The authors thank Colleen Van Kirk for mouse retina and cerebellum samples, and Peter Gregory for figure generation. The authors also thank Dr. Allison Gillaspy and the Laboratory for Molecular Biology and Cytometry Research core for access to the MiSeq. This work was supported by NIH grants DA029405, EY021716, and AG026607 and the Donald W. Reynolds Foundation.

从组织1.核酸分离注：联合隔离DNA和RNA从同一组织样品提供了机会，收集的DNA进行分析后生和配对的RNA为基因表达分析。这里给出的例子中是从实验性的组织，但对不同类型的样品或其它分离方法的各种途径柱纯化的形式都可以使用。的主要要求是对高度纯化的核酸而不污染蛋白质或有机溶剂。 <ol><li>选择一块新鲜冷冻或新鲜组织进行机械均化并放置在冰上。存储或转移组织，以2.0毫升的圆底离心管中。 </li><li>添加1个5毫米的不锈钢珠含组织各管。添加适当量的裂解缓冲液，根据制造商的细胞数或组织重量的建议。 </li><li>使用珠磨机机械均质化，在均化组织30赫兹进行30秒。 注：频率和机械均质化的持续时间依赖于组织类型。较软的组织将彻底均匀使用推荐的设置，而更严格的组织要求设置的优化。 </li><li>使用硅胶离心柱在室温下制造商的方案进行DNA和RNA的分离。 <ol><li>洗脱的RNA在50μl不含RNA酶的水并置于冰上。使用RNA通过定量PCR基因表达分析。 </li><li>洗脱在100μlTE缓冲液中的DNA。使用洗脱液以增加DNA的浓度和产量重复洗脱的DNA断列。利用DNA进行有针对性的甲基化定量分析。 </li></ol></li><li>使用标准的分光光度计吸光度在230nm，260nm处，280处和320处评估的DNA和RNA的质量。 注：A260 / A280的比例应为〜1.8-2高质量的DNA。 A260 / A280的比例应为〜2的高品质的RNA。过量吸光度在230nm印度语的存在茨污染物的分离步骤。 </li><li>质量通过根据制造商的说明使用毛细管电泳芯片进一步检查的RNA。 注：高品质的RNA会产生RNA完整性数（RIN）&gt; 8。在除了rRNA的峰的电泳额外峰的存在表明样品污染。 </li><li>根据制造商的协议量化DNA和RNA的荧光测定法。 注意：使用荧光检测是更为敏感和特异单独量化核酸比分光光度法。 </li><li>商店中分离的DNA和RNA在-80℃。 </li></ol> 2.目标识别和引物设计<ol><li>确定感兴趣的基因组区域（S）以被BSAS基于适当的参照基因组序列分析。差异表达基因的基因启动子甲基化是定量的共同目标。 </li><li> 在硅片亚硫酸氢盐转换准备对于测序后比对ED的参考基因组的读取通过改变.fasta序列文件在文本编辑器。在5&#39;-3&#39;方向，取代非CpG的胞嘧啶与胸腺嘧啶的（ 图2）。 </li><li>设计底漆集从亚硫酸氢盐转化的DNA扩增感兴趣的区域。 <ol><li>选择并复制感兴趣的未转化的区域，在5&#39;-3&#39;方向，成亚硫酸氢特异性PCR引物设计程序（ 图3）。 注：最优亚硫酸氢盐PCR扩增子长度为每扩增子250-400碱基对作为亚硫酸氢盐处理的DNA片段，它是难以扩增大&gt; 400bp的区域。如果，例如，1 kb的区域是感兴趣，多个引物对，可设计成覆盖该区域。设计扩增子为等于bp的尺寸，便于池。避免扩增长度&lt;250基点，因为这些可能是长度不足以库生成。最佳的引物长度为BSAS是&gt;每个引物20基点。 </li><li>前锋选择55-65℃，Tm值范围和最大Tm值之间的差异和反向1-2℃的引物。 </li><li>选择的引物对，最好覆盖感兴趣的区域。不要选择含有CpG位点或者直接相邻CpG位点的引物，因为这会导致偏见的PCR反应。使用标准的PCR引物，可以从任意数量的学术或商业供应商订购。 </li><li>重建在RNA酶/ DNA酶的游离水冻干引物以100微米的工作原液。 </li></ol></li><li>商店的PCR引物在-20℃下。稀释100μM的引物股票10μM工作股票PCR反应。 </li></ol> 3.重亚硫酸盐特异PCR优化注：对于基因组DNA的亚硫酸氢盐转化，可用一个号码为亚硫酸氢盐转化不同的商业试剂盒。选择最适合的计划实验套件或协议。 &lt;OL&gt; <li>基因组DNA之间的200毫微克〜2微克使用。为优化实验，运行多个转化反应，以便有足够的亚硫酸氢盐转化的DNA用于多个基站的PCR反应。 </li><li>使用小（ 如 10微升）洗脱的亚硫酸氢盐转化的DNA量保持高浓度的DNA。 注：1-2微升亚硫酸氢盐转化的DNA足以用于优化PCR反应，如果1微克基因组DNA，用于转换输入。 </li><li>通过放大亚硫酸氢特异性PCR亚硫酸氢盐转化的DNA。 BS-PCR反应需要使用Taq酶能够放大亚硫酸氢盐转化的DNA。 <ol><li>组装为单个扩增子的靶扩增优化以下反应。对于多个样品，组装在一个96孔PCR板的反应。 25微升2倍反应缓冲液 0.5微升dNTP混合物 5微升10μM正向引物（最终1μM） 5微升10μM反向引（最终1μM） 2微升模板亚硫酸氢盐转化DNA 0.4微升（5 U /微升）DNA聚合酶 12.1微升DNA酶自由水（最终反应体积50微升） </li><li>密封PCR板用适当的粘合剂或热封膜。 </li><li>将反应在适当的热循环仪，使用下列循环条件与热盖： <ol><li>执行初始变性，在95℃下进行10分钟。 </li><li>变性在95℃下30秒。 </li><li>被用于退火30秒，在引物的特异性T 米 。在开始的退火温度少数℃以下的底漆优化反应的Tm值。 注：最佳的退火温度也可使用梯度热循环仪，以测试一个温度范围内进行测定。 </li><li>执行一个延伸72℃30秒。延长更长的扩增子的延长时间。 </li><li>重复步骤3.3.3.2-3.3.3.4为35个循环，以TAL的初步优化。更高的循环数目可能是必要的，但通常应该避免，以防止克隆扩增，将创建反应工件。 </li><li>执行一个最终延伸在72℃7分钟。 </li><li>保持反应在4℃。 </li></ol></li><li>可视化扩增通过PAGE电泳。或者，根据制造商的协议使用的毛细管电泳的DNA芯片。 <ol><li>混合24微升含6微升5×加载染料在冰上和旋涡的PCR反应。 </li><li>使1升的1×TBE缓冲液通过混合双蒸2 200毫升5×TBE的运行缓冲液和800毫升O.稀释DNA阶梯，每孔0.5微克DDH 2 O的负载和染料的终浓度。 </li><li>负载井30微升PCR反应或阶梯。 </li><li>运行凝胶在200 V的〜45分钟。停止凝胶运行，当第一染料前沿到达凝胶的末端。 </li><li>染色的凝胶用5微克/毫升溴化乙锭通过混合5微升的10毫克/毫升在100ml双蒸2 O的覆盖整个凝胶，并让孵育〜5分钟。 </li><li>图像通过在482纳米的激发波长的UV光盒或多峰成像器的凝胶。 </li><li>在参考了梯子，大小的PCR扩增子，并通过寻找多个频带比预期的扩增子大小等（ 图4A和C）确定该反应的特异性。 </li></ol></li><li>曾经为BS-PCR最佳条件已经确定，对实验样品进行BS-PCR。 <ol><li>净化从剩余的引物和其他反应组分的扩增子。执行二氧化硅柱纯化，SPRI珠粒，或基于凝胶的清理和定量。 </li><li>洗脱扩增子在30微升TE缓冲液中。 </li><li>量化使用荧光检测使用制造商的协议扩增。 </li><li>如果存在，每个样品的多个扩增子，它们集中在每体积重量相等在-20℃下量和存储。 </li></ol></li></ol></li></ol> 4. NGS文库制备和图书馆QC 注：BSAS NGS文库制备采用了简化的转座子介导的协议与双索引（见材料清单上的文库制备试剂盒的选择详细信息）。这种方法提供了一个非常迅速，高通量路径用于文库构建已验证并显示了小至在亚硫酸氢盐测序应用16,19没有偏压。双索引允许对样品比单一索引的较高多路复用。其他图书馆编制的风格可能是可能的，但还没有经过测试。 <ol><li>准备从BS PCR扩增NGS库在96孔PCR板。稀释扩增至0.2纳克/μL作最后输入的1纳克，或5微升0.2纳克/微升稀释质量。执行清理使用SPRI珠生成库。用50-90微升SPRI珠隔离库瓦特第i此协议。的SPRI珠粒的量取决于文库的靶的尺寸和所需的测序反应循环数。 注：使用微孔板摇床或板状涡旋重悬珠。 <ol><li>洗脱库脱珠转移到新的96孔板，并密封与储存在-20℃的热封膜。 </li></ol></li><li>确定库（ 图4B和D）的大小和浓度。 <ol><li>运行库上的毛细管电泳的DNA根据制造商的协议上浆芯片。 <ol><li>使用高灵敏度的毛细管电泳的DNA检测，以确定检测到的库的峰的平均粒径和摩尔浓度的根据制造商的协议的估计。 </li></ol></li><li>量化库通过qPCR，使用被设计针对在生成的文库，并使用标准的具有已知浓度的接头序列的引物。 <ol><li>运行的qPCR重根据制造商的协议使用绝对定量的设置上的实时仪器操作。 </li><li>使用库的大小和摩尔定量从定量PCR来计算摩尔浓度的库。 </li></ol></li></ol></li><li>稀释库利用10毫摩尔Tris-CL与0.1％Tween-20的pH为8.5 4纳米。商店库在96孔PCR板在-20℃下密封带热封膜。 </li></ol> 5.测序文库使用台式序<ol><li>解冻并根据制造商的说明制备试剂。 </li><li>冰上解冻4纳米库。 </li><li>使在1.5ml微量离心管中加入1ml 0.2N的NaOH中。 </li><li>在等体积量池4nM的库，如果多个库被使用。 </li><li>稀释，并根据制造商的说明，以所期望的最终摩尔浓度变性汇集库。 <ol><li>不断稀释变性冰上，直到准备汇集库加载的试剂盒。 </li></ol></li><li>生成样品表含样品名称和相应的索引信息，并指定只生成.fastq文件。 <ol><li>在序相应的样品表文件夹中加载样品板。 </li></ol></li><li>添加共有600微升稀释变性库试剂盒对应得很好。 </li><li>运行的测序反应来完成。 </li></ol> 6.甲基化定量数据分析注：可用于NGS数据分析，包括商业和开源的多个程序。上运行的程序的细节可以与每个特定的包中找到。一般说明在随该软件包的特定命令的每一步都给出。 （见材料清单在这个协议中使用的软件的详细信息）。 <ol><li>进口.fastq.gz文件到合适的本身quencing分析管道用于读修剪保留质量分数（Qscores）。 （在这个例子程序中选择“导入”，选择用来产生读测序方法，选择.fastq.gz读取导入文件，并保留Qscores从.fastq文件读通过取消在“常规”放弃质量得分“微调应用选项​​“，选择一个位置为导入读取，并选择”完成“。） <ol><li>修剪并保留读取使用的NGS数据分析管道以下设置。 （对于此程序中选择“修剪序列”下“NGS核心工具”，然后选择读取进行修剪。） <ol><li>只保留读取包含≥Q30分数。 （对于此程序中选择“裁剪使用质量得分”，并设置上限0.001）。 </li><li>只保留读取包含≤1暧昧的核苷酸。 （对于此程序中选择“修剪暧昧核苷酸”，并设置1。&#39;模棱两可的最大数量“选择一个位置来保存修剪读取，并选择”完成“。） </li></ol></li><li>地图修剪读取从2.2 在硅片亚硫酸氢盐转化基因参考。 （对于示例软件选择“地图读取参考”下“NGS核心工具”，选择修剪读取被映射，并选择“下一步”，选择要转换的参考序列，并选择在“参考屏蔽&#39;无遮拦”，选择“下一个&#39;。） <ol><li>分配最高刑罚为不匹配，插入和删除。设置参数，使得所读取的长度的100％被要求以90％的序列同一性映射。例如软件，分配的不匹配，插入和删除一个比分3。分配用于读出长度映射到参考的最低分数的分数为1，并指定为0.9的分数之间映射的读取和参照同一性的最低分数。 </li><li&gt;丢弃或忽略非特异性读取映射到参考。这些可以读取同样映射到多个区域。例如软件中选择“忽略”下的“非特异性匹配处理”，然后选择“下一步”。 </li><li>用于对准输出生成每个样本作为一个独立的文件中。例如软件，选择“创建独立的读取映射”下“输出选项”和“保存”“结果处理”项下。选择一个保存位置读取映射，并选择“完成”。 </li></ol></li><li>运行变量调用工作流上的映射读取。对于例如软件，选择“概率变检测”下“重测序分析”中，选择读映射进行分析，并选择“下一步”。选择“忽略非特异性匹配”下“读过滤器”，然后选择“下一步”。 <ol><li>确保序列处于DEP次方≥千X通过在&#39;意义&#39;设置&#39;最小覆盖“到1000。 </li><li>确保该变种呼叫存在于正向和反向的读取。例如软件，选择“需要在这两个前沿存在和反向看台上&#39;下&#39;变过滤器”，然后选择“下一步”。 </li><li>出口对齐，变种，叫数据作为注解表。例如软件，选择“创建注释表”下“输出选项”，然后选择一个位置来保存变量表文件。选择“完成”。 </li></ol></li></ol></li><li>使用在从变体表文件感兴趣区域注明CpG部位的变体频率计算5-MC百分比。胞嘧啶在CG的基准C中的频率是5-MC百分比。 注：使用变种，呼吁高度甲基化的CpG的算法，更换的CpG胞嘧啶与胸腺嘧啶。这将identiFY映射胞嘧啶的作为变体，导致胞嘧啶频率的CpG的。 </li><li>通过首先计算C的百分比不是在基准（C）的CpG部位确定亚硫酸氢盐转化效率（BSC）。乘非CpG的C&#39;S由总T的映射（T MP）的数量由全碳的映射在参考T的（C MPT）的比例，以及鸿沟。 100减去计算出的​​频率是库（等式1）的亚硫酸氢盐转化效率（BSC）。 </li></ol><img alt="式（1）" fo:content-width="1.5in" src="/files/ftp_upload/52488/52488eq1.jpg" />公式1亚硫酸氢盐转化效率。 注意：虽然哺乳动物基因组包含非常低量的非CpG的胞嘧啶甲基化（用干细胞20的异常可能），植物基因组含有一个显著量。为了确定硫酸氢转换效率在这些referencê基因组，一个合适的途径是测序线粒体基因组或叶绿体基因组或已知的非甲基化的基因存在于植物的基因组的部分21，并如上所述计算的转换效率。亚硫酸氢盐转化效率应该在大多数情况下≥98％，与未转化的C&#39;S由于非CpG甲基化可能。

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The authors have nothing to disclose.

BSAS允许集中，准确的DNA甲基化与定量两个样本数量和指标数量的高吞吐量的能力。此外，该库一代使用转座子介导的tagmentation需要较少的输入DNA，更快，包含了比传统的剪切和随后的适配器连接技术更少的步骤。这些改进在现有的方法允许任何感兴趣的目标进行精确基层DNA甲基化分析。此外，BSAS为确认已使用的全基因组亚硫酸氢钠22或23捕捉方法确定的感兴趣区域（差异甲基化区域（的DMR），CpG ​​岛，监管区）的新方法。采用正交确认的方法和更精确的定量方法，提高了表观基因研究的严格分析。实现了与BSAS高阅读深度允许在一个窄的置信区间准确定量，再质保的义务在精确的定量分析16。此外，为了在单次实验运行许多样品的能力可以增加对整个基因组的方法确认，这将增加功率统计样本大小;目前许多后生研究的一个弱点。重要的是，BSAS提供一个碱基特异性CpG甲基化的定量，其允许可检验的假设为后生研究的产生。假设如在特定的反应元件增加甲基化将导致一个特定的转录因子降低的结合。 BSAS，结合配对mRNA表达数据，提供了用于在一个单一的一块组织或细胞群的获得两种表观遗传调节和表达的方法。调控和表达的配对测量，也增加了科学严谨和结果的影响。 在BSAS协议中的关键步骤包括引物设计，扩增优化和生成库/QC。 PCR偏见介绍，如果引不正确的设计和放大，在不同的效率8。使用甲基化的标准，如酶促产生100％和0％的胞嘧啶甲基化的标准，可以用于确定程度，如果有的话，甲基化定量的偏差，并且是一个适当的方法控制量化甲基化16时。类似地，应注意优化PCR反应，用于产生单个高质量扩增子时服用。如果没有扩增子是本使用在协议详述所建议的准则，优化步骤包括增加的PCR循环数或bsDNA输入量。如果有多个PCR产物，包括引物二聚体，优化步骤包括减小bsDNA输入量，降低了PCR引物的摩尔浓度的输入，提高退火温度，或降低PCR循环数。这些优化过程的一个或一个组合产生足够的结果，以产生一个单一的集锦。H-质量的扩增子。另外，重新设计引物对引物不产生一个扩增子的好办法。对于这些地区的地方重新设计不适合或没有可能，退化引物包括胞嘧啶的和胸腺嘧啶的在正向引物CpG位点和腺嘌呤的和鸟嘌呤的在反向引CpG位点可能工作。然而，这些引物组还需要进一步优化，以确保无PCR的偏差发生，这个协议的范围之外的处理。质量库是成功的非常重要的。确保QC措施进行了测序前确定库的规模，质量和数量。测序反应是容易发生故障与低质量的库的输入。偏压中的甲基化的定量可以通过确保甲基化频率存在于正向和反向测序读数来缓解。此外，基地调整至CpG位点的质量分数应≥Q30高协nfidence的定量。虽然其他人已经表明以前很少在亚硫酸氢盐转化的DNA 19 tagmentation反应没有偏差，保证上述指标可以确认低偏置甲基化定量。 BSAS目前在CpG位点的甲基化测量，但是，这种方法的未来的扩展将允许的CpG 5- HMC的成对测量用加入了如引入钾高钌酸铵重亚硫酸盐转化24之前实验步骤5-MC和5- HMC区分。这将增加BSAS效用的影响通过允许配对甲基化，既5-MC和5- HMC，和表达数据，以确定DNA甲基化的基因表达的调节作用。转座子介导的文库制备的PCR扩增子并导致降低测序深度在扩增区域的端部。因此，高的兴趣的区域的设计应该驻留在扩增子的中间。怎么样以往，因为BSAS生成测序阅读深度&gt; 1000的X，5-MC定量精度邻近扩增区域的端部测量仍然很高。

自的天然存在的DNA的修饰中胞嘧啶甲基1的形式中，第一报告它已经过了半个世纪。胞嘧啶甲基化是其中基环上的5 个碳具有一个甲基（5-MC），最常在哺乳动物基因组中的CpG二核苷酸基序中产生的修饰的胞嘧啶核苷酸碱基。的5-MC在基因的启动子功能的存在通常与转录抑制有关，而没有与转录活性相关联。 巨大进展已在我们的DNA甲基化在发展3，后生轮廓4，癌症发病5,6，和其他一些研究领域transgeneration传播中的作用的理解。许多的这些进步已经走在过去的几年中作为新的方法已经被开发来分析DNA甲基化7。同的出现和下一代测序（NGS）技术的普及，现在有许多方法来跨越整个基因组或基因组8的大区域轮廓的DNA甲基化。这些方法打开发现研究，量化DNA甲基化模式和DNA甲基化的差异的可能性。除了这些假设产生的方法，有针对性/假设检验的DNA甲基化的定量方法显著需求。 焦磷酸测序，甲基化特异性PCR，和亚硫酸氢盐转化的DNA直接Sanger测序一直是最常用的方法为目标区域的分析（ 即 ，单一的基因的启动子区域或CpG岛）9-12。所有这些方法都依赖于亚硫酸氢盐转化率，其中，非甲基化胞嘧啶的转化成尿嘧啶的化学脱氨反应，而甲基化胞嘧啶的保持完好13,14。的bisulfite- PCR扩增转换后的DNA结果更换尿嘧啶与胸腺嘧啶15允许差异甲基化读出的基差。而非常有用，所述限制于这些方法包括低定量准确，短的读取长度，以及低样品通量。为了解决这些限制我们开发我们称之为亚硫酸氢盐测序扩增子（BSAS）16的方法。该方法的目标是能够分析所关注目标的基因组区域中的大量的高定量准确的样品。 BSAS使用的现有方法的元素（亚硫酸氢盐转化率和区域特异性PCR扩增），并将其与简单下一代文库构建（座子介导的）17和台式NGS 18（ 图1）。这种方法提供了一个更定量和更高的吞吐量的方法，研究在任何感兴趣的区域中的胞嘧啶甲基化。在这里，我们提出了方法进行详细描述，并进行故障排除和质量检查BSAS过程的方法。

<table border="0" cellpadding="0" cellspacing="0" width="840">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:304px;">Name</td>
			<td style="width:255px;">Company</td>
			<td style="width:97px;">Cat #</td>
			<td style="width:185px;">Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agilent 2100 Bioanlyzer</td>
			<td>Agilent</td>
			<td>G29393AA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agilent RNA 6000 Nano kit</td>
			<td>Agilent</td>
			<td>5067-1511</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agilent High Sensitivity DNA kit</td>
			<td>Agilent</td>
			<td>5067-4626</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Typhoon 9200</td>
			<td>Amersham/GE Healthcare Life Sciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Agencourt AMPure XP - PCR Purification</td>
			<td>Beckman Coulter</td>
			<td>A63880</td>
			<td>5 mL</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PowerPac Basic Power Supply</td>
			<td>Bio Rad</td>
			<td>164-5050</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">twin.tech PCR plate 96&#160;</td>
			<td>Eppendorf</td>
			<td>951020362</td>
			<td>semi-skirted</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heatsealing Film</td>
			<td>Eppendorf</td>
			<td>0030127838</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heatsealing Foil</td>
			<td>Eppendorf</td>
			<td>0030127854</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heat Sealer</td>
			<td>Eppendorf</td>
			<td>5390000.024</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.1-10ul Tips</td>
			<td>Eppendorf</td>
			<td>0030073.002</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-200ul Tips</td>
			<td>Eppendorf</td>
			<td>0030073.045</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50-1000ul Tips</td>
			<td>Eppendorf</td>
			<td>0030073.100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MixMate&#160;</td>
			<td>Eppendorf</td>
			<td>5353000.014</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1.5 mL Microcentrifuge Tubes</td>
			<td>Eppendorf</td>
			<td>0030121.023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge 5424</td>
			<td>Eppendorf</td>
			<td>5424000.010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2.0 mL Microcentrifuge Tube</td>
			<td>Eppendorf</td>
			<td>022363352</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nextera XT DNA Sample Preparation Kit</td>
			<td>Illumina</td>
			<td>FC-131-1024</td>
			<td>24 rxn</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nextera XT Index Kit</td>
			<td>Illumina</td>
			<td>FC-131-1001</td>
			<td>24 indexes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MiSeq Desktop Sequencer</td>
			<td>Illumina</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MiSeq Reagent Kit v2</td>
			<td>Illumina</td>
			<td>MS-102-2002</td>
			<td>300cycle</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">KAPA SYBR FAST Universal qPCR kit</td>
			<td>KAPA Biosystems</td>
			<td>KK4824</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Quant-iT PicoGreen dsDNA Assay kit</td>
			<td>Life Technologies</td>
			<td>P7589</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Quant-iT RiboGreen RNA Assay kit</td>
			<td>Life Technologies</td>
			<td>R11490</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ProFlex 96-well PCR system</td>
			<td>Life Technologies</td>
			<td>4484075</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Novex 6% TBE DNA gel</td>
			<td>Life Technologies</td>
			<td>EC6261BOX</td>
			<td>10-well</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Novex TBE Running Buffer</td>
			<td>Life Technologies</td>
			<td>LC6675</td>
			<td>5X (1 L)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Novex Hi-Density TBE Sample Buffer</td>
			<td>Life Technologies</td>
			<td>LC6678</td>
			<td>5X (10 mL)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1 Kb Plus DNA Ladder</td>
			<td>Life Technologies</td>
			<td>10787-018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xcell SureLock Mini-Cell</td>
			<td>Life Technologies</td>
			<td>EI0001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UltraPure 10mg/mL Ethidium Bromide</td>
			<td>Life Technologies</td>
			<td>15585-011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">7900HT Fast Real-Time PCR system</td>
			<td>Life Technologies</td>
			<td>4329001</td>
			<td>384well block</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DynaMag-96 Side Skirted</td>
			<td>Life Technologies</td>
			<td>12027</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SpectroMax M2 Multi-Mode Microplate Reader</td>
			<td>Molecular Devices/VWR</td>
			<td>89429-532</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">AllPrep DNA/RNA Mini Kit</td>
			<td>Qiagen</td>
			<td>80204</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stainless Steel Beads</td>
			<td>Qiagen</td>
			<td>69989</td>
			<td>5 mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">CLC Genomics Workbench</td>
			<td>Qiagen</td>
			<td></td>
			<td>Software for methylation pipeline</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAQuick PCR Purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td>50 rxn</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TissueLyser II</td>
			<td>Retsch/Qiagen</td>
			<td>85300</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nuclease-Free Water</td>
			<td>Sigma</td>
			<td>W4502</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TRIS hydrochloride</td>
			<td>Sigma</td>
			<td>PHG0002-100G</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tween-20</td>
			<td>Sigma</td>
			<td>P9416-50ML</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NanoDrop 2000</td>
			<td>Thermo Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">384-well Full-Skirted Plate, Standard</td>
			<td>Thermo Scientific</td>
			<td>AB-1384</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Absolute qPCR Seal</td>
			<td>Thermo Scientific</td>
			<td>AB-1170</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EZ DNA Methylation-Lightning kit</td>
			<td>Zymo Research</td>
			<td>D5030</td>
			<td>50 rxn</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ZymoTaq DNA Polymerase</td>
			<td>Zymo Research</td>
			<td>E2001</td>
			<td>50 rxn</td>
		</tr>
	</tbody>
</table>

targeted dna methylation analysis by next-generation sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

BSAS读取正确对准到转换后的参考序列将类似于图5。CpG二核苷酸可以清楚地被识别和甲基化状态可以通过观察在所述映射中读取CpG部位碱呼叫来估计。例如，对于甲基化控制，0％甲基化将导致所有的读取映射到CpG部位含有T的（ 图5A）。 100％甲基化的控制，将导致所有的读取映射读取到CpG部位含有C的（ 图5B）。 胞嘧啶频率（C / c + T）在CpG位点的定量产生原始样品中的甲基化频。从全基因组的甲基化对照（n = 3 /甲基化比率）产生的代表性标准曲线示出了在各个甲基化率（ 图6）的甲基化的定量的非线性，以及在定量精度。作为该方法中的总的一个例子，RNA和DNA共 - 异<html>从小鼠小脑和视网膜（N = 4 /组）迟来。视紫红质的表达，选择性地表达在视网膜组织，使用qPCR测量。表达仅在视网膜（ 图7A）进行检测。使用BSAS，CpG甲基化水平进行定量的视紫红质启动子区。相比，在视网膜（P &lt;0.001，参数t检验）（ 图7B）&lt;15％整个启动子区域的甲基化的累积水平&gt; 80％，在小脑。从站点特定的甲基化定量BSAS甲基化定量分析的好处，并且甲基化水平可以在任何给定的基因组区域的CpG一个特定的基础上进行比较。相比，视网膜样品（P &lt;0.001，参数t检验在各CpG位点）（ 图7C）横跨视紫红质启动子CpG甲基化水平显著高于小脑样本。 1highres.jpg“/&gt; 图1：BSAS方法的示意图 。在该方法中，基因组DNA是亚硫酸氢盐转化成修改非甲基化胞嘧啶向尿嘧啶。接着，在PCR期间这些尿嘧啶变为胸腺嘧啶。 PCR扩增是针对感兴趣的区域，高度丰富为只是这些序列。将所得的PCR扩增子通过一个简单的过程tagmentation制成双索引库。随后文库进行测序在工作台上的下一代序器和测序读数映射到胞嘧啶的是在一个碱基特异性的方式来确定的，在硅片转换参考序列和百分比的甲基化。 <img alt="图2" src="/files/ftp_upload/52488/52488fig2highres.jpg" /> 图2： 在亚硫酸氢硅片的关心区域的转换的兴趣从任何基因组参考的任何区域可SELEC特德在硅片亚硫酸氢盐转化。非CpG的胞嘧啶替换为胸腺嘧啶的在参考序列。的CpG的胞嘧啶保持在亚硫酸氢盐转化的胞嘧啶参考。 <img alt="图3" src="/files/ftp_upload/52488/52488fig3highres.jpg" /> 图3：引物放置亚硫酸氢盐PCR引物被设计针对在硅片亚硫酸氢盐转化的参考序列。引物被设计成使得它们不重叠的CG二核苷酸或设计邻近的CG二核苷酸。 <img alt="图4" src="/files/ftp_upload/52488/52488fig4highres.jpg" /> 图4：高，质量差的PCR扩增和测序文库的例子代表电泳痕迹和凝胶显示（A）的转座子介导的库代扩增的理想选择用一个高点。浓度产物。从该扩增子产生的（B）中示出了库的高浓度，甚至尺寸分布。（C）的质量差的PCR扩增子可以是低的大小，浓度和包含多个产品和/或引物二聚体。这些质量低劣的扩增子将导致（D）中失败，文库产生具有低浓度和低粒度分布。 <img alt="图5" src="/files/ftp_upload/52488/52488fig5highres.jpg" /> 图5：从甲基化对照测序输出实例。 （A） 在硅片转换的参考序列与CpG位点以红色突出显示。 0％甲基化控制映射到读取选定区域显示胸腺嘧啶（贪婪亮点）映射在CpG位点。（B）100％的甲基化控制映射读取显示胞嘧啶的（蓝色高亮显示）映射在CpG位点。 图6：在一系列的甲基化甲基化控制定量全基因组的甲基化控制从0％混合在甲基化率（N = 3 /比）至100％用BSAS定量。绘制定量预计与量化表明甲基化控制定量的线性度。在所有的控制，有高精度的甲基化定量分析。 <img alt="图7" src="/files/ftp_upload/52488/52488fig7highres.jpg" /> 图7：从组织样品配对DNA甲基化和基因表达分析的实施例。 （A）中从小鼠小脑和视网膜组织视紫红质的相对mRNA表达。（B）的视紫红质的平均启动子甲基小脑和视网膜的DNA（*** P &lt;0.001，参数t-检验）之间定量BSAS。 &lt;STRONG&gt; C）CpG位点特异的甲基化水平的跨越视紫红质启动子（-244至+6，相对于转录起始位点[TSS]）在小鼠小脑和视网膜的DNA。

Watch this Scientific Journal Video about Targeted DNA Methylation Analysis by Next-generation Sequencing at JoVE.com

Targeted DNA Methylation Analysis by Next-generation Sequencing

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

有针对性的DNA甲基化分析通过新一代测序

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

targeted-dna-methylation-analysis-by-next-generation-sequencing

Research

JoVE Journal

Biology

36.9K 次观看.  University of Oklahoma College of Medicine. Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

视频: Targeted DNA Methylation Analysis by Next-generation Sequencing

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA methyltransferases is viable, thus providing a system to study how loss of DNA methylation in a genome affects growth and development. Imprinting refers to differential expression of maternal and paternal alleles and plays an important role in reproduction development in both mammal and plants. DNA methylation is critical for determining whether the maternal or paternal alleles of an imprinted gene is expressed or silenced. In flowering plants, there is a double fertilization event in reproduction: one sperm cell fertilizes the egg cell to form embryo and a second sperm fuses with the central cell to give rise to endosperm. Endosperm is the tissue where imprinting occurs in plants. MEDEA, a SET domain Polycomb group gene, and FWA, a transcription factor regulating flowering, are the first two genes shown to be imprinted in endosperm and their expression is controlled by DNA methylation and demethylation in plants. In order to determine imprinting status of a gene and methylation pattern in endosperm, we need to be able to isolate endosperm first. Since seed is tiny in Arabidopsis, it remains challenging to isolate Arabidopsis endosperm and examine its methylation. In this video protocol, we report how to conduct a genetic cross, to isolate endosperm tissue from seeds, and to determine the methylation status by bisulfite sequencing.

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

Imprinting is a phenomenon in plant and mammal reproduction. DNA methylation plays an important role in mechanisms of imprinting. Isolating endosperm and determining methylation status of imprinted genes in Arabidopsis can be difficult. In this protocol, we describe how to isolate endosperm and determine methylation by bisulfite sequencing.

印记基因的DNA甲基化的测定拟南芥胚乳

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

科研

生物学

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

DNA甲基化：亚硫酸氢修改和分析

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.

A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.

荧光显微镜筛选和新一代测序：参与细胞器完整性的基因鉴定的有用工具

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

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

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

的16S核糖体RNA基因的扩增子的新一代测序

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

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

增强的减少表示亚硫酸氢盐测序的DNA甲基化的评估，在碱基对决议

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

逆转录病毒整合位点的扩增，新一代测序和基因组DNA制图

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to poorly folding proteins or random open reading frames. We have developed a strategy where, by using TEM-1 &#946;-lactamase (the enzyme conferring ampicillin resistance) on a genomic scale, we can select collections of correctly folded protein domains from the coding portion of the DNA of any intronless genome. The protein fragments obtained by this approach, the so called &#34;domainome&#34;, will be well expressed and soluble, making them suitable for structural/functional studies.
By cloning and displaying the &#34;domainome&#34; directly in a phage display system, we have showed that it is possible to select specific protein domains with the desired binding properties (e.g., to other proteins or to antibodies), thus providing essential experimental information for gene annotation or antigen identification.
The identification of the most enriched clones in a selected polyclonal population can be achieved by using novel next-generation sequencing technologies (NGS). For these reasons, we introduce deep sequencing analysis of the library itself and the selection outputs to provide complete information on diversity, abundance and precise mapping of each of the selected fragment. The protocols presented here show the key steps for library construction, characterization, and validation.

The protocols described allow the construction, characterization and selection (against the target of choice) of a &#34;domainome&#34; library made from any DNA source. This is achieved by a research pipeline that combines different technologies: phage display, a folding reporter and next generation sequencing with a web tool for data analysis.

Interactome: Domainome 图书馆建设、噬菌体展示和下一代测序的验证与选择协议

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to ...

Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective and widely available vaccines are lacking for a majority of these diseases, necessitating the development of novel disease mitigation strategies. To this end, a promising avenue of disease control involves targeting the vector microbiome, the community of microbes inhabiting the vector. The vector microbiome plays a pivotal role in pathogen dynamics, and manipulations of the microbiome have led to reduced vector abundance or pathogen transmission for a handful of vector-borne diseases. However, translating these findings into disease control applications requires a thorough understanding of vector microbial ecology, historically limited by insufficient technology in this field. The advent of next-generation sequencing approaches has enabled rapid, highly parallel sequencing of diverse microbial communities. Targeting the highly-conserved 16S rRNA gene has facilitated characterizations of microbes present within vectors under varying ecological and experimental conditions. This technique involves amplification of the 16S rRNA gene, sample barcoding via PCR, loading samples onto a flow cell for sequencing, and bioinformatics approaches to match sequence data with phylogenetic information. Species or genus-level identification for a high number of replicates can typically be achieved through this approach, thus circumventing challenges of low detection, resolution, and output from traditional culturing, microscopy, or histological staining techniques. Therefore, this method is well-suited for characterizing vector microbes under diverse conditions but cannot currently provide information on microbial function, location within the vector, or response to antibiotic treatment. Overall, 16S next-generation sequencing is a powerful technique for better understanding the identity and role of vector microbes in disease dynamics.

Here we present a next-generation sequencing protocol for 16S rRNA sequencing which enables identification and characterization of microbial communities within vectors. This method involves DNA extraction, amplification and barcoding of samples through PCR, sequencing on a flow-cell, and bioinformatics to match sequence data to phylogenetic information.

下一代 16S rRNA 扩增子测序的蜱菌群鉴定

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective ...

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

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

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

使用两步PCR和下一代16S rRNA基因测序进行微生物群分析

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

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high quality and quantity DNA from a well-preserved individual mosquito sufficient for downstream whole genome sequencing. However, reliance on an expensive automated DNA extraction instrument can be prohibitive for many laboratories. Here, the study provides a budget-friendly magnetic-bead-based DNA extraction protocol, which is suitable for low to medium throughput. The protocol described here was successfully tested using individual Aedes aegypti mosquito samples. The reduced costs associated with high quality DNA extraction will increase the application of high throughput sequencing to resource limited labs and studies.

Described here is a DNA extraction protocol using magnetic beads to produce high quality DNA extractions from mosquitoes. These extractions are suitable for a downstream next-generation sequencing approach.

用于下一代测序的基于磁珠的蚊子DNA提取协议

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high ...

Methods Article

有针对性的DNA甲基化分析通过新一代测序