We give special thanks to Ezra Lyon, Eliot Zhu, Michele Wing, Esko Kautto and Eric Samorodnitsky for technical support. We would also like to thank Jenny Badillo for her administrative support for our team. We acknowledge the Ohio Supercomputer Center (OSC) for providing disk space, processing capacity, and support to run our analyses. We thank the Comprehensive Cancer Center (CCC) at The Ohio State University Wexner Medical Center for their administrative support of this work. S.R. and Team are supported by the American Cancer Society (MRSG-12-194-01-TBG), a Prostate Cancer Foundation Young Investigator Award, NHGRI (UM1HG006508-01A1), Fore Cancer Research Foundation, American Lung Association, and Pelotonia.

注意:此协议说明了同时处理和4个样品的分析。这种方法是用RNA从细胞中分离，新鲜冷冻组织和福尔马林固定，石蜡包埋的组织的（FFPE）兼容。该协议始于50 - 从RNA输入每个样本的1000纳克（250纳克推荐）。 1. rRNA的消耗和RNA议事碎片<ol><li> rRNA的消耗 <ol><li>在室温下除去洗脱，素，片段组合，rRNA的去除组合，rRNA的结合缓冲液和重悬浮缓冲液从-20℃并解冻。从4℃除去的洗脱缓冲液，rRNA的去除珠和RNA / cDNA的特定顺磁性珠粒，并把到室温。 </li><li>添加0.25微克RNA​​对PCR管。稀释用不含核酸酶的超纯水的总RNA，以10微升总终体积。添加5微升的rRNA结合缓冲液，以每管再加入的rRNA去除混合5微升，轻轻pipettË向上和向下混合。在与盖的热循环仪的地方管预加热至100℃，程序热循环仪于68℃5分钟以变性RNA。 </li><li> RNA变性后，从热循环仪取出试管并在室温下孵育1分钟。涡rRNA基因去除珠管大力重悬珠。添加35微升的rRNA去除珠新管和转移RNA变性反应（20微升）添加到含有的rRNA去除珠管。 </li><li>调整吸管45微升和吸管迅速向上和向下20X混合。在RT孵育管1分钟。然后放置在磁性支架管在室温下放置1分钟。转移上清液至新标记的PCR试管中在磁性支架再次将这些试管在室温下放置1分钟。这将确保没有珠粒转移。转移上清至新标记的PCR管。 </li><li>涡流的RNA / cDNA的特定顺磁珠，并添加99微升的珠每管。轻轻吸管整个音量上下10倍混合。如果有降级-RNA起，加193微升充分混合的RNA / cDNA的顺磁性的珠子，每管。孵育在室温下进行15分钟。然后将试管在室温下的磁性支架5分钟。取下并丢弃上清液。 </li><li>加入200μl新鲜配制的70％乙醇（乙醇）的。保持磁架管和照顾到不打扰珠。在室温下孵育30秒，然后取出并丢弃上清液。允许管放置在室温下5 - 10分钟进行干燥。 </li><li>离心机解冻室温洗脱缓冲液在600×g下5秒。添加11微升洗脱缓冲液对每个管和轻轻吸取上下10倍混合。在RT孵育管2分钟。放置在磁性支架管在室温下5分钟。将上清转移的8.5μl到新标记的PCR试管中。 </li></ol></li><li> rRNA基因RNA耗尽的碎片 <ol><li>加入8.5微升埃尔UTE，8.5微升黄金和8.5微升片段组合，以每管。吸管轻轻向上和向下10倍混合。地方管在与下面的程序热循环仪:预加热盖至100℃，94℃8分钟，然后4℃保持。 注意:此步骤是为了产生155碱基对的平均插入片段大小。如果RNA样品的平均片段大小低于200基点，跳过此步骤并继续执行第一链cDNA合成。 </li></ol></li></ol> 2. cDNA合成<ol><li> 合成第一链cDNA <ol><li>从-20°C取出第一链合成的混合，在室温下解冻。 </li><li>预编程热循环仪，使用以下设置:预热盖选项和设定为100℃，然后25℃10分钟，42℃15分钟，70℃15分钟，并保持在4℃下。程序保存为"合成第一链。" </li><li>在600＃离心机解冻-第一链合成混合管215;克5秒。将1微升逆转录酶与9微升第一链合成混合。 </li><li>添加8微升第一链合成的和逆转录酶混合到每个管中，轻轻地吸取上下6倍混合。使用固定速度的迷你台式离心机在600 XG 4秒的离心管中，使液体底部。将管在热循环仪，并选择合成第一链。当热循环仪达到4℃，除去管和立即进行合成第二链cDNA。 </li></ol></li><li> 合成cDNA第二链 <ol><li>预热热循环仪，以16℃下与盖子预先加热至30℃。解冻第二链预混并在冰上再悬浮缓冲。在事先从4℃除去的顺磁性珠的瓶子，静置至少30分钟，以使它们到室温。 </li><li>添加5μl的重悬浮缓冲液，以每个PCR管中。离心机第二链预混600×g下5秒，并添加20微升到每个PCR管中。地方管在16℃，1小时预热的热循环仪。当完成温育后，从热循环仪取出，并允许管来室温。 </li><li>涡流的顺磁性珠粒，直到它们被充分分散。添加90微升充分混合的顺磁珠到每个管中。轻轻吸取整个音量上下10倍混合。在室温下孵育管15分钟。在室温下的磁性支架5分钟地方管。 </li><li>取下并丢弃135微升上清液然后离开磁力架管进行乙醇洗。加入200μl新鲜制备的80％的乙醇，以每管，而不会干扰所述珠，然后在室温下孵育30秒。取下并丢弃所有的上清液从每个。重复一共有两个80％的乙醇洗涤。 </li><li>让试管在室温下静置5 - 10分钟以在磁性支架干燥。离心解冻室temperatur在600×克5秒ê悬浮缓冲液。从磁性底座取出PCR管。添加17.5微升悬浮缓冲液到每个PCR管，轻轻吸取整个音量上下10倍到调匀。孵育管在室温下2分钟。 </li><li>在室温下5分钟的磁性支架地方管，然后转移15微升上清液（双股cDNA）至0.2ml的PCR条管中。 注意:这是如cDNA可被储存在-20℃下长达7天的安全停止点。 </li></ol></li></ol> 3.文库制备<ol><li> 腺苷酸3&#39;端 <ol><li>在室温下拆下-20°C和解冻A尾组合。预编程热循环仪使用以下设置:选择预热盖选项和设定为100℃，然后将在37℃30分钟，70℃5分钟，并保持在4℃。这个程序保存为"ATAIL70"。 </li><li>加入2.5微升再悬浮的缓冲到每个管中。然后加入12.5微升解冻A尾组合。轻轻吸取整个音量上下10倍到调匀。将管在热循环仪，并选择ATAIL70。当热循环仪的温度是在4℃下，从热循环仪取出PCR管并立即进行结扎适配器。 </li></ol></li><li> 结扎适配器 <ol><li>除去合适的RNA适配器管，从-20℃停止连接缓冲液和重悬浮缓冲液并在室温下解冻。直到指示在协议中这样做，不要从-20°C除去连接混合物管。从4℃除去的顺磁性珠的瓶子，静置至少30分钟，使至室温。 </li><li>预加热热循环仪，以30℃，并选择预加热盖选项和设定为100℃。离心600×克5秒解冻RNA适配器管。在使用前立即从-20℃取出连接混合物管存储。 </li><li>添加2.5微升的再悬浮缓冲液中向每个样品管中。加入2.5微升连接混合物。返回连接混合物管-20°C使用后立即存储。添加2.5微升解冻的RNA适配器索引的向每个样品管中。轻轻吸取整个音量上下10倍到调匀。 </li><li>使用固定速度的迷你台式离心机在600×g的时间为4秒离心管中。管放置在预热的热循环仪。盖上盖子，在30℃下孵育10分钟。 </li><li>除去从热循环仪管和添加5微升停止连接缓冲液到每个管灭活结扎。轻轻吸取整个音量上下10倍到调匀。 </li><li> 2.2.4，使用42微升的混合顺磁珠，并丢弃79.5微升上清 - 如2.2.3所述重复洗。与在磁性支架上的管，让在室温下进行5样品风干 - 10分钟。 </li><li>拆下的PCR条管磁架，并添加52.5微升悬浮缓冲液每管。轻轻吸取整个音量上下10倍到调匀。在室温下孵育管2分钟。放置在RT磁性支架上的管5分钟，或直至所述液体是明确的。转移上清液加入50μl来自每个管新0.2毫升的PCR条管中。注意不要打扰珠。 </li><li> 2.2.4，用50微升的混合顺磁珠，并丢弃95微升上清 - 如2.2.3所述重复洗。与在磁性支架上的管，让在RT样品风干5 - 10分钟。 </li><li>从磁性底座上卸下的PCR条管和添加22.5微升重悬浮缓冲液到每个管中。轻轻吸取整个音量上下10倍到调匀。 </li><li>孵育在RT管2分钟。放置在磁性支架上的管，在室温下放置5分钟或直至液体是明确的。转移20微升上清液从每个管到一个新0.2毫升PCR条管。注意不要打扰珠。这是一种安全的停止点和cDNA可以储存在-20℃达7天。 </li></ol></li></ol> 4.图书馆放大<ol><li> 丰富DNA片段 <ol><li>在室温下取出PCR主混合物，PCR引物鸡尾酒和再悬浮缓冲从-20°C的存储和解冻。除去从4℃贮存的顺磁性珠的瓶中，静置至少30分钟，使至室温。 </li><li>预编程热循环仪使用以下设置:选择预热盖选项和设定为100℃，然后在98℃设定的初始变性在98℃，30秒，变性的15个循环持续10秒，退火在60℃下30秒，和延伸72℃30秒，在72℃下的最后一个扩展周期为5分钟，并保持在4℃。程序保存为"PCR"。 </li><li>离心解冻PCR主在600×g下5秒混合和PCR引物的管。添加5微升的解冻PCR引物，以每个样品管中。加入25微升解冻PCR扩增预混每个样品管中。轻轻吸取整个音量上下10倍到调匀。 </li><li>请将加盖PCR条管的预编程热循环仪。盖上盖子，运行PCR程序。当PCR完成后，从热循环仪取出试管，并保持在冰上。 </li><li> 2.2.4，用50微升的混合顺磁珠，并丢弃95微升上清 - 如2.2.3所述重复洗。与在磁性支架上的管，让在室温下进行5样品风干 - 10分钟。 </li><li>从磁性支架移除管和添加32.5微升重悬浮缓冲液到每个样品管中。轻轻吸取整个音量上下10倍到调匀。孵育在室温下进行2分钟。放置在磁性支架上的PCR试管在室温下5分钟，或直至所述液体是明确的。然后换乘30微升苏pernatant新鲜0.2毫升PCR管。 </li><li>使用荧光计7执行cDNA的定量和使用毛细管电泳系统8确定的cDNA质量。 注意:这是一个安全的停止点和cDNA可以储存在-20℃达7天。注:该库被认为是一种RNA测序文库。 后续步骤导致捕获库。 </li></ol></li></ol> 5.杂交，捕获和测序<ol><li> 复用杂交 <ol><li>从-20°C删除自定义水合探头，婴儿床-1 DNA，阻断通用寡核苷酸，以及适配器特异性阻断寡核苷酸和解冻冰。 </li><li>在低绑定1.5mL管中，并结合500 ng的DNA（每个样品125纳克时，复用4个样本），5微升婴儿床-1 DNA（1微克/微升），1UL通用阻断寡核苷酸，0.5微升P7（6个核苷酸）适配器特定阻断寡核苷酸（该量可能需要根据多路复用conditi进行调整项）和0.5微升P7（8核苷酸）适配器特异性阻断寡聚（该量可能需要根据多路复用条件）进行调整。 </li><li>将样品管在真空浓缩器具有面向旋转的相反方向帽打开。在45℃下20分钟，或直至所述液体完全蒸发干燥的内容。 </li><li>干重悬内容与8.5微升2X杂交缓冲液，3.4微升杂交A组份和1.1微升无核酸酶的水。允许再悬浮和涡每2.5分钟10分钟。再悬浮的物质转移至0.2mL的PCR管并在95℃下孵育10分钟在热循环。 </li><li>从热循环仪中删除的杂交样品管，加入2微升重新悬浮在1.5皮摩尔/微升的浓度定制探针。备选地，加入4微升重新悬浮在0.75皮摩尔/微升的浓度定制探针。孵育杂交反应过夜 - 在65℃下（16 24小时）。 注:探头从不同的供应商处购买之间可以使用和制造商的说明应遵循。杂交步骤的持续时间也可以变化。 </li></ol></li><li> 珠制备及捕获 <ol><li>从4℃除去的链霉抗偶联的顺磁珠瓶并在室温下平衡30分钟。稀释10倍的洗涤缓冲液（I，II，III和严格）和2.5倍珠洗涤液以创建1X工作方案。 </li><li>等分试样140微升1×洗涤缓冲液的我成到一个新的1.5毫升管。的1倍严格缓冲热量全部量，并在65℃下1×洗涤缓冲液I的等分试样在至少2小时的热块。 </li><li>等份加入100μl的每捕获蛋白链菌素偶联的顺磁珠到1.5毫升管。放置在磁铁和弃上清。添加每100微升磁珠200μl的珠洗涤缓冲液并涡旋10秒。在磁铁将用于2 - 5分钟或直到上清液是显而易见的。一旦上清液是明确的，放弃它，并重新泥炭一次，总共两次洗涤的。 </li><li>拆除珠子洗涤缓冲液后，加入等体积的珠洗涤液作为初始起点体积（ 即 100微升一个捕获）。重悬并转移到0.2mL的PCR管中。将管磁架为2 - 5分钟或直到上清液是显而易见的。弃上清。 </li><li>既在在65℃的热循环仪的杂交样品和珠，杂交混合物转移到珠管和移液管上下10倍混合。孵育在65℃下45分钟，涡旋，并使用一个固定的速度（6.0 XG）台式微型离心机降速样品4秒。 </li></ol></li><li> 珠洗 <ol><li>从热循环仪中取出捕获管，加入100微升预先加热1×洗涤缓冲液I至管，涡旋10秒以混合。将混合物转移到一个新的低绑定1.5mL管中。放置管中磁分离齿条和允许2 - 5分钟进行分离，或直至上清液是明确的。 ðiscard上清。 </li><li>加入200μl预热1X严格的洗涤液和移液器上下10次混合。孵育在65℃下5分钟。把试管磁分离架上，并允许2 - 3分钟分离或直到上清液是显而易见的。弃上清。重复严格洗涤一次，总共两次洗涤的。 </li><li>加入200μl室温1×洗涤缓冲液I和涡旋2分钟以混合。放置管在磁性分离架上并允许2 - 5分钟进行分离，或直至上清液是明确的。弃上清。 </li><li>加入200μl室温1×洗涤缓冲液II和涡旋1分钟以混合。放置管在磁性分离架上并允许2 - 5分钟进行分离，或直至上清液是明确的。弃上清。 </li><li>加入200μl室温1×洗涤缓冲液III和涡旋30秒以混合。放置管中磁分离机架允许2 - 5分钟进行分离，或直至上清液清楚了。弃上清。 </li><li>从磁分离机架上卸下该管，并添加20微升不含核酸酶的水重悬珠子。吹打向上和向下10倍调匀。 </li></ol></li><li> 拍摄后的PCR扩增 <ol><li>从4℃除去顺磁珠，在室温下平衡30分钟。 </li><li>在RT除去-20℃并解冻的热启动PCR预拌（2×）和PCR引物混合，然后置于冰上。 27.5微升2倍热启动PCR预拌与PCR引物1的2.75微升至2.75微升PCR引物2相结合的图书馆准备扩增预混（这些卷1杂交库加过量10％）。 </li><li>设置通过加入珠20微升加与文库扩增主混合物的30微升的50微升的总体积捕获的DNA在PCR管进行反应。帽筒正确，涡旋混合。 4秒的离心管使用一个固定的速度迷你卡在6.0×g的LE-离心机。 <ol><li>设置下述PCR程序: - 在98℃变性12个周期为15秒，退火65选择预热盖选项和设定为100℃，然后将初始变性在98℃45秒，10 ℃30秒和延伸72℃60秒，在72℃下的最后一个扩展周期为60秒并保持在4℃。 </li></ol></li><li>从热循环仪取出样本，加入75微升顺珠。拌匀，并在室温孵育15分钟。 </li><li>放置在磁体管在室温下2 - 3分钟，然后除去上清液。通过加入200微升80％乙醇，温育30秒，然后除去上清液洗上磁珠。重复总共两次80％洗涤。 </li><li>在RT孵育5 - 10分钟，以允许珠粒干燥。不要过度干燥，开裂。珠悬浮在22微升的Tris-EDTA pH值8.0（1X TE解决方案），并允许为洗脱3分钟。将样品在磁铁3 - 5分钟，日恩转移20微升洗脱的产品来新低绑定1.5mL管中，以确保没有珠结转。 </li><li>执行使用荧光计7捕获的cDNA的定量和使用毛细管电泳系统8确定捕获的cDNA质量。 </li></ol></li><li> 桌面序时的加载过程9 <ol><li>稀捕获cDNA文库利用10毫摩尔Tris-氯pH 8.5的具有0.1％Tween 20的解冻10N的NaOH和杂交缓冲在冰上4 1nM的最终浓度。使用前约30分钟，在解冻水RT桌面序V2试剂盒盒1。不要填写上面的最大加载线10。请注意，这是一个测序平台的具体程序和政策有可能根据制造商的说明而变化。 </li><li>通过在离心管（始终准备好新鲜）20微升10 N氢氧化钠980微升核酸游离水结合准备的0.2N氢氧化钠1毫升。通过稀释PhiX库（库控制）至4纳米组合2微升10与3微升的10mM三Cl pH 8.5的纳米库控制的具有0.1％Tween 20。 </li><li> 5微升4nm的图书馆与5微升0.2N的氢氧化钠和旋涡简要相结合的混合最终变性图书馆和图书馆的控制。使用固定速度小型台式离心机在6.0×g下4秒离心管中。孵育在RT 5分钟以变性库。 </li><li>添加990微升预冷的杂交缓冲液含有10微升变性库的管中。这导致了20 PM库。标记变性20 PM库与日期，并可以在-20℃储存长达3周。 </li><li>混合375微升20日下午库控制的225微升预冷杂交缓冲液稀释库控制导致12.5分库控制。倒置几次到该溶液中混合。 </li><li>结合594微升变性最终库6微升下午12时05分变性库控制和旋涡混合的。将结合SAMP乐库和磁带库控制预留冰上直到样品准备加载到桌面序试剂盒。 </li></ol></li></ol> 6.数据分析<ol><li> 序列质量评估 <ol><li>计算使用序列质量评估11原始序列数据（FASTQ文件）的质量。 注意:此步骤有助于评估数据之前，进一步进行下游分析。该软件具有内置参数运行，并产生一组度量为每个FASTQ文件。 </li></ol></li><li> 对准 <ol><li>对齐序列读取（FASTQ文件）的参考人类基因组hg19并使用Tophat2 12转录（2.0.10版本），同时提供成绩单称为一个GTF文件。输出是在所谓的BAM文件的二进制对准格式的形式。 </li><li>执行包括排序，并使用Samtools 13（版本0.1.19索引后处理步骤）的BAM文件。执行重复的标记，重新排序SAM，插入尺寸计算，并使用工具皮卡德14（1.84版）增加或更换读取组。 </li></ol></li><li> RNA测序质量评估 <ol><li>计算使用RNA测序质量评估RNA测序数据的一系列的质量控制指标。输入到这个软件是从Tophat2对准一个BAM文件15。输出是一个HTML文件，该目录总读取次数，重复的，映射的读百分比和rRNA的百分比， 等等 ，等等。 </li></ol></li><li> 变异召唤 <ol><li>使用STAR（版本2.4.0）16对齐，然后调用单核苷酸变异使用GATK的（版本3.3-0）HaplotypeCaller 17。遵循GATK BAM后处理步骤和过滤标准标志，并从输出中删除误报。 </li></ol></li><li> 基因表达 <ol><li>计算基因表达USING袖扣从燕尾服套装软件18（版本2.1.1）。 注意:输入来自Tophat2对齐工具BAM文件。输出是在同种型，基因和转录物水平，其中，表达作为FPKM（每百万映射读取片段每千碱基）计算产生的。 </li></ol></li><li> 融合呼叫 <ol><li>呼叫使用ChimeraScan 19（0.4.5版本），高顶礼帽融合20个自定义和TRUP 21每个样品的融合。注释使用Oncofuse 22（版本1.0.9b2）域的融合。 </li></ol></li></ol>

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B., 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=Quantitative+gene+profiling+of+long+noncoding+RNAs+with+targeted+RNA+sequencing.">Quantitative gene profiling of long noncoding RNAs with targeted RNA sequencing.</a> Nat Methods. 12, 339-342 (2015).</li><li>Cieslik, M., 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+use+of+exome+capture+RNA-seq+for+highly+degraded+RNA+with+application+to+clinical+cancer+sequencing.">The use of exome capture RNA-seq for highly degraded RNA with application to clinical cancer sequencing.</a> Genome Res. , (2015).</li><li>Cabanski, C. R., 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=cDNA+hybrid+capture+improves+transcriptome+analysis+on+low-input+and+archived+samples.">cDNA hybrid capture improves transcriptome analysis on low-input and archived samples.</a> J Mol Diagn. 16, 440-451 (2014).</li><li>Costa, C., Gimenez-Capitan, A., Karachaliou, N., Rosell, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+molecular+screening:+from+the+RT-PCR+to+the+RNA-seq.">Comprehensive molecular screening: from the RT-PCR to the RNA-seq.</a> Transl Lung Cancer Res. 2, 87-91 (2013).</li><li>Zhao, W., 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+RNA-Seq+by+poly+(A)+capture,+ribosomal+RNA+depletion,+and+DNA+microarray+for+expression+profiling.">Comparison of RNA-Seq by poly (A) capture, ribosomal RNA depletion, and DNA microarray for expression profiling.</a> BMC Genomics. 15, 419 (2014).</li></ol>

S.R. receives funding from Novartis and Ariad Pharmaceuticals for conduct of clinical trials. S.R. immediate family members own stock in Johnson and Johnson.

RNA测序Capture是RNA测序和芯片之间的中间策略评估转录的选定部分接近。捕获的优点包括在桌面序器，高吞吐量，和基因组改变的检测降低的成本，快速的周转时间。可适于表征非编码RNA 23，检测单核苷酸的方法变体4-6，检查RNA剪接，并确定基因融合或结构重排24。此外，这种方法可应用于已经经过用福尔马林固定并包埋在石蜡块24,25临床或处理的样本。 <p class=...

Whole transcriptome or RNA sequencing (RNAseq) is an unbiased sequencing method to assess all RNA products. The goal of targeted RNAseq (Capture) is a focused evaluation of selected transcripts with increased sensitivity, dynamic range, reduced cost or scale, and increased throughput compared to standard RNAseq. Similar to standard RNAseq, targeted enrichment approaches can be used to evaluate gene expression, multiple RNA species such as mRNA, microRNA (miRNA), lncRNA1, other noncoding RNAs2, gene fusions3, and mutations4-6.
Capture involves hybridization of complementary oligonucleotides to enrich cDNA libraries for sequencing. The rationale for RNAseq Capture is similar to microarray approaches where complementary oligonucleotides or probes are hybridized to samples and then measured for relative abundance. For microarray technologies, expression is based on relative signal measured for transcripts binding to these probes. Microarrays are thus limited by range, potential background noise from non-specific binding, and cross-hybridization of probes. Furthermore, arrays have limited dynamic range for low and highly expressed transcripts compared to RNAseq1. Microarrays are widely utilized due to their reduced cost and high throughput capacity compared to RNAseq. 
Here, we demonstrate a method for RNAseq Capture that offers a middle ground between RNAseq and microarray approaches for evaluating the transcriptome. RNAseq Capture has intermediate throughput, greater dynamic range and sensitivity, and is scaled for fast turnaround on desktop sequencers. RNAseq Capture also requires reduced computational resources in terms of storage space and data processing.

<table border="0" cellpadding="0" cellspacing="0" width="870">
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			<td style="width:143px;">217004</td>
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			<td>E7023-6X500ML</td>
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			<td height="20" style="height:20px;width:407px;">Thermoblock 24 X 1.5ml</td>
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			<td style="width:143px;">21516-166</td>
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			<td height="20" style="height:20px;width:407px;">MiSeq Reagent Kit v2 (300-cycles)</td>
			<td style="width:153px;">Illumina</td>
			<td style="width:143px;">MS-102-2002</td>
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			<td height="20" style="height:20px;width:407px;">MiSeq Desktop Sequencer</td>
			<td style="width:153px;">Illumina</td>
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			<td height="40" style="height:40px;width:407px;">TruSeq Stranded Total RNA Kit with RiboZero Gold SetA</td>
			<td style="width:153px;">Illumina</td>
			<td style="width:143px;">RS-122-2301</td>
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			<td height="20" style="height:20px;">25 rxn xGen&#174; Universal Blocking Oligo - TS-p5</td>
			<td style="width:153px;">IDT</td>
			<td>127040822</td>
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			<td height="20" style="height:20px;">25 rxn xGen&#174; Universal Blocking Oligo - TS-p7(6nt)</td>
			<td style="width:153px;">IDT</td>
			<td>127040823</td>
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			<td height="20" style="height:20px;">25 rxn xGen&#174; Universal Blocking Oligo - TS-p7(8nt)</td>
			<td style="width:153px;">IDT</td>
			<td>127040824</td>
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			<td height="20" style="height:20px;">Agencourt&#174; AMPure&#174; XP - PCR Purification beads&#160;</td>
			<td>Beckman-Coulter</td>
			<td>A63880</td>
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			<td height="20" style="height:20px;">Dynabeads&#174; M-270 Streptavidin</td>
			<td>Life Technologies</td>
			<td>65305</td>
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			<td>Roche Applied Science</td>
			<td>05480647001</td>
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			<td height="20" style="height:20px;">Qubit&#174; Assay Tubes&#160;</td>
			<td>Life Technologies</td>
			<td>Q32856</td>
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			<td>Life Technologies</td>
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			<td>Roche NimbleGen&#160;</td>
			<td>05634261001 or 05634253001&#160;</td>
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			<td>Life Technologies</td>
			<td>Q32866</td>
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			<td>Life Technologies</td>
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			<td>Roche Applied Science</td>
			<td>05634253001</td>
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			<td height="20" style="height:20px;">SuperScript II Reverse Transcription 200U/ul</td>
			<td>Life Technologies</td>
			<td>18064-014</td>
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			<td height="20" style="height:20px;">D1000 ScreenTape</td>
			<td>Agilent Technol. Inc.</td>
			<td>5067-5582</td>
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			<td height="20" style="height:20px;">Agencourt RNAClean XP -40ml</td>
			<td>Beckman Coulter Inc</td>
			<td>A63987</td>
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			<td height="20" style="height:20px;">RNA ScreenTape</td>
			<td>Agilent Technol. Inc.</td>
			<td>5067-5576</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">RNA ScreenTape Ladder</td>
			<td>Agilent Technol. Inc.</td>
			<td>5067-5578</td>
			<td></td>
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			<td height="20" style="height:20px;">RNA ScreenTape Sample Buffer</td>
			<td>Agilent Technol. Inc.</td>
			<td>5067-5577</td>
			<td></td>
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			<td height="20" style="height:20px;">Sodium Hydroxide</td>
			<td>Sigma</td>
			<td align="left">72068-100ML</td>
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			<td height="20" style="height:20px;">DynaBeads MyOne Streptavidin T1</td>
			<td>Life Technologies</td>
			<td>65602</td>
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			<td>12331D</td>
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			<td>Chloroform</td>
			<td>Sigma</td>
			<td>C2432-1L</td>
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			<td height="20" style="height:20px;">KAPA HotStart ReadyMix</td>
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			<td>KK2602</td>
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			<td style="width:153px;">Agilent Technol. Inc.</td>
			<td>5067- 5583</td>
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targeted rna sequencing assay to characterize gene expression and genomic alterations

RNA测序（RNA测序）是可以被用于检测和表征的基因表达，突变，基因融合，和非编码RNA一个通用的方法。标准RNA测序需要30 - 亿测序读出并可以包括多个RNA产物如mRNA和非编码RNA。我们证明有针对性的RNA测序（捕获）如何允许在使用桌面序选定的RNA产品聚焦研究。 RNA测序采集可以表征可能以其它方式使用传统的RNA测序方法错过没有注释，低或瞬时表达成绩单。在这里，我们描述了从细胞系，核糖体RNA耗尽，cDNA合成，制备条形码库，杂交和靶向转录物的捕获和多重测序上的桌面序的RNA的提取。我们也勾勒出计算分析管道，其中包括质量控制评估，校准，检测融合，基因表达定量和单国统会的鉴定leotide变种。该测定允许有针对性的转录测序来表征的基因表达，基因融合，和突变。

在RNA测序捕捉的概略突出关键步骤示于图1。与已知的突变四癌细胞系被用来证明RNA测序捕捉技术的有效性（K562与ABL1融合，LC2与RET融合，EOL1与PDGFRalpha融合和RT- 4 FGFR3融合）。这四个样品汇集在一起​​，并进行测序2倍于台式机音序器，生成FASTQ文件100个基点的读取。 1）质量控制评估，2）对准至人类转录，3）基因表达的定量，4）?...

Watch this Scientific Journal Video about Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations at JoVE.com

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

有针对性的RNA测序分析表征基因表达和基因组改变

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding ...

targeted-rna-sequencing-assay-to-characterize-gene-expression-genomic

Research

JoVE Journal

Biology

10.3K 次观看.  The Ohio State University. We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

视频: Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

The Preparation of Primary Hematopoietic Cell Cultures From Murine Bone Marrow for Electroporation

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell  electroporation system and Gene Pulser  electroporation buffer were specifically developed to transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells.This video demonstrates how to establish primary hematopoietic cell cultures from murine bone marrow, and then prepare them for electroporation in the MXcell system. We begin by isolating femur and tibia. Bone marrow from both femur and tibia are then harvested and cultures are established. Cultured bone marrow cells are then transfected and analyzed.

This procedure describes how to establish primary hematopoietic cell cultures from murine bone marrow and is followed by transfection using the Gene Pulser MXCell electroporation system.

主电穿孔的小鼠骨髓造血细胞培养制备

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

科研

生物学

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

从胚胎斑马鱼和cDNA合成的基因表达分析的RNA分离

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

使用基因脉冲MXcell电穿孔系统主细胞的转染效率高

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

使用自动细胞计数器简化基因表达研究：在Namalwa细胞的IL - 4依赖的基因表达的siRNA击倒

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

石蜡包埋及冷冻的部分果蝇成人肌肉

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

在体外重组活跃T。谷盗端粒酶

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

条形码酵母图书馆竞争力的基因屏幕

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

由外显子捕获和大规模平行测序检测体细胞遗传学改变在肿瘤标本

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

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.