Name: purification of high molecular weight genomic dna from powdery mildew for long-read sequencing
Uploaded: 2017-03-31T13:00:00.000+00:00
Duration: 6 min 56 s
Description: Watch this Scientific Journal Video about Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing at JoVE.com

This protocol was developed in support of a Joint Genome Institute-Community Sequencing Project (#1657). The work was supported in part by the Philomathia Foundation, the National Science Foundation (#0929226) and the Energy Biosciences Institute to S. Somerville; and by Swiss National Science Foundation (#310030_163260) to B. Keller. We would like to thank our colleagues, M. Figureroa and M. Miller (University of Minnesota), R. Panstruga (RWTH Aachen University), C. Pedersen (University of Copenhagen), P. Spanu (Imperial College), J. Taneja (University of California Berkeley), M. Wildermuth (University of California Berkeley), R. Wise (Iowa State University) and S. Xiao (University of Maryland) for their generous advice and for volunteering their protocols as we developed the protocol described here.

1.真菌材料的制备<ol><li>成长白粉病<ol><li>生长在生长室的植物在以下条件下:22℃白天温度，20℃夜间温度，80％相对湿度，14-H昼长和125μE/米2 / s的光强度（由荧光灯提供）。 </li><li>感染的黄瓜植物（ 黄瓜苜蓿为Bush Champion）与高氏cichoracearum应变UCSC1如所描述的18，29。 </li></ol></li><li>收获白粉病分生孢子<ol><li>从感染的植物收获的分生孢子在7 - 接种后10天使用小真空与在线过滤器（11微米），其上的分生孢子积聚。施加轻柔的压力，运行真空喷嘴沿叶片的表面以收集分生孢子。 注:G. cichoracearum的平均产量为20mg分生孢子从一个重INFE在面积约130厘米2反恐执行黄瓜叶。 </li><li>刷约150mg的分生孢子（约375μL体积，或分生孢子从7-8感染黄瓜叶子）从所述过滤器的清洁上纸的片材。从这里，分生孢子转移到2个毫升冷冻管具有两个32分之5英寸直径预冷（在液氮中）钢研磨球。立即返回管到液氮中。商店管在-80℃下。 </li></ol></li><li>砸开分生孢子球磨<ol><li>闪光冻结球磨机的铝板在液氮中。装载冷冻管进入球磨机架。在30个循环/秒（室温）剧烈摇晃以在球磨机中进行30秒和分生孢子钢球的冷冻管。立即重新冷冻在液氮中的冷冻管。 注意:首先，通过显微镜在研磨过程中在100X放大率访问分生孢子的样品为细胞壁破裂的效率。如果需要，研磨另外的1 - 2轮的30秒30个循环/ S，BUT保持轮数降到最低。一旦最佳条件已建立通过显微镜在这个阶段分生孢子破裂的常规评估是不必要的。 </li></ol></li></ol> 2.基因组DNA纯化<ol><li>分生孢子裂解<ol><li>工作迅速，避免融化的分生孢子，从冷冻管用磁铁取出钢珠。直到形成淤浆10秒-加入700μl65℃的裂解缓冲液（ 表1）并涡旋5。 </li><li>添加300μL预温（65℃）5％（V / V）十二烷基肌氨酸钠和轻轻颠倒以混合（每3秒1点反转）五次。在65°C孵育管30分钟;在10，20和30分钟轻轻颠倒3次。使用宽孔移液管尖端，转移到新的2毫升的离心管中全部溶液。在所有后续步骤，通过温和，缓慢翻转混合，并用宽口移液管尖端转移含DNA的溶液。 </li></ol></li><li> DNA提取我<ol><li>加入1个体积的氯形式:异戊醇（24:1体积/体积），以裂解溶液和轻轻颠倒以混合五次。孵育在室温下10分钟，轻轻翻转到在中途点再次在温育结束时混合五次和。离心机在室温下以14,000×g的15分钟。使用大口径的移液管尖端，小心地转移水层到新的2ml微量离心管中;避免包括从所述界面材料。 </li></ol></li><li> DNA沉淀我<ol><li>加入1倍体积室温100％异丙醇（约750微升），并轻轻颠倒以混合六次。离心机在室温下以14,000×g的15分钟。小心取出上清液并丢弃。 </li><li>添加450μL预冷却（-20℃）70％乙醇。离心机在室温下以14,000×g下5分钟。小心取出上清液并丢弃。离心机中以1000×g离心3秒钟，小心地取出用细枪头上清液并丢弃。空气干燥15分钟将沉淀吨室温。不要干燥时间超过15分钟。 </li><li>重悬300μL的TE颗粒（10毫摩尔Tris-CL，1mM的乙二胺四乙酸，pH 8.0）中。在4℃下孵育过夜。轻轻一抖重悬任何残留的材料，没有去成的解决方案。 </li></ol></li><li>以除去RNA污染，加入10μL的RNA酶A（10毫克/毫升）。轻轻倒转混合三次。离心1000 XG为3秒。孵育在37℃下2小时。 </li><li> DNA提取II <ol><li>添加300μL苯酚:氯仿:异戊醇（25:24:1体积/体积）并轻轻颠倒以混合五次。在室温下孵育10分钟，轻轻倒置在半路标记再次在温育结束时混合五次和。在以14,000×g下室温离心15分钟。使用宽孔移液管尖端，将上清转移至新的1.5毫升离心管中。 </li></ol></li><li> DNA沉淀II <ol><li>添加0.01体积的3M乙酸钠pH 5.2（approximatelY 3微升），轻轻颠倒混合五倍。加入2.5倍体积预冷（-20℃）的100％乙醇（约750微升）。轻轻颠倒混合五倍。在-20℃下孵育过夜</li><li>离心机在4℃下30分钟，在14,000×克。小心取出上清液并丢弃。 </li><li>添加450μL预冷却（-20℃）70％乙醇。离心机在4℃下5分钟，在14,000×g下。小心取出上清液并丢弃。离心1000 XG为3秒。小心地取出用细移液管尖，弃上清。 </li><li>空气干燥沉淀，在室温下30-60分钟。重悬27.5微升TE沉淀。在4℃下孵育过夜。轻轻一抖重悬任何残留的材料，没有去成的解决方案。 </li><li>等分试样2.5μL到22.5μLTE（终体积25微升）用于质量控制测试（见下文）。商店的DNA样品在4℃下，直到提交用于测序。对于长期储存，储存样本在-80℃下，并尽量减少冻融循环，以防止剪切。 注意:在此步骤作为所述高分子量的基因组DNA可以吹打难以正确地吸取时要小心，并确保"QC等分试样"是基因组DNA样品的精确表示是非常重要的。 </li></ol></li></ol> 3.质量控制<ol><li>样品纯度<ol><li>使用TE空白，通过在小容积分光光度计加载QC等分试样的1μL评估DNA的样品纯度。记录A260 / A280和A260 / A230读数。纯DNA将具有约1.8和A260 / A230比值在1.8和2.2之间的A260 / A280比值。 </li></ol></li><li>基因组DNA的定量<ol><li>使用根据制造商的说明书商业双链DNA定量测定试剂盒进行QC等分试样的定量。 </li></ol></li><li> DNA质量评价我<ol><li>负载大约60纳克基因组DNA和一个在1×TAE（40毫摩尔Tris，20mM乙酸和1mM EDTA，pH 8.0）中的0.7％琼脂糖凝胶DNA梯。在70 V代表40运行凝胶装置 - 45分钟，直到基因组DNA带是大约从梯井和频带分离2厘米是显而易见的。这些条件可能需要基于可用凝胶电泳装置进行调整。 </li></ol></li><li> DNA质量评估II <ol><li>负载上在0.5×TBE（44.5毫摩尔Tris，44.5毫摩尔硼酸，1mM EDTA中，pH值8.3）和1％琼脂糖凝胶约60纳克基因组DNA和高分子量DNA梯（适于脉冲场凝胶电泳） 80 kbp的波形脉冲场凝胶电泳协议16小时 - 在0.5X TBE缓冲液中运行使用5运行。 </li></ol></li><li>细菌，真菌和植物污染物的评估<ol><li>进行聚合酶链式反应（PCR）扩增，使用引物和扩增条件列表中的核糖体基因的适当的内转录间隔区（ITS）区域编在表2中 。负载10μL在凝胶上得到的反应的。 注:表示白粉病ITS区域应与真菌ITS引物扩增带。从该扩增子和与细菌或植物的引物产生的扩增子的任何DNA应当测序，以确定污染的来源。只有白粉病ITS序列应在真菌扩增子被发现。 </li></ol></li></ol>

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The authors have nothing to disclose.

为了从专性活体营养性白粉病菌获得纯的，高分子量的基因组DNA中，先前所描述的方法的修改版本被开发30。使用该优化的协议的平均产量是每150毫克分生孢子7微克DNA，与现有协议中得到的产率加倍。另外，平均尺寸从大约20 kbp的增加到超过48.5 kbp的。该协议是在黄瓜- G. cichoracearum系统进行了优化。为了获得在其他白粉病最好的质量和纯度最高的DNA或专性活体营养性真菌?...

<html>白粉病是一组专性活体营养真菌植物致病菌，当合起来，为植物病害全球1的最大原因。有白粉病的超过900种描述，已经在分类学上分为家庭Erisyphaceae 2中的五个部落。两个原因，一是经济上的重要性，他们用自己的主机发展的亲密关系，白粉病病一直为&gt;百年研究的课题。感染后，白粉菌引起的细胞结构，代谢及宿主分子生物学剧变，受益这种病原体。然而，白粉病的研究是特别具有挑战性的，因为它们专生活方式，并且在纯培养的真菌生长的尚未描述3，4，5，"外部参照"&gt; 6，7，8。白粉病的可靠，稳定的遗传转化还尚未完成，虽然瞬时转化已经在一些物种中9，10的报道。 白粉病基因组测序和组装已经证明，很难因数量的基因组本身的特性。白粉病基因组是大的（120 - 180 MBP）相对于其它真菌的基因组，和包括60 - 90％的均匀分布的重复元件11。这些元件包括非长末端重复，以及未分类的重复元件。单白粉病品种， 白粉病的F两个专化型 。 SP。 大麦和f。 SP。 小麦（BGH和白粉菌，分别地）以及葡萄白粉病葡萄白粉菌，有蜂ñ测序，以及其他几个人的基因组草案已经完成12，13，14。基因组的重复性质取得了组装困难，并且已完成的基因组BGH组装到6989个supercontigs具有2 MB 12的L50。 尽管有大量的基因组中，白粉病似乎具有少量的蛋白质编码基因，分别在BGH和白粉菌预测，5845倍6540的基因。测序白粉病似乎也缺少了在其他真菌，这是对他们的寄主植物的真菌生存11，12，13，14依赖性一致至关重要至少99核心基因。 近端粒，着丝粒，核糖体RN重复序列A基因阵列和在可转座元件富集区域是从短读测序策略组装不良和低于限额在基因组组件15常常。这样的区域被认为是负责许多发生在基因组序列的间隙，并且这适用于白粉病与它们广泛重复元件16的膨胀。高塑性的基因组区域在这种重复区域3经常发现。它们充当的染色体重排的一个位点和通常编码在强选择压力的基因，如编码效应蛋白的基因和编码次生代谢的酶的基因。在单分子长读测序技术的进步提供了跨越基因组15的重复区域测序潜在的解决方案。例如，Faino 等。 （2015）发现，其中包括长序列读技术和光学米apping允许他们产生的间隙少的基因组序列为植物病原真菌黄萎病大丽花的两个菌株，三倍基因组中的重复DNA序列的比例，增加基因注释的数量（和减少的部分的数目或丢失的基因注解）和揭示基因组重排17。 为了采用这些长读测序技术，高浓度的高品质的基因组DNA，以最小的尺寸&gt; 20 kbp的，是必要的。在这里，我们描述了我们的分生孢子收集，高分子量DNA的纯化方法从分生孢子和我们使用白粉病品种高氏cichoracearum质量控制评估生长在黄瓜18。该协议是基于B·凯勒实验室集团（苏黎世大学，苏黎世瑞士）13，19开发的协议和包括几个修饰导致增加的DNA产量和DNA&gt; 48.5千碱基对大小的比例较高。该协议还包括基于来自能源联合基因组的美国国防部研究所20，21，22项建议的质量控制措施。 包括在裂解缓冲液中的各试剂，并且对于每个纯化步骤的基本原理的功能，在亨利（2008）23所描述的。用于从植物和微生物组织高分子量的基因组DNA的分离可用公开的方案本协议24，25，26，27，28的设计期间进行了协商。

<table><tbody><tr><td>MM400 Ball Mill</td><td>Retsch</td><td>20.745.0001</td><td>Or equivalent ball mill</td></tr><tr><td>2 mL tubes for ball milling</td><td>Sarstedt&amp;nbsp;</td><td>72.694.007</td><td></td></tr><tr><td>5/32&quot; stainless steel milling balls</td><td>OPS Diagnostics</td><td>GBSS 165-5000-01</td><td></td></tr><tr><td>Microprocessor controlled 280 Series Water Bath Preset to 65 &amp;deg;C&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>2825</td><td>Or equivalent water bath</td></tr><tr><td>Microprocessor controlled 280 Series Water Bath Preset to 37 &amp;deg;C</td><td>Thermo Fisher Scientific</td><td>2825</td><td>Or equivalent water bath</td></tr><tr><td>Eppendorf 5417R refrigerated microcentrifuge</td><td>Krakeler Scientific&amp;nbsp;</td><td>38-022621807</td><td>Or equivalent microcentrifuge</td></tr><tr><td>Chlorform:IAA (24:1 v/v)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>C0549</td><td>Hazardous in case of skin contact or inhalation. Wear nitrile gloves, protective eyewear, lab coat and work in chemical hood</td></tr><tr><td>Phenol:Chloroform:IAA&lt;br /&gt; (25:24:1 v/v)</td><td>Thermo Fisher Scientific</td><td>15593031</td><td>Hazardous in case of skin contact or inhalation. Wear nitrile gloves, protective eyewear, lab coat and work in chemical hood</td></tr><tr><td>100% Isopropanol</td><td>Sigma-Aldrich&amp;nbsp;</td><td>W292907</td><td></td></tr><tr><td>100% Ethanol</td><td>Sigma-Aldrich&amp;nbsp;</td><td>34923</td><td>Chill to -20 &amp;deg;C prior use</td></tr><tr><td>Sodium Acetate</td><td>Sigma-Aldrich&amp;nbsp;</td><td>S2889</td><td></td></tr><tr><td>Rnase, Dnase-free (10 mg/mL)</td><td>Thermo Fisher Scientific</td><td>EN0531</td><td></td></tr><tr><td>Potassium metabisulfite</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P2522</td><td></td></tr><tr><td>Sodium Lauryl Sacroinate</td><td>Sigma-Aldrich&amp;nbsp;</td><td>L9150</td><td></td></tr><tr><td>Tris base</td><td>Sigma-Aldrich&amp;nbsp;</td><td>10708976001</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>EDS</td><td></td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>S9888</td><td></td></tr><tr><td>Cetyltrimethyl ammonium bromide (CTAB)</td><td>Sigma-Aldrich&amp;nbsp;</td><td>H6269</td><td></td></tr><tr><td>Uvex by Honeywell Futura Goggles S345C, Uvextreme</td><td>Staples</td><td>423263</td><td>Or equivalent eyewear</td></tr><tr><td>High Five COBALT nitrile gloves, LARGE</td><td>Neta Scientific</td><td>HFG-N193</td><td>Or equivalent gloves</td></tr><tr><td>GelRed Nucleic Acid Gel Stain 10,000x in water</td><td>Biotium</td><td>41003</td><td></td></tr><tr><td>Ethidium Bromide 10 mg/mL</td><td>Biotium</td><td>40042</td><td>Hazardous in case of skin contact. Wear nitrile gloves and protective eyewear</td></tr><tr><td>Agarose Ultrapure Bioreagent</td><td>VWR International LLC</td><td>JT4063</td><td></td></tr><tr><td>GeneRuler 1 kb Plus DNA Ladder</td><td>Thermo Fisher Scientific</td><td>FERSM1332</td><td></td></tr><tr><td>8 - 48 kb CHEF DNA size standards</td><td>Bio-Rad</td><td>170-3707</td><td></td></tr><tr><td>Pippin Prep</td><td>Life Technologies Corporation</td><td>4472172</td><td>Or equivalent pulse-field gel aparatus</td></tr><tr><td>Whatman qualitative filter paper, Grade 1</td><td>Sigma-Aldrich&amp;nbsp;</td><td>WHA1001042</td><td>Or equivalent growth chamber</td></tr><tr><td>Percival Growth Chamber</td><td>Percival</td><td>AR66LXC9</td><td></td></tr><tr><td>NanoDrop 8000 UV-Vis Spectrophotometer</td><td>Thermo Fisher Scientific</td><td>ND-8000-GL</td><td>Or equivalent spectrophotometer</td></tr><tr><td>Quant-iT PicoGreen dsDNA Assay Kit</td><td>Thermo Fisher Scientific</td><td>P11496</td><td></td></tr><tr><td>TAE Buffer (Tris-acetate-EDTA) (50x)</td><td>Thermo Fisher Scientific</td><td>B49</td><td>Dilute 1/50 with water before use (to 1x)</td></tr><tr><td>TE Buffer</td><td>Thermo Fisher Scientific</td><td>12090015&amp;nbsp;</td><td></td></tr><tr><td>Tris-Borate-EDTA, 10x Solution (Electrophoresis)</td><td>Thermo Fisher Scientific</td><td>BP13334</td><td>Dilute 1/10 with water before use (to 1x)</td></tr><tr><td>Liquid Nitrogen</td><td></td><td></td><td>Liquid nitrogen (-196 &amp;deg;C) is a freezing hazard. It will also expand rapidly upon warming and should not keep in a tightly closed container</td></tr></tbody></table>

purification of high molecular weight genomic dna from powdery mildew for long-read sequencing

白粉病菌是一类重要经济价值的植物病原真菌。相对知之甚少的分子生物学和这些病原体的遗传学，部分原因是由于缺乏发达的遗传和基因组资源。这些有机体具有大的，重复的基因组，这取得了基因组测序和装配极其困难。在这里，我们描述了从一个白粉病物种， 高氏cichoracearum高分子量的基因组DNA的集合，提取，纯化和质量控制评估方法。所描述的协议包括孢子，随后优化的苯酚/氯仿基因组DNA提取的机械破坏。典型的产率是每150个毫克分生孢子7微克DNA。正在使用此过程中分离的基因组DNA是适合长时间读测序（ 即 ，&gt; 48.5 kbp的）。质量控制措施，以确保尺寸，收量，以及基因组DNA的纯度也在该方法中描述的。这里所描述的质量的基因组DNA的测序将允许组件和多个白粉病的基因组，这反过来将导致更好地理解的比较和改进了这一农业病原体的控制。

60ng从G.纯化的基因组DNA的代表性实例cichoracearum使用凝胶电泳在琼脂糖凝胶上运行，并用脉冲场凝胶电泳在图1和2，分别被示出。基因组DNA制备物通，勉强通过和失败的质量控制的代表。基因组DNA制备物，充分通过质量控制是理想的使用长读基因组测序方法测序。制剂，该制剂通过轻微的质量控制是可接受的，但制剂，充...

Watch this Scientific Journal Video about Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing at JoVE.com

Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing

Described here is a method for the extraction, purification, and quality control of genomic DNA from the obligate biotrophic fungal pathogen, powdery mildew, for use in long-read genome sequencing.

超高分子量基因组DNA的分离纯化白粉病对长读测序

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics ...

purification-high-molecular-weight-genomic-dna-from-powdery-mildew

Research

JoVE Journal

Genetics

11.6K 次观看.  University of California Berkeley. Described here is a method for the extraction, purification, and quality control of genomic DNA from the obligate biotrophic fungal pathogen, powdery mildew, for use in long-read genome sequencing.

视频: Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic fragments. Organellar genomes also exist in admixtures within a given cell or tissue type (heteroplasmy), and an abundance of subtypes may change throughout development or when under stress (sub-stoichiometric shifting). Next-generation sequencing (NGS) technologies are required to obtain deeper understanding of organellar genome structure and function. Traditional sequencing studies use several methods to obtain organellar DNA: (1) If a large amount of starting tissue is used, it is homogenized and subjected to differential centrifugation and/or gradient purification. (2) If a smaller amount of tissue is used (i.e., if seeds, material, or space is limited), the same process is performed as in (1), followed by whole-genome amplification to obtain sufficient DNA. (3) Bioinformatics analysis can be used to sequence the total genomic DNA and to parse out organellar reads. All these methods have inherent challenges and tradeoffs. In (1), it may be difficult to obtain such a large amount of starting tissue; in (2), whole-genome amplification could introduce a sequencing bias; and in (3), homology between nuclear and organellar genomes could interfere with assembly and analysis. In plants with large nuclear genomes, it is advantageous to enrich for organellar DNA to reduce sequencing costs and sequence complexity for bioinformatics analyses. Here, we compare a traditional differential centrifugation method with a fourth method, an adapted CpG-methyl pulldown approach, to separate the total genomic DNA into nuclear and organellar fractions. Both methods yield sufficient DNA for NGS, DNA that is highly enriched for organellar sequences, albeit at different ratios in mitochondria and chloroplasts. We present the optimization of these methods for wheat leaf tissue and discuss major advantages and disadvantages of each approach in the context of sample input, protocol ease, and downstream application.

The comparison and optimization of two plant organellar DNA enrichment methods are presented: traditional differential centrifugation and fractionation of the total gDNA based on methylation status. We assess the resulting DNA quantity and quality, demonstrate performance in short-read next-generation sequencing, and discuss the potential for use in long-read single-molecule sequencing.

适用于下一代测序的植物细菌DNA富集方法的优化与比较分析

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic ...

科研

遗传学

Nanopore DNA Sequencing for Metagenomic Soil Analysis

This article describes the steps for construction of a DNA library from soil, preparation and use of the nanopore flow cell, and analysis of the DNA sequences identified using computer software. Nanopore DNA sequencing is a flexible technique that allows for rapid microbial genome sequencing to identify bacterial and viral species, to characterize bacterial strains, and to detect genetic mutations that confer resistance to antibiotics. The advantages of nanopore sequencing (NS) for life sciences include its low complexity, reduced cost, and rapid real-time sequencing of purified genomic DNA, PCR amplicons, cDNA samples, or RNA. NS is an example of "strand sequencing" which involves sequencing DNA by guiding a single stranded DNA molecule through a nanopore that is inserted into a synthetic polymer membrane. The membrane has an electrical current applied across it, so as the individual bases pass through the nanopore the electrical current is disrupted to varying degrees by the four nucleotide bases. The identification of each nucleotide occurs by detecting the characteristic modulation of the electrical current by the different bases as they pass through the nanopore. The NS system consists of a handheld, USB powered portable device and a disposable flow cell that contains a nanopore array. The portable device plugs into a standard laptop computer that reads and records the DNA sequence using computer software.

Nanopore technology for sequencing biomolecules has wide applications in the life sciences, including identification of pathogens, food safety monitoring, genomic analysis, metagenomic environmental monitoring, and characterization of bacterial antibiotic resistance. In this article, the procedure for metagenomic soil DNA sequencing for species identification using the nanopore sequencing technology is demonstrated.

基因土壤分析的纳米 DNA 测序

This article describes the steps for construction of a DNA library from soil, preparation and use of the nanopore flow cell, and analysis of the DNA ...

环境科学

Novel Sequence Discovery by Subtractive Genomics

Subtractive genomics can be used in any research where the goal is to identify the sequence of a gene, protein, or general region that is embedded in a larger genomic context. Subtractive genomics enables a researcher to isolate a target sequence of interest (T) by comprehensive sequencing and subtracting out known genetic elements (reference, R). The method can be used to identify novel sequences such as mitochondria, chloroplasts, viruses, or germline restricted chromosomes, and is particularly useful when T cannot be easily isolated from R. Beginning with the comprehensive genomic data (R + T), the method uses Basic Local Alignment Search Tool (BLAST) against a reference sequence, or sequences, to remove the matching known sequences (R), leaving behind the target (T). For subtraction to work best, R should be a relatively complete draft that is missing T. Since sequences remaining after subtraction are tested through quantitative Polymerase Chain Reaction (qPCR), R does not need to be complete for the method to work. Here we link computational steps with experimental steps into a cycle that can be iterated as needed, sequentially removing multiple reference sequences and refining the search for T. The advantage of subtractive genomics is that a completely novel target sequence can be identified even in cases in which physical purification is difficult, impossible, or expensive. A drawback of the method is finding a suitable reference for subtraction and obtaining T-positive and negative samples for qPCR testing. We describe our implementation of the method in the identification of the first gene from the germline-restricted chromosome of zebra finch. In that case computational filtering involved three references (R), sequentially removed over three cycles: an incomplete genomic assembly, raw genomic data, and transcriptomic data.

The purpose of this protocol is to use a combination of computational and bench research to find novel sequences that cannot be easily separated from a co-purifying sequence, which may be only partially known.

减法基因组学的新序列发现

Subtractive genomics can be used in any research where the goal is to identify the sequence of a gene, protein, or general region that is embedded in a ...

Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae

Chromatin immunoprecipitation sequencing (ChIP-seq) is a powerful and widely used molecular technique for mapping whole genome locations of transcription factors (TFs), chromatin regulators, and histone modifications, as well as detecting entire genomes for uncovering TF binding patterns and histone posttranslational modifications. Chromatin-modifying activities, such as histone methylation, are often recruited to specific gene regulatory sequences, causing localized changes in chromatin structures and resulting in specific transcriptional effects. The rice blast is a devastating fungal disease on rice throughout the world and is a model system for studying fungus-plant interaction. However, the molecular mechanisms in how the histone modifications regulate their virulence genes in Magnaporthe oryzae remain elusive. More researchers need to use ChIP-seq to study how histone epigenetic modification regulates their target genes. ChIP-seq is also widely used to study the interaction between protein and DNA in animals and plants, but it is less used in the field of plant pathology and has not been well developed. In this paper, we describe the experimental process and operation method of ChIP-seq to identify the genome-wide distribution of histone methylation (such as H3K4me3) that binds to the functional target genes in M. oryzae. Here, we present a protocol to analyze the genome-wide distribution of histone modifications, which can identify new target genes in the pathogenesis of M. oryzae and other filamentous fungi.

Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae

Here, we present a protocol to analyze the genome-wide distribution of histone modifications, which can identify new target genes in the pathogenesis of M. oryzae and other filamentous fungi.

使用麦格纳波特奥里扎伊的色度免疫沉淀测序方法对希斯通修饰分布进行全基因组分析

Chromatin immunoprecipitation sequencing (ChIP-seq) is a powerful and widely used molecular technique for mapping whole genome locations of transcription ...

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

超高分子量基因组DNA的提取土壤和沉积物

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

生物学

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

单细胞基因组测序的超高通量微流控平台

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

生物工程

Ultra-long Read Sequencing for Whole Genomic DNA Analysis

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes and analysis of complex structural variants. Nanopore platforms perform single-molecule sequencing by directly measuring the current changes mediated by DNA passage through the pores and can generate hundreds of kilobase (kb) reads with minimal capital cost. This platform has been adopted by many researchers for a variety of applications. Achieving longer sequencing read lengths is the most critical factor to leverage the value of nanopore sequencing platforms. To generate ultra-long reads, special consideration is required to avoid DNA breakages and gain efficiency to generate productive sequencing templates. Here, we provide the detailed protocol of ultra-long DNA sequencing including high molecular weight (HMW) DNA extraction from fresh or frozen cells, library construction by mechanical shearing or transposase fragmentation, and sequencing on a nanopore device. From 20-25 &#181;g of HMW DNA, the method can achieve N50 read length of 50-70 kb with mechanical shearing and N50 of 90-100 kb read length with transposase mediated fragmentation. The protocol can be applied to DNA extracted from mammalian cells to perform whole genome sequencing for the detection of structural variants and genome assembly. Additional improvements on the DNA extraction and enzymatic reactions will further increase the read length and expand its utility.

Long-read sequences greatly facilitate the assembly of complex genomes and characterization of structural variation. We describe a method to generate ultra-long sequences by nanopore-based sequencing platforms. The approach adopts an optimized DNA extraction followed by modified library preparations to generate hundreds of kilobase reads with moderate coverage from human cells.

用于全基因组 DNA 分析的超长读数测序

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes ...

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

超高分子量DNA提取微生物席

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.

Using the cystic fibrosis airway as an example, the manuscript presents a comprehensive workflow comprising a combination of metagenomic and metatranscriptomic approaches to characterize the microbial and viral communities in animal-associated samples.

从复杂的动物相关样品测序宏基因组和Metatranscriptomes:净化不纯

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

从真核细胞染色体外的环状DNA基因组范围内的净化

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...