </td><td> 12.5微升</td><td> 1X </td></tr><tr><td>的浓缩CoralLoad，10倍</td><td> 2.5微升</td><td> 1X </td></tr><tr><td> 25毫米氯化镁2（可选） </td><td>变量</td><td> ≥1.5毫</td></tr><tr><td> Q-解决方案，5（可选） </td><td> 5微升</td><td> 1X </td></tr><tr><td>引物A /底漆B </td><td>变量/变量</td><td> 0.2μM/0.2微米</td></tr><tr><td> RNase-free的水</td><td>变量</td><td> - </td></tr><tr><td> 总成交量（后加入模板DNA） </td><td> 25微升 </td><td></td></tr></tbody></table> 表1中。 PCR反应混合物。 <table border="1"><tbody><tr><td></td><td> ＆NBSP; </td><td></td><td> 补充意见 </td></tr><tr><td>最初的PCR活化步骤</td><td> 15分钟</td><td> 95°C </td><td> HotStartTaq DNA聚合酶被激活</td></tr><tr><td> 3步法循环:变性</td><td> 30秒</td><td> 94°C </td><td></td></tr><tr><td>退火</td><td> 30秒</td><td> 60°C 56°C </td><td>对于基因组DNA 亚硫酸氢盐转化的DNA </td></tr><tr><td>延期</td><td> 30秒</td><td> 72°C </td><td></td></tr><tr><td> 的周期数 </td><td> 45 </td><td></td><td></td></tr><tr><td> 最后延伸 </td><td> 10分钟</td><td> 72°C </td><td></td></tr></tbody></table> 表2中。 PCR循环规范。

Watch this Scientific Journal Video about Pyrosequencing for Microbial Identification and Characterization at JoVE.com

Pyrosequencing for Microbial Identification and Characterization

焦磷酸测序法是一种通用的技术，可用于确定细菌种类，区分细菌的菌株，并检测抗微生物药物抗性的基因突变，有利于微生物的基因组测序。在这段视频中，微生物扩增子，扩增子的焦磷酸测序，DNA序列分析的程序将被证明。

焦磷酸测序的微生物鉴定和表征

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate ...

pyrosequencing-for-microbial-identification-and-characterization

Research

JoVE Journal

Biology

47.0K 次观看.  Johns Hopkins University. 焦磷酸测序法是一种通用的技术，可用于确定细菌种类，区分细菌的菌株，并检测抗微生物药物抗性的基因突变，有利于微生物的基因组测序。在这段视频中，微生物扩增子，扩增子的焦磷酸测序，DNA序列分析的程序将被证明。

视频: Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

焦磷酸测序：一个准确的基因分型的简单方法

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

科研

生物学

Isolation, Propagation, and Identification of Bacterial Species with Hydrocarbon Metabolizing Properties from Aquatic Habitats

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous catalytic enzymes and are naturally capable of metabolizing hydrocarbons. Scientists harness biodiversity in aquatic ecosystems to isolate bacteria with biodegradation and bioremediation potential. Such isolates from the environment provide a rich set of metabolic pathways and enzymes, which can be further utilized to scale up the degradation process at an industrial scale. In this article, we outline the general process of isolation, propagation, and identification of bacterial species from aquatic habitats and screen their ability to utilize hydrocarbons as the sole carbon source in vitro using simple techniques. The present protocol describes the isolation of various bacterial species and their subsequent identification using the 16S rRNA analysis. The protocol also presents steps for characterizing the hydrocarbon degrading potential of bacterial isolates. This protocol will be useful for researchers trying to isolate bacterial species from environmental habitats for their biotechnological applications.

We present the process of isolating, propagating, and characterizing hydrocarbon-degrading bacteria from aquatic habitats. The protocol outlines bacterial isolation, identification by the 16S rRNA method, and testing of their hydrocarbon-degrading potential. This article would help researchers in characterizing microbial biodiversity in environmental samples, and specifically screen for microbes with bioremediation potential.

从水生栖息地分离、繁殖和鉴定具有碳氢化合物代谢特性的细菌种类

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous ...

环境科学

Identification of Rare Bacterial Pathogens by 16S rRNA Gene Sequencing and MALDI-TOF MS

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in immunocompromised patients. In most cases only few data, mostly published as case reports, are available which investigate the role of such pathogens as an infectious agent. Therefore, in order to clarify the pathogenic character of such microorganisms, it is necessary to conduct epidemiologic studies which include large numbers of these bacteria. The methods used in such a surveillance study have to meet the following criteria: the identification of the strains has to be accurate according to the valid nomenclature, they should be easy to handle (robustness), economical in routine diagnostics and they have to generate comparable results among different laboratories. Generally, there are three strategies for identifying bacterial strains in a routine setting: 1) phenotypic identification characterizing the biochemical and metabolic properties of the bacteria, 2) molecular techniques such as 16S rRNA gene sequencing and 3) mass spectrometry as a novel proteome based approach. Since mass spectrometry and molecular approaches are the most promising tools for identifying a large variety of bacterial species, these two methods are described. Advances, limitations and potential problems when using these techniques are discussed.

Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and molecular techniques (16S rRNA gene sequencing) permit the identification of rare bacterial pathogens in routine diagnostics. The goal of this protocol lies in the combination of both techniques which leads to more accurate and reliable data.

由16S rRNA基因测序和MALDI-TOF MS罕见的细菌病原鉴定

There are a number of rare and, therefore, insufficiently described bacterial pathogens which are reported to cause severe infections especially in ...

免疫与感染

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 ...

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

的16S核糖体RNA基因的扩增子的新一代测序

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

高效的核酸提取和16S rRNA基因序列的细菌群落特征

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...

Genotyping Single Nucleotide Polymorphisms in the Mitochondrial Genome by Pyrosequencing

Mutations in the mitochondrial genome (mtDNA) have been associated with maternally inherited genetic diseases. However, interest in mtDNA polymorphisms has increased in recent years due to the recently developed ability to produce models by mtDNA mutagenesis and a new appreciation of the association between mitochondrial genetic aberrations and common age-related diseases such as cancer, diabetes, and dementia. Pyrosequencing is a sequencing-by-synthesis technique that is widely employed across the mitochondrial field for routine genotyping experiments. Its relative affordability when compared to massive parallel sequencing methods and ease of implementation make it an invaluable technique in the field of mitochondrial genetics, allowing for the rapid quantification of heteroplasmy with increased flexibility. Despite the practicality of this method, its implementation as a means of mtDNA genotyping requires the observation of certain guidelines, specifically to avoid certain biases of biological or technical origin. This protocol outlines the necessary steps and precautions in designing and implementing pyrosequencing assays for use in the context of heteroplasmy measurement.

Pyrosequencing assays enable the robust and rapid genotyping of mitochondrial DNA single nucleotide polymorphisms in heteroplasmic cells or tissues.

通过焦磷酸测序对线粒体基因组中的单核苷酸多态性进行基因分型

Mutations in the mitochondrial genome (mtDNA) have been associated with maternally inherited genetic diseases. However, interest in mtDNA polymorphisms ...

医学

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. ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

使用两步PCR和下一代16S rRNA基因测序进行微生物群分析

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

Isolation, Characterization, and Total DNA Extraction to Identify Endophytic Fungi in Mycoheterotrophic Plants

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as any other vital resource, the fungi with which these plants intimately associate are essential for them. Hence, some of the most relevant techniques in studying mycoheterotrophic species are the ones that enable the investigation of associated fungi, especially those inhabiting roots and subterranean organs. In this context, techniques for identifying culture-dependent and culture-independent endophytic fungi are commonly applied. Isolating fungal endophytes provides a means for morphologically identifying them, analyzing their diversity, and maintaining inocula for applications in the symbiotic germination of orchid seeds. However, it is known that there is a large variety of non-culturable fungi inhabiting plant tissues. Thus, culture-independent molecular identification techniques offer a broader cover of species diversity and abundance. This article aims to provide the methodological support necessary for starting two investigation procedures: a culture-dependent and an independent one. Regarding the culture-dependent protocol, the processes of collecting and maintaining plant samples from collection sites to laboratory facilities are detailed, along with isolating filamentous fungi from subterranean and aerial organs of mycoheterotrophic plants, keeping a collection of isolates, morphologically characterizing hyphae by slide culture methodology, and molecular identification of fungi by total DNA extraction. Encompassing culture-independent methodologies, the detailed procedures include collecting plant samples for metagenomic analyses and total DNA extraction from achlorophyllous plant organs using a commercial kit. Finally, continuity protocols (e.g., polymerase chain reaction [PCR], sequencing) are also suggested for analyses, and techniques are presented here.

The current article aims to provide detailed and adequate protocols for the isolation of plant-associated endophytic fungi, long-term preservation of isolates, morphological characterization, and total DNA extraction for subsequent molecular identification and metagenomic analyses.

分离、表征和总 DNA 提取以鉴定真菌异养植物中的内生真菌

Mycoheterotrophic plants present one of the most extreme forms of mycorrhizal dependency, having totally lost their autotrophic capacity. As essential as ...