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>

<ol>
	<li>Agrios, G. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant Pathology. , (1969).</li><li>Braun, U., Cook, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Taxonomic manual of the Erysiphales (Powdery Mildews). , (2012).</li><li>Both, M., Csukai, M., Stumpf, M. P. H., Spanu, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiles+of+Blumeria+graminis+indicate+dynamic+changes+to+primary+metabolism+during+development+of+an+obligate+biotrophic+pathogen.">Gene expression profiles of Blumeria graminis indicate dynamic changes to primary metabolism during development of an obligate biotrophic pathogen.</a> Plant Cell. 17 (7), 2107-2122 (2005).</li><li>Glawe, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+powdery+mildews:+A+review+of+the+world's+most+familiar+(yet+poorly+known)+plant+pathogens.">The powdery mildews: A review of the world's most familiar (yet poorly known) plant pathogens.</a> Annu Rev Phytopathol. 46, 27-51 (2008).</li><li>Fabro, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+expression+profiling+Arabidopsis+at+the+stage+of+Golovinomyces+cichoracearum+haustorium+formation.">Genome-wide expression profiling Arabidopsis at the stage of Golovinomyces cichoracearum haustorium formation.</a> Plant Physiol. 146 (3), 1421-1439 (2008).</li><li>H&#252;ckelhoven, R., Panstruga, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+biology+of+the+plant-powdery+mildew+interaction.">Cell biology of the plant-powdery mildew interaction.</a> Curr Opin Plant Biol. 14 (6), 738-746 (2011).</li><li>Micali, C. O., Neumann, U., Grunewald, D., Panstruga, R., O'Connell, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+of+a+specialized+plant-fungal+interface+during+host+cell+internalization+of+Golovinomyces+orontii+haustoria.">Biogenesis of a specialized plant-fungal interface during host cell internalization of Golovinomyces orontii haustoria.</a> Cell Microbiol. 13 (2), 210-226 (2011).</li><li>Chandran, D., Inada, N., Hather, G., Kleindt, C. K., Wildermuth, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+microdissection+of+Arabidopsis+cells+at+the+powdery+mildew+infection+site+reveals+site-specific+processes+and+regulators.">Laser microdissection of Arabidopsis cells at the powdery mildew infection site reveals site-specific processes and regulators.</a> Proc Natl Acad Sci. 107 (1), 460-465 (2010).</li><li>Spanu, P. D., Panstruga, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Powdery+mildew+genomes+in+the+crosshairs.">Powdery mildew genomes in the crosshairs.</a> New Phytol. 195 (1), 20-22 (2012).</li><li>Vela-Corc&#237;a, D., Romero, D., Tor&#233;s, J. A., De Vicente, A., P&#233;rez-Garc&#237;a, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+transformation+of+Podosphaera+xanthii+by+electroporation+of+conidia.">Transient transformation of Podosphaera xanthii by electroporation of conidia.</a> BMC Microbiol. 15 (20), (2015).</li><li>Hacquard, S., Francis, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Advances in Botanical Research. 70, 109-142 (2014).</li><li>Spanu, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+expansion+and+gene+loss+in+powdery+mildew+fungi+reveal+tradeoffs+in+extreme+parasitism.">Genome expansion and gene loss in powdery mildew fungi reveal tradeoffs in extreme parasitism.</a> Science. 330 (6010), 1543-1546 (2010).</li><li>Wicker, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+wheat+powdery+mildew+genome+shows+the+unique+evolution+of+an+obligate+biotroph.">The wheat powdery mildew genome shows the unique evolution of an obligate biotroph.</a> Nat Genet. 45 (9), 1092 (2013).</li><li>Jones, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+genomic+structural+variation+in+the+grape+powdery+mildew+pathogen,+Erysiphe+necator.">Adaptive genomic structural variation in the grape powdery mildew pathogen, Erysiphe necator.</a> BMC Genomics. 15, 1081 (2014).</li><li>Thomma, B. P. H. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mind+the+gap;+seven+reasons+to+close+fragmented+genome+assemblies.">Mind the gap; seven reasons to close fragmented genome assemblies.</a> Fungal Genet Biol. 90, 24-30 (2016).</li><li>Parlange, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+major+invasion+of+transposable+elements+accounts+for+the+large+size+of+the+Blumeria+graminis+f.sp.+tritici+genome.">A major invasion of transposable elements accounts for the large size of the Blumeria graminis f.sp. tritici genome.</a> Funct Integr Genomics. 11, 671-677 (2011).</li><li>Faino, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+real-time+sequencing+combined+with+optical+mapping+yields+completely+finished+fungal+genome.">Single-molecule real-time sequencing combined with optical mapping yields completely finished fungal genome.</a> mBio. 6, (2015).</li><li>Adam, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+Erysiphe+cichoracearum+and+E.+cruciferarum+and+a+survey+of+360+Arabidopsis+thaliana+accessions+for+resistance+to+these+two+powdery+mildew+pathogens.">Comparison of Erysiphe cichoracearum and E. cruciferarum and a survey of 360 Arabidopsis thaliana accessions for resistance to these two powdery mildew pathogens.</a> Mol Plant Microbe In. 12 (12), 1031-1043 (1999).</li><li>Bourras, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+avirulence+loci+and+allele-specific+effector+recognition+control+the+PM3+race-specific+resistance+of+wheat+to+powdery+mildew.">Multiple avirulence loci and allele-specific effector recognition control the PM3 race-specific resistance of wheat to powdery mildew.</a> Plant Cell. 27 (10), 2991-3012 (2015).</li><li>Henry, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Plant genotyping II: SNP technology. , (2008).</li><li>Healey, A., Furtado, A., Cooper, T., Henry, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol:+a+simple+method+for+extracting+next-generation+sequencing+quality+genomic+DNA+from+recalcitrant+plant+species.">Protocol: a simple method for extracting next-generation sequencing quality genomic DNA from recalcitrant plant species.</a> Plant Methods. 10 (21), (2014).</li><li>Fang, G., Hammar, S., Grumet, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quick+and+inexpensive+method+for+removing+polysaccharides+from+plant+genomic+DNA.">A quick and inexpensive method for removing polysaccharides from plant genomic DNA.</a> Biotechniques. 13 (1), 52-54 (1992).</li><li>Jordon-Thaden, I. E., Chanderbali, A. S., Gitzendanner, M. A., Soltis, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+CTAB+and+TRIzol+protocols+improve+RNA+extraction+from+chemically+complex+Embryophyta.">Modified CTAB and TRIzol protocols improve RNA extraction from chemically complex Embryophyta.</a> Appl Plant Sci. 3 (5), 1400105 (2015).</li><li>Murray, M. G., Thompson, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+isolation+of+high+molecular+weight+plant+DNA.">Rapid isolation of high molecular weight plant DNA.</a> Nucleic Acids Res. 8 (19), 4321-4325 (1980).</li><li>Greco, M., S&#225;ez, C. A., Brown, M. T., Bitonti, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+effective+method+for+high+quality+co-extraction+of+genomic+DNA+and+total+RNA+from+low+biomass+Ectocarpus+siliculosus,+the+model+brown+alga.">A simple and effective method for high quality co-extraction of genomic DNA and total RNA from low biomass Ectocarpus siliculosus, the model brown alga.</a> PLoS One. 9 (5), e96470 (2014).</li><li>Wilson, I. W., Schiff, C. L., Hughes, D. E., Somerville, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+trait+loci+analysis+of+powdery+mildew+disease+resistance+in+the+Arabidopsis+thaliana+accession+Kashmir-1.">Quantitative trait loci analysis of powdery mildew disease resistance in the Arabidopsis thaliana accession Kashmir-1.</a> Genetics. 158 (3), 1301-1309 (2001).</li></ol>

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，可能需要这种协议的其他修改。在未来，该协议可被用于获得高分子量DNA适合于从其他专性活体营养性真菌的长读取DNA测序，尽管可能需要进一步修改。 平衡高DNA产量和提取的DNA的高分子量是一个问题这个协议的优化期间。步骤包括涡旋，以便最小化所述DNA的剪切被淘汰，并通过倒置轻轻地混合所取代。这种变化提高了提取的DNA的质量而不显著降低产率。另外，在开始时除去钢球相比离开球在溶液中，直至溶解步骤结束时，细胞裂解步骤显著增加的DNA产率。 无G. cichoracearum分生孢子保持在步骤1.3球磨过程后完好无损。为了获得高分子量的基因组DNA的良好的产率，这是至关重要的，在此步骤中的真菌细胞壁被破坏。分生孢子完整性可显微镜来评估，并且可能需要对其他真菌种类附加球磨或破坏的替代方法。 有些真菌孢子，特别是布氏白粉菌，含有可续颜料氨化DNA的提取和纯化的尝试。在这些情况下，我们建议，包括如在Bourras 等人概括的DNA过滤步骤。 （2015）协议19，并用TE在过滤步骤之后的附加洗涤步骤。在添加过滤步骤导致DNA降解一个小而显著增加，并且如果过度色素沉着保持所述第二DNA沉淀步骤后只应被包括在内。这些颜料中，如果允许保留在溶液中，可与DNA的质量和数量的以后的分析和干扰可能与测序反应干扰。 该协议包括一个传统的琼脂糖凝胶电泳步骤和脉冲场凝胶电泳步骤。首先是DNA的分数即&gt; 20千碱基和运行于&lt;20 kbp的涂片分数的初始评估。对于下游测序反应的粉状密耳所需的目的露基因组，DNA的级分&lt;20 kbp的应该是最小的（ 见图1， 泳道2 与 泳道4）。脉冲场凝胶电泳评估提供的大小为DNA&gt; 20千碱基对的更好的估计可以通过大小来分离。 &gt; 48.5千碱基对的DNA是理想的在随后的测序反应（ 见图2， 泳道4）的使用。 在这些实验中使用的真菌材料在生长室中繁殖和污染，使用严格隔离的协议和控制人员的限制。正因为如此，与其他的非白粉病病原体污染是最小的，并使用特定的真菌引物仅回收G. cichoracearum序列扩增的ITS区域的测序。这可能不是在这些CA从外地网站或缺乏相同的隔离控制温室收集白粉病的情况下，和污染评估ES将是高品质的基因组从所提取的DNA的组件的关键。 各真菌DNA提取协议没有一个修改是唯一重要的。而在许多步骤轻微的修改导致了本文报道的高分子量白粉病DNA的相对高的产率。

<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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>MM400 Ball Mill</td>
			<td>Retsch</td>
			<td>20.745.0001</td>
			<td>Or equivalent ball mill</td>
		</tr>
		<tr>
			<td>2mL tubes for ball milling</td>
			<td>Sarstedt&#160;</td>
			<td>72.694.007</td>
			<td></td>
		</tr>
		<tr>
			<td>5/32&#34; 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&#176;C&#160;</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&#176;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&#160;</td>
			<td>38-022621807</td>
			<td>Or equivalent microcentrifuge</td>
		</tr>
		<tr>
			<td>Chlorform:IAA (24:1 v/v)</td>
			<td>Sigma-Aldrich&#160;</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 (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&#160;</td>
			<td>W292907</td>
			<td></td>
		</tr>
		<tr>
			<td>100% Ethanol</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>34923</td>
			<td>Chill to -20&#176;C prior use</td>
		</tr>
		<tr>
			<td>Sodium Acetate</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>S2889</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase, Dnase-free (10mg/ml)</td>
			<td>Thermo Fisher Scientific</td>
			<td>EN0531</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium metabisulfite</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>P2522</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Lauryl Sacroinate</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>L9150</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>10708976001</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>EDS</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>Cetyltrimethyl ammonium bromide (CTAB)</td>
			<td>Sigma-Aldrich&#160;</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 10mg/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>&#160;Agarose Ultrapure Bioreagent</td>
			<td>VWR International LLC</td>
			<td>JT4063</td>
			<td></td>
		</tr>
		<tr>
			<td>GeneRuler 1kb Plus DNA Ladder</td>
			<td>Thermo Fisher Scientific</td>
			<td>FERSM1332</td>
			<td></td>
		</tr>
		<tr>
			<td>8-48kb CHEF DNA size standards</td>
			<td>Bio-Rad</td>
			<td>170-3707</td>
			<td></td>
		</tr>
		<tr>
			<td>Pippin Prep&#160;&#160;</td>
			<td>Life Technologies Corporation</td>
			<td>4472172</td>
			<td>Or equivalent pulse-field gel aparatus</td>
		</tr>
		<tr>
			<td>Whatman&#160;qualitative filter paper, Grade 1</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>WHA1001042</td>
			<td>Or equivalent growth chamber</td>
		</tr>
		<tr>
			<td>Percival&#160; 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&#160;</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 &#176;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，充分传递是优选的。失败的质量控制的筹备工作时间不长，读取的基因组测序方法可以接受的。 <img alt="图1" src="/files/ftp_upload/55463/55463fig1.jpg" /> 图1:纯化基因组DNA的琼脂糖凝胶电泳。 第1道: </str翁&gt;的DNA梯，与频带范围从20 kbp的至75bp的; 泳道2:通过质量控制的基因组DNA; 泳道3:这被认为边际基因组DNA;和泳道4:基因组DNA失败质量控制。可接受的，并且边缘的基因组DNA样品具有运行高于20千碱基的单一条带以最小的涂抹在&lt;20千碱基。失败的质量控制基因组DNA样品已涂抹低于20 kbp的，并且可以包括低分子量的片段在约100bp（*）。脉冲场凝胶电泳需要可接受的，并且边缘的DNA样品之间进行区分。溴化乙锭加入到凝胶电泳之前以可视化的基因组DNA。将样品在60 V电泳50分钟。凝胶用凝胶记录系统在UV光下成像。 <img alt="图2" src="/files/ftp_upload/55463/55463fig2.jpg" /> 数字2:脉冲场凝胶电泳纯化白粉病基因组DNA（PFGE）。 泳道1:分子量标准，与48.5 kbp的最高频带和在8.1千对碱基的最低水平。 泳道2和5:分子量标准示出（在凝胶图像的底部）20，10，7,5，4，3，2和1.5千对碱基的带。 泳道3:边缘DNA样品，并在尺寸为20个48.5千碱基之间多数的DNA。 泳道4:可接受的DNA样品，与大多数DNA&gt; 48.5 kbp的（*）的。将样品在75伏用于使用5-80 kbp的PFGE波形程序15个小时运行。 DNA染色染料加入到该凝胶电泳后，将凝胶用凝胶记录系统在UV光下成像。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 试剂 </td><td> 终浓度 </td></tr><tr><td>钾代谢isulfite（K 2 S 2 O 5） </td><td> 0.25M的</td></tr><tr><td>的Tris缓冲液pH 7.5 </td><td> 0.2M的</td></tr><tr><td>乙二胺四乙酸（EDTA） </td><td> 50毫</td></tr><tr><td>氯化钠（NaCl） </td><td> 2M的</td></tr><tr><td>十六烷基三甲基溴化铵（CTAB） </td><td> 2％V / V </td></tr></tbody></table> 表1: 裂解缓冲液 的制备 。带来至5mL的与卫生署2 O的体积<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 目标 </td><td> 底漆名称 </td><td fo:width="2.8in"> 引物序列 </td><td> 的Tm（℃） </td><td> 预计产品尺寸（BP） </td></tr><tr><td rowspan="2">真菌ITS2 </td><td> FW（ITS9） </td><td> GAACGCAGCRAAIIGYGA </td><td> 56-61.3 </td><td rowspan="2"> 336 </td></tr><tr><td> RV（ITS4） </td><td> TCCTCCGCTTATTGATATGC </td><td> 52.1 </td></tr><tr><td rowspan="2">细菌16S V4 </td><td> FW（515F） </td><td> GTGCCAGCMGCCGCGGTAA </td><td> 63.8-66.5 </td><td rowspan="2"> 331 </td></tr><tr><td> RV（805R） </td><td> GGACTACHVGGGTWTCTAAT </td><td> 46.9-53.8 </td></tr><tr><td rowspan="2">细菌16S V4，V5 </td><td> FW（515F-Y） </td><td> GTGYCAGCMGCCGCGGTAA </td><td> 60.8-66.5 </td><td rowspan="2"> 450 </td></tr><tr><td> RV（926R） </td><td> CCGYCAATTYMTTTRAGTTT </td><td> 43.9-53.8 </td></tr><tr><td rowspan="2">真菌18S V4 </td><td> FW </td><td> CCAGCASCYGCGGTAATTCC </td><td> 58.7-61.5 </td><td rowspan="2"> 431 </td></tr><tr><td> RV </td><td> ACTTTCGTTCTTGATYRA </td><td> 43.2-48.3 </td></tr><tr><td rowspan="2">黄瓜PDS（LOC101204524） </td><td> CsPDS˚F </td><td> GTGAGTAAGGTTACCGTTTGGGGCTTATCCCAAT </td><td> 63.8 </td><td rowspan="2"> 750 </td></tr><tr><td> CsPDS [R </td><td> GCGTGAGCTCGGTACTCTCATCCACTCTTGCAC </td><td> 66.6 </td></tr></tbody></table> 表2:污染评估引物。

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 ...

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Nanyang Technological University

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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

Methyl-binding DNA capture Sequencing for Patient Tissues

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable development and differentiation of different tissue types. Dysregulation of this process is often the hallmark of various diseases like cancer. Here, we outline one of the recent sequencing techniques, Methyl-Binding DNA Capture sequencing (MBDCap-seq), used to quantify methylation in various normal and disease tissues for large patient cohorts. We describe a detailed protocol of this affinity enrichment approach along with a bioinformatics pipeline to achieve optimal quantification. This technique has been used to sequence hundreds of patients across various cancer types as a part of the 1,000 methylome project (Cancer Methylome System).

Here we present a protocol to investigate genome wide DNA methylation in large scale clinical patient screening studies using the Methyl-Binding DNA Capture sequencing (MBDCap-seq or MBD-seq) technology and the subsequent bioinformatics analysis pipeline.

为患者组织甲基结合DNA序列捕获

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

科研

遗传学

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 ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

核密度分析的天然染色质沉淀测序文库的生成

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

G2-seq: A High Throughput Sequencing-based Technique for Identifying Late Replicating Regions of the Genome

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have been directed toward elucidation of the location and timing of initiation of genome duplication rather than its completion. However, it is critical that we understand regions of the genome that are last to complete replication, because these regions suffer elevated levels of chromosomal breakage and mutation, and they have been associated with both disease and aging. Here we describe how we have extended a technique that has been used to monitor replication initiation to instead identify those regions of the genome last to complete replication. This approach is based on a combination of flow cytometry and high throughput sequencing. Although this report focuses on the application of this technique to yeast, the approach can be used with any cells that can be sorted in a flow cytometer according to DNA content.

We describe a technique for combining flow cytometry and high throughput sequencing to identify late replicating regions of the genome.

G2-seq: 一种基于高通量测序技术的基因组晚期复制区域识别方法

Numerous techniques have been developed to follow the progress of DNA replication through the S phase of the cell cycle. Most of these techniques have ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

植物标本标本的鲁棒 DNA 分离与高通量测序库建设

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

基于纠错 DNA 和 RNA 测序的稀有事件检测方法

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

人骨髓间充质干细胞细胞外微 rna 的分离、测序和分析

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

纳米测序全血 mRNA 测序

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...

Identification of Circular RNAs using RNA Sequencing

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

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

使用RNA测序识别循环RNA

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

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise heterozygous genetic modifications is challenging due to CRISPR-CAS9 mediated indel formation in the second allele. Here, we demonstrate a protocol to help overcome this difficulty by using two repair templates in which only one expresses the desired sequence change, while both templates contain silent mutations to prevent re-cutting and indel formation. This methodology is most advantageous for gene editing coding regions of DNA to generate isogenic control and mutant human stem cell lines for studying human disease and biology. In addition, optimization of transfection and screening methodologies have been performed to reduce labor and cost of a gene editing experiment. Overall, this protocol is widely applicable to many genome editing projects utilizing the human pluripotent stem cell model.

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem cells.

在人类多能干细胞中使用CRISPR-CAS9生成定义的基因组修饰

Human pluripotent stem cells offer a powerful system to study gene function and model specific mutations relevant to disease. The generation of precise ...