Name: methyl-binding dna capture sequencing for patient tissues
Uploaded: 2016-10-31T12:30:00.000+00:00
Duration: 8 min 40 s
Description: Watch this Scientific Journal Video about Methyl-binding DNA capture Sequencing for Patient Tissues at JoVE.com

这项工作是由CPRIT研究培训奖RP140105支持，以及部分由美国国立卫生研究院（NIH）授予R01 GM114142和威廉与艾拉·欧文斯医学研究基金会的支持。

所有的组织得到以下机构审查委员会委员的批准，并在所有参与者同意双方分子分析和后续研究。该协议是由人类研究委员会在得克萨斯大学健康科学中心大学圣安东尼奥的批准。 1.甲基结合DNA捕获（MBDCap） <ol><li> 样品采集和DNA提取 <ol><li>从患者的石蜡包埋组织标本收集大块肿瘤和正常组织样本。 </li><li>使用商业DNA mini试剂盒来分离从根据制造商的协议的石蜡包埋的组织样品的基因组DNA。 </li><li>使用具有根据制造商的协议在这里描述的方法的甲基化的DNA富集试剂盒。 </li></ol></li><li> 初始卷边洗 注意:珠粒用来偶联用MBD-生物素蛋白，它检测在基因组上的DNA甲基化。珠通常被暂停在原液。使用下列步骤来洗掉这种液体。 <ol><li>轻轻吹打起来悬浮的链霉珠粒（见材料表 ）的库存和向下，得到均匀的悬浮液。不要震荡混合珠。 </li><li>对于输入DNA之一微克，珠与1X绑定/洗涤液的90微升管ADD10微升。旋转1分钟。不要震荡混合。 </li><li>放置管（多个）上进行1分钟的磁架。 </li><li>用移液器除去液体并丢弃该液体。 </li><li>添加1个绑定/洗涤缓冲液250微升至珠和旋转1分钟。 </li><li>重复步骤1.2.3 - 1.2.5两次。 </li></ol></li><li> 耦合MBD-生物素蛋白的小珠 <ol><li>对于输入DNA的1.2微克，加MBD-生物素蛋白7微升（3.5微克），每管。 </li><li>添加1个绑定/洗涤缓冲液，93微升的蛋白质，使100微升的最终体积。 </li><li>混合珠蛋白混合物于室温旋转混合器1小时。 </li></ol></li><li> 支离破碎的DNA（输入） <ol><li>通过在96微升的TE缓冲液中，24 5倍的微升绑定/洗涤缓冲液中加入总基因组DNA的1.2微克声处理的DNA，使120微升的溶液。使用30秒接通电源和超声在30秒关闭电源，20 - 25倍。 </li><li>根据制造商的协议350bp的 - 用生物分析仪系统与市售的高灵敏度的DNA试剂盒以检查片段的大小是在150的范围内运行超声处理的DNA溶液1微升。 </li></ol></li><li> 洗MBD-珠 <ol><li>上放置装有磁性机架MBD-珠1分钟的管。 </li><li>去除并丢弃用吸管的液体不接触珠。 </li><li>与1X绑定/洗涤液250微升重悬珠。 </li><li>混合珠上在RT旋转混合器5分钟。 </li><li>重复步骤1.5.1 - 1.5.4两次。 </l我&gt; </ol></li><li> 支离破碎的DNA俘获反应（1微克输入DNA） <ol><li>该DNA /缓冲混合物转移到含有MBD-珠的管中。 </li><li>混合MBD-珠与DNA上的旋转混合器，在室温1小时。另外，在4℃混合O / N </li></ol></li><li> 从珠中除去非捕获的DNA <ol><li>混合该DNA和MBD-珠后，将在该磁性架管1分​​钟，从而集中所有的珠粒在管的内壁上。 </li><li>用吸管除去上层液体，并将其保存在干净的DNA酶的离心管作为nonmethylated DNA。储存在冰这个样本。 </li><li>添加1个绑定/洗涤缓冲液200微升至珠洗珠。 </li><li>混合珠在旋转混合器3分钟。 </li><li>放置在管上1分钟磁性机架。 </li><li>用吸管取出液体，并将其保存在离心管。 </li><li>对于帽TURE≤1微克输入DNA的反应，重复步骤1.7.3 - 1.7.6在总共2个洗分数。 </li></ol></li><li> 单次洗脱 <ol><li>混合使用高盐缓冲液低盐缓冲液（1:1），以获得洗脱缓冲液（1,000毫摩尔NaCl）。 </li><li>悬浮在洗脱缓冲液（1,000毫摩尔NaCl）的200μL珠。 </li><li>孵育在旋转混合器珠粒3分钟。 </li><li>放置在管上1分钟磁性机架。 </li><li>随着管处的磁架，去除液体用吸管没有枪头触及珠，并将其保存在干净的DNA酶的1.5 ml离心管。储存在冰这个样本。 </li><li>重复步骤1.8.1 - 1.8.4一次，收集在同一个管中的第二样品（总体积将400微升）。存放在冰合并样本。 </li></ol></li><li> 乙醇沉淀（DNA清理） <ol><li>到每个noncaptured，洗涤和洗脱馏分FR嗡前面的步骤中，添加1μL的肝糖（20微克/微升，包括在试剂盒），3M醋酸钠1/10的样品体积，pH为5.2（ 例如 ，每400 40微升样品的微升）和100％的2样品体积乙醇（ 例如 ，每400 800μL样品μL）。总体积为1,241微升。 </li><li>拌匀，并在-80孵化℃下至少2小时。 </li><li>离心管15分钟，在11363 XG在4℃ </li><li>小心弃去上清液而不扰乱沉淀。 </li><li>加入冷的70％乙醇500微升。 </li><li>离心管5分钟，在11363 XG在4℃ </li><li>小心弃去上清液而不扰乱沉淀。 </li><li>重复步骤1.9.6 - 1.9.7一次，并删除任何剩余的残留上清。 </li><li>风干〜5分钟沉淀。不要完全干燥沉淀。 </li><li>重悬DNA沉淀中的游离DNA酶的水或其它37.5微升缓冲区适当的音量。 </li><li>置于冰上的DNA或DNA储存在-20 度或以下，直至进一步使用。 </li><li>检查的DNA的总量超过20纳克，（ 例如 ，使用荧光定量系统，见材料数据表 ）。 </li></ol></li></ol> 2.测序<ol><li>使用来自MBDCap方法回收用于测序的DNA片段。对于待测序的每个样品，20的总量 - 需要用于文库制备40纳克的DNA。 </li><li>对于DNA的序列库准备使用一个完全自动化的文库构建系统（见材料表 ），并按照制造商提供的标准协议。 400个基点文库制备 - 尺寸200，选择的DNA片段。自动化文库制备系统允许标准化文库制备过程和整个样本的变化减少因手工准备。 </li><li>使用适配器总理RS并稀释至100倍的浓度。如此，使用10μL的各样品。 </li><li>以下步骤2.2，取出反应，并使用荧光定量系统（见材料表 ）量化的DNA序列文库。 <ol><li>建立PCR扩增步骤。 PCR主混合物的选择是改善基因组的GC富集区的PCR效率。向PCR管中，添加15微升的DNA SEQ文库，25μL的PCR主混合物（见材料表 ），2微升的PCR引物混合物（见材料表 ），以及8微升H 2 O </li></ol></li><li>按照从PCR扩增预混制造商PCR的建议，改变循环时间和温度不同的原料:98℃下45秒，98℃下15秒，65℃下30秒，72℃下30×周期s，然后1分钟72 摄氏度 。保持在4℃ <ol><li> PERFORM足够的周期，2落得 - 10纳米文库DNA（8 - 10次）。 </li></ol></li><li>清理用1 PCR反应:1的比例的，根据制造商的协议PCR纯化珠粒（见材料表 ）。 </li><li> 30μL洗脱缓冲液 - 20洗脱。 </li><li>条码每个样品池4个样品汇集成的流动池的一个车道。使用测序标准的50个基点的单读协议（见材料表 ）。 </li></ol> 3.生物信息学分析注意:进一步处理从测序获得的原始FASTQ文件来执行的质量控制和映射所述短DNA序列（读取）到基因组中。 <ol><li>用蝴蝶结短读对齐，对每个样品FASTQ文件基因组映射工具映射读取到参考基因组。多个读映射工具可用于执行该步骤，并且可以根据个人的优先使用。 <ol><li>允许最多在whol 2个错配Ë同时映射到参考基因组的读取。报告最多的每次读20路线最好。例如，使用领结-v 2 --best -k 20 --chunkmbs 200 &lt;ebwt&gt; &lt;fastqFile&gt; &lt;alignFile&gt;。 </li></ol></li><li>使用LONUT 9来恢复乘法映射读取提高富集区域的检测。对于每个读，至多一个对准将当它是足够接近上述任何称为峰回收。例如，使用:perl的lonut.pl -g hg19 -w 250 -p 99 &lt;alignFile&gt; &lt;outputPath&gt;。 LONUT将执行以下步骤。 <ol><li>分割成路线唯一地映射读取和乘法映射读取。在唯一映射峰电话使用读取峰值主叫带11。 -w在示例命令250和-p 99选项是带。 </li><li>联合收割机唯一映射读取与恢复的读取从LONUT获得，合并的读出，在床的格式，将在进一步的分析中使用。计算COMBIN数编读为未来的正常化。 </li></ol></li><li>斌读取。提取读取使用perl脚本感兴趣区域:perl的methyPipeline.pl &lt;sampleInfoFile&gt; &lt;refGeneFile&gt; -up &lt;向上游延伸长度&gt; -dn &lt;向下游延伸长度&gt;。在sampleInfoFile列出的每个床档，Perl脚本执行以下任务。 注:见补充编码文件的Perl脚本。 <ol><li>斌读入固定的容器大小， 例如 ，100个基点，24条染色体。 </li><li>对于refGeneFile指定的每个基因，提取各地转录起始位点（TSS）的垃圾桶，直至达到选项-up和-dn指定延伸长度。例如，提取就是上行和下行的TSS的4kb的该区域中的数据。在块大小为100bp，该提取的数据有81箱，而中心区段对应到TSS。 </li><li>对于反义链上的基因，翻转所提取的区域从左至右，使得上游箱总是PLA土木工程署左侧用perl脚本。 </li><li>进一步分裂TSS±4 KB区域分为三个子区:左，中上游4 kb的上游2 kb的;中东，上行和下行2 KB;右，下游2 KB到4 KB。 </li><li>对于每个样品，除以计数的在由组合映射读取在3.3.3计算总数每个仓读取。正常化将消除样品特定阅读差异，并允许比较不同样本的读取。 </li></ol></li><li>运行bash脚本（如在脚本文件中指示），以归一化执行统计测试读取从左侧区域获得的，在描述为在侧翼区的区域，以检测正常和病例组之间差异甲基化。 注:请参阅的bash脚本的补充编码文件。基于被差异甲基化的区域，有七个组合: 整个区域:整个TSS±4 kb的区域差异甲基化 MiddleLeft:在这两个中间和左侧区域差异甲基化 MiddleRight:在这两个中间和右侧区域差异甲基化 侧翼:在左，右区域差异甲基化 左图:在左侧区域差异甲基化只 右:在右侧区域差异甲基化只 中东:在中间区域差异甲基化只</li><li>使用相同的bash脚本以可视化的差异甲基化龙卷风情节。绘制一个单独的面板上的超和低甲基化的基因，并如分别在3.4中描述，排序在该区域组合的顺序。 </li></ol>

<ol>
	<li>Trimarchi, M. P., Mouangsavanh, M., Huang, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+epigenetics:+a+perspective+on+the+role+of+DNA+methylation+in+acquired+endocrine.">Cancer epigenetics: a perspective on the role of DNA methylation in acquired endocrine.</a> Chin. J. Cancer. 30, 749-756 (2011).</li><li>Nair, S. 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=Comparison+of+methyl-DNA+immunoprecipitation+(MeDIP)+and+methyl-CpG+binding+domain+(MBD)+protein+capture+for+genome-wide+DNA+methylation+analysis+reveal+CpG+sequence+coverage+bias.">Comparison of methyl-DNA immunoprecipitation (MeDIP) and methyl-CpG binding domain (MBD) protein capture for genome-wide DNA methylation analysis reveal CpG sequence coverage bias.</a> Epigenetics. 6, 34-44 (2011).</li><li>Zuo, T., Tycko, B., Liu, T. M., Lin, J. J., Huang, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+in+DNA+methylation+profiling.">Methods in DNA methylation profiling.</a> Epigenomics. 1, 331-345 (2009).</li><li>Clark, C., 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+comparison+of+the+whole+genome+approach+of+MeDIP-seq+to+the+targeted+approach+of+the+Infinium+HumanMethylation450+BeadChip((R))+for+methylome+profiling.">A comparison of the whole genome approach of MeDIP-seq to the targeted approach of the Infinium HumanMethylation450 BeadChip((R)) for methylome profiling.</a> PLoS One. 7, e50233 (2012).</li><li>Walker, D. 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=DNA+methylation+profiling:+comparison+of+genome-wide+sequencing+methods+and+the+Infinium+Human+Methylation+450+Bead+Chip.">DNA methylation profiling: comparison of genome-wide sequencing methods and the Infinium Human Methylation 450 Bead Chip.</a> Epigenomics. , 1-16 (2015).</li><li>Huang, Y. W., Huang, T. H., Wang, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+DNA+methylomes+from+microarray+to+genome-scale+sequencing.">Profiling DNA methylomes from microarray to genome-scale sequencing.</a> Technol. Cancer Res. Treat. 9, 139-147 (2010).</li><li>Serre, D., Lee, B. H., Ting, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MBD-isolated+Genome+Sequencing+provides+a+high-throughput+and+comprehensive+survey+of+DNA+methylation+in+the+human+genome.">MBD-isolated Genome Sequencing provides a high-throughput and comprehensive survey of DNA methylation in the human genome.</a> Nucleic Acids Res. 38, 391-399 (2010).</li><li>Brinkman, A. 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=Whole-genome+DNA+methylation+profiling+using+MethylCap-seq.">Whole-genome DNA methylation profiling using MethylCap-seq.</a> Methods. 52, 232-236 (2010).</li><li>Wang, 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=LOcating+non-unique+matched+tags+(LONUT)+to+improve+the+detection+of+the+enriched+regions+for+ChIP-seq+data.">LOcating non-unique matched tags (LONUT) to improve the detection of the enriched regions for ChIP-seq data.</a> PLoS One. 8, e67788 (2013).</li><li>Gu, 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=CMS:+a+web-based+system+for+visualization+and+analysis+of+genome-wide+methylation+data+of+human+cancers.">CMS: a web-based system for visualization and analysis of genome-wide methylation data of human cancers.</a> PLoS One. 8, e60980 (2013).</li><li>Lan, X., Bonneville, R., Apostolos, J., Wu, W., Jin, V. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=W-ChIPeaks:+a+comprehensive+web+application+tool+for+processing+ChIP-chip+and+ChIP-seq+data.">W-ChIPeaks: a comprehensive web application tool for processing ChIP-chip and ChIP-seq data.</a> Bioinformatics. 27, 428-430 (2011).</li><li>Jadhav, R. 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=Genome-wide+DNA+methylation+analysis+reveals+estrogen-mediated+epigenetic+repression+of+metallothionein-1+gene+cluster+in+breast+cancer.">Genome-wide DNA methylation analysis reveals estrogen-mediated epigenetic repression of metallothionein-1 gene cluster in breast cancer.</a> Clin. Epigenetics. 7, 13 (2015).</li><li>Hsu, Y. 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=Promoter+hypomethylation+of+EpCAM-regulated+bone+morphogenetic+protein+gene+family+in+recurrent+endometrial+cancer.">Promoter hypomethylation of EpCAM-regulated bone morphogenetic protein gene family in recurrent endometrial cancer.</a> Clin. Cancer Res. 19, 6272-6285 (2013).</li><li>Wang, Y. V., 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=Roles+of+Distal+and+Genic+Methylation+in+the+Development+of+Prostate+Tumorigenesis+Revealed+by+Genome-wide+DNA+Methylation+Analysis.">Roles of Distal and Genic Methylation in the Development of Prostate Tumorigenesis Revealed by Genome-wide DNA Methylation Analysis.</a> Sci. Rep. , (2015).</li><li>Plongthongkum, N., Diep, D. H., Zhang, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+profiling+of+DNA+modifications:+cytosine+methylation+and+beyond.">Advances in the profiling of DNA modifications: cytosine methylation and beyond.</a> Nat Rev Gen. 15, 647-661 (2014).</li><li>Riebler, A., 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=BayMeth:+improved+DNA+methylation+quantification+for+affinity+capture+sequencing+data+using+a+flexible+Bayesian+approach.">BayMeth: improved DNA methylation quantification for affinity capture sequencing data using a flexible Bayesian approach.</a> Genome Biol. 15, R35 (2014).</li></ol>

该协议是在添黄医生和Victor金博士在得克萨斯大学健康科学中心在圣安东尼奥大学的实验室开发的。

所述MBDCap-SEQ技术是亲和富集的方法3，用大量的患者15调查队列时视为一个成本有效的替代方案。这里介绍的管道描述了从样品采购到数据分析和解释一个全面的方法。其中最重要的步骤是建立一个PCR扩增过程，以改善在GC富集区域的基因组中的PCR效率，这是在DNA甲基化发生。此外，它是必不可少的，以确保测序后，每个样品具有至少20多万唯一映射读取到基因组中。此覆盖范围预计将提供足够的?...

通过DNA甲基化的基因的表观遗传调控是确定在主体1由不同的组织类型的稳定分化的细胞命运所需的基本机制之一。这个过程的失调已经知道，引起各种疾病，包括癌症2。 该方法主要涉及对DNA中3的CpG二核苷酸的胞嘧啶残基的加入甲基团。目前有用于调查此机制，每一个都具有如在许多研究中2-8概述自身优势几个不同的技术。在这里，我们将讨论这些技术称为甲基结合DNA测序捕获（MBDCap-SEQ），在这里我们使用一个亲和富集技术来识别DNA的甲基化区域之一。该技术是建立在以富集含有甲基化CpG位点的基因组DNA片段的MBD2蛋白的甲基结合能力。我们利用商业甲基化DNA富集试剂盒这些甲基化区域的隔离。我们的实验室已筛选使用这种技术数百患者样品的，在这里我们提供了全面优化的协议，它可以被用来研究大患者群。 正如任何下一代测序技术明显，MBDCap-SEQ也需要以横跨所述样本以准确定量的甲基化水平的特定生物信息学方法。已经有许多最近的努力来优化测序数据9,10的正常化和分析过程的研究。在这个协议中，我们展示的实施独特的读恢复的方法，这些方法中的一种 - LONUT - 其次是每个样品的线性正常化，以实现跨越大量的患者样本公正的比较。

<table><tbody><tr><td>Methylminer DNA enrichment Kit</td><td>Invitrogen</td><td>ME10025</td><td></td></tr><tr><td>Dynabeads M-280 Streptavidin</td><td>Invitrogen</td><td>112-05D</td><td></td></tr><tr><td>Bioruptor Plus Sonication Device</td><td>diagenode</td><td>B01020001</td><td></td></tr><tr><td>3 M sodium acetate, pH 5.2</td><td>Sigma</td><td>S7899</td><td>100 mL</td></tr><tr><td>SPRIworks Fragment Library System I</td><td>Beckman Coulter</td><td>A50100</td><td>Fully automated library construction system</td></tr><tr><td>Adapter Primers</td><td>Bioo Scientific</td><td>514104</td><td>PCR primer mix</td></tr><tr><td>Qubit</td><td>Invitrogen</td><td>Q32854</td><td>Fluorometric Quantitation System</td></tr><tr><td>PCR master mix</td><td>KAPA scientific</td><td>KK2621</td><td>PCR master mix</td></tr><tr><td>AMPure XP</td><td>Beckman Coulter</td><td>A63881</td><td>PCR Purification beads</td></tr><tr><td>EB Buffer</td><td>Qiagen</td><td>19086</td><td></td></tr><tr><td>HiSeq 2000 Sequencing System</td><td>Illumina</td><td></td><td></td></tr></tbody></table>

methyl-binding dna capture sequencing for patient tissues

甲基化是必要的表观遗传修饰的DNA，这是负责为不同组织类型的稳定发展和分化所需的基因的精确调节中的一个。这个过程的失调经常是各种疾病，如癌症的标志。在这里，我们勾勒出近期测序技术之一，甲基结合DNA测序捕获（MBDCap-SEQ），用于为大型患者群的各种正常和疾病组织进行量化甲基化。我们描述一个生物信息学管道，以达到最佳的量化沿着这条亲和富集方法的详细的协议。这一技术已被用于测序数百名患者在各种癌症类型为1000甲基化项目（癌症甲基化系统）的一部分。

我们已经使用MBDCap-SEQ研究DNA甲基化的改变在大量的病人来自不同癌症类型包括乳腺癌12，子宫内膜13，前列腺14，和肝癌等。这里，我们证明从最近公布的12乳腺癌研究的一些信息。在这种情况下，我们使用了全基因组测序的方法，以确定被差动在肿瘤在不同的基因组区域的甲基化相对于正常CpG岛。调查显示，在总共调查了位于基因启动子（N = 13,081）CpG岛，相对于普通19.5％，表现...

Watch this Scientific Journal Video about Methyl-binding DNA capture Sequencing for Patient Tissues at JoVE.com

Methyl-binding DNA capture Sequencing for Patient Tissues

在这里，我们提出了一个协议，调查使用甲基结合DNA测序捕获（MBDCap-SEQ或MBD-SEQ）技术以及随后的生物信息学分析管道的大规模临床病人的筛查研究全基因组DNA甲基化。

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 ...

methyl-binding-dna-capture-sequencing-for-patient-tissues

Universidad San Sebastian

Research

JoVE Journal

Genetics

8.6K 次观看.  University of Texas Health Science Center at San Antonio. 在这里，我们提出了一个协议，调查使用甲基结合DNA测序捕获（MBDCap-SEQ或MBD-SEQ）技术以及随后的生物信息学分析管道的大规模临床病人的筛查研究全基因组DNA甲基化。 

视频: Methyl-binding DNA capture Sequencing for Patient Tissues

Comprehensive DNA Methylation Analysis Using a Methyl-CpG-binding Domain Capture-based Method in Chronic Lymphocytic Leukemia Patients

The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during cancer development and progression. Despite this, the global epigenetic regulation of lncRNAs and repetitive sequences in cancer has not been well investigated, particularly in chronic lymphocytic leukemia (CLL). This study focuses on a unique approach: the immunoprecipitation-based capture of double-stranded, methylated DNA fragments using methyl-binding domain (MBD) proteins, followed by next-generation sequencing (MBD-seq). CLL patient samples belonging to two prognostic subgroups (5 IGVH mutated samples + 5 IGVH unmutated samples) were used in this study. Analysis revealed 5,800 hypermethylated and 12,570 hypomethylated CLL-specific differentially methylated genes (cllDMGs) compared to normal healthy controls. Importantly, these results identified several CLL-specific, differentially methylated lncRNAs, repetitive elements, and protein-coding genes with potential prognostic value. This work outlines a detailed protocol for an MBD-seq and bioinformatics pipeline developed for the comprehensive analysis of global methylation profiles in highly CpG-rich regions using CLL patient samples. Finally, a protein-coding gene and an lncRNA were validated using pyrosequencing, which is a highly quantitative method to analyze CpG methylation levels to further corroborate the findings from the MBD-seq protocol.

This work describes an optimized methyl-CpG-binding domain (MBD) sequencing protocol and a computational pipeline to identify differentially methylated CpG-rich regions in chronic lymphocytic leukemia (CLL) patients.

在慢性淋巴细胞性白血病患者中使用甲基CpG结合域捕获法进行综合DNA甲基化分析

The role of long noncoding RNAs (lncRNAs) in cancer is coming to the forefront due to growing interest in understanding their mechanistic functions during ...

科研

遗传学

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

线粒体 DNA 甲基化的精确检测方法

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

Methylated DNA Immunoprecipitation

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on gene expression, and is involved with normal development as well as disease 1-4. Thus, the ability to discriminate between methylated DNA and non-methylated DNA is essential for generating methylation profiles for such studies. Methylated DNA immunoprecipitation (MeDIP) is an efficient technique for the extraction of methylated DNA from a sample of interest 5-7. A sample of as little as 200 ng of DNA is sufficient for the antibody, or immunoprecipitation (IP), reaction. DNA is sonicated into fragments ranging in size from 300-1000 bp, and is divided into immunoprecipitated (IP) and input (IN) portions. IP DNA is subsequently heat denatured and then incubated with anti-5'mC, allowing the monoclonal antibody to bind methylated DNA. After this, magnetic beads containing a secondary antibody with affinity for the primary antibody are added, and incubated. These bead-linked antibodies will bind the monoclonal antibody used in the first step. DNA bound to the antibody complex (methylated DNA) is separated from the rest of the DNA by using a magnet to pull the complexes out of solution. Several washes using IP buffer are then performed to remove the unbound, non-methylated DNA. The methylated DNA/antibody complexes are then digested with Proteinase K to digest the antibodies leaving only the methylated DNA intact. The enriched DNA is purified by phenol:chloroform extraction to remove the protein matter and then precipitated and resuspended in water for later use. PCR techniques can be used to validate the efficiency of the MeDIP procedure by analyzing the amplification products of IP and IN DNA for regions known to lack and known to contain methylated sequences. The purified methylated DNA can then be used for locus-specific (PCR) or genome-wide (microarray and sequencing) methylation studies, and is particularly useful when applied in conjunction with other research tools such as gene expression profiling and array comparative genome hybridization (CGH) 8. Further investigation into DNA methylation will lead to the discovery of new epigenetic targets, which in turn, may be useful in developing new therapeutic or prognostic research tools for diseases such as cancer that are characterized by aberrantly methylated DNA 2, 4, 9-11.

This video demonstrates the protocol for methylated DNA immunoprecipitation (MeDIP). MeDIP is a two day procedure that selectively extracts methylated DNA fragments from a genomic DNA sample using antibodies with specificity for 5 -methylcytosine (anti-5 mC).

甲基化的DNA免疫沉淀

The identification of DNA methylation patterns is a common procedure in the study of epigenetics, as methylation is known to have significant effects on ...

生物学

Optimized Analysis of DNA Methylation and Gene Expression from Small, Anatomically-defined Areas of the Brain

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to the display of physiological and behavioural phenotypes. Such environmental programming is likely to increase the susceptibility to metabolic, cardiovascular and mental diseases 3,4.
DNA methylation and histone modifications are considered key processes in the mediation of the gene-environment dialogue and appear also to underlay environmental programming 5. In mammals, DNA methylation typically comprises the covalent addition of a methyl group at the 5-position of cytosine within the context of CpG dinucleotides.
CpG methylation occurs in a highly tissue- and cell-specific manner making it a challenge to study discrete, small regions of the brain where cellular heterogeneity is high and tissue quantity limited. Moreover, because gene expression and methylation are closely linked events, increased value can be gained by comparing both parameters in the same sample.
Here, a step-by-step protocol (Figure 1) for the investigation of epigenetic programming in the brain is presented using the 'maternal separation' paradigm of early life adversity for illustrative purposes. The protocol describes the preparation of micropunches from differentially-aged mouse brains from which DNA and RNA can be simultaneously isolated, thus allowing DNA methylation and gene expression analyses in the same sample.

A streamlined workflow to study DNA methylation and gene expression changes upon early-life stress is shown. Starting from maternal separation of newborn mice and isolation of discrete brain tissues, we represent a protocol to simultaneously isolate DNA and RNA from brain tissue punches for subsequent bisulfite sequencing and RT-PCR analysis.

优化小，解剖定义的大脑区域的DNA甲基化和基因表达分析

Exposure to diet, drugs and early life adversity during sensitive windows of life 1,2 can lead to lasting changes in gene expression that contribute to ...

神经科学

Genome-Wide Analysis of DNA Methylation in Gastrointestinal Cancer

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is a useful measure to provide an accurate analysis of the methylation status of gastrointestinal (GI) malignancies. Given the multiple potential translational uses of DNA methylation analysis, practicing clinicians and others new to DNA methylation studies need to be able to understand step by step how these genome-wide analyses are performed. The goal of this protocol is to provide a detailed description of how this method is used for the biomarker identification in GI malignancies. Importantly, we describe three critical steps that are needed to obtain accurate results during genome-wide analysis. Clearly and concisely written, these three methods are often lacking and not noticeable to those new to epigenetic studies. We used 48 samples of a GI malignancy (gastric cancer) to highlight practically how genome-wide DNA methylation analysis can be performed for GI malignancies.

Herein, we describe a procedure for genome-wide analysis of DNA methylation in gastrointestinal cancers. The procedure is of relevance to studies that investigate relationships between methylation patterns of genes and factors contributing to carcinogenesis in gastrointestinal cancers.

胃肠癌DNA甲基化基因组全谱分析

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is ...

癌症研究

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 ...

Selective Capture of 5-hydroxymethylcytosine from Genomic DNA

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene expression, maintenance of genome integrity, parental imprinting, X-chromosome inactivation, regulation of development, aging, and cancer1. Recently, the presence of an oxidized 5-mC, 5-hydroxymethylcytosine (5-hmC), was discovered in mammalian cells, in particular in embryonic stem (ES) cells and neuronal cells2-4. 5-hmC is generated by oxidation of 5-mC catalyzed by TET family iron (II)/&alpha;-ketoglutarate-dependent dioxygenases2, 3. 5-hmC is proposed to be involved in the maintenance of embryonic stem (mES) cell, normal hematopoiesis and malignancies, and zygote development2, 5-10. To better understand the function of 5-hmC, a reliable and straightforward sequencing system is essential. Traditional bisulfite sequencing cannot distinguish 5-hmC from 5-mC11. To unravel the biology of 5-hmC, we have developed a highly efficient and selective chemical approach to label and capture 5-hmC, taking advantage of a bacteriophage enzyme that adds a glucose moiety to 5-hmC specifically12.
Here we describe a straightforward two-step procedure for selective chemical labeling of 5-hmC. In the first labeling step, 5-hmC in genomic DNA is labeled with a 6-azide-glucose catalyzed by &beta;-GT, a glucosyltransferase from T4 bacteriophage, in a way that transfers the 6-azide-glucose to 5-hmC from the modified cofactor, UDP-6-N3-Glc (6-N3UDPG). In the second step, biotinylation, a disulfide biotin linker is attached to the azide group by click chemistry. Both steps are highly specific and efficient, leading to complete labeling regardless of the abundance of 5-hmC in genomic regions and giving extremely low background. Following biotinylation of 5-hmC, the 5-hmC-containing DNA fragments are then selectively captured using streptavidin beads in a density-independent manner. The resulting 5-hmC-enriched DNA fragments could be used for downstream analyses, including next-generation sequencing.
Our selective labeling and capture protocol confers high sensitivity, applicable to any source of genomic DNA with variable/diverse 5-hmC abundances. Although the main purpose of this protocol is its downstream application (i.e., next-generation sequencing to map out the 5-hmC distribution in genome), it is compatible with single-molecule, real-time SMRT (DNA) sequencing, which is capable of delivering single-base resolution sequencing of 5-hmC.

Described is a two-step labeling process using &beta;-glucosyltransferase (&beta;-GT) to transfer an azide-glucose to 5-hmC, followed by click chemistry to transfer a biotin linker for easy and density-independent enrichment. This efficient and specific labeling method enables enrichment of 5-hmC with extremely low background and high-throughput epigenomic mapping via next-generation sequencing.

从基因组DNA选择性捕获的5  - 羟甲基

5-methylcytosine (5-mC) constitutes ~2-8% of the total cytosines in human genomic DNA and impacts a broad range of biological functions, including gene ...

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among traditional technologies, RNA interference suffers from partial target abrogation and the requirement for life-long treatments. In contrast, artificial nucleases can impose stable gene inactivation through induction of a DNA double strand break (DSB), but recent studies are questioning the safety of this approach. Targeted epigenetic editing via engineered transcriptional repressors (ETRs) may represent a solution, as a single administration of specific ETR combinations can lead to durable silencing without inducing DNA breaks.
ETRs are proteins containing a programmable DNA-binding domain (DBD) and effectors from naturally occurring transcriptional repressors. Specifically, a combination of three ETRs equipped with the KRAB domain of human ZNF10, the catalytic domain of human DNMT3A and human DNMT3L, was shown to induce heritable repressive epigenetic states on the ETR-target gene. The hit-and-run nature of this platform, the lack of impact on the DNA sequence of the target, and the possibility to revert to the repressive state by DNA demethylation on demand, make epigenetic silencing a game-changing tool. A critical step is the identification of the proper ETRs&#39; position on the target gene to maximize on-target and minimize off-target silencing. Performing this step in the final ex vivo or in vivo preclinical setting can be cumbersome.
Taking the CRISPR/catalytically dead Cas9 system as a paradigmatic DBD for ETRs, this paper describes a protocol consisting of the in vitro screen of guide RNAs (gRNAs) coupled to the triple-ETR combination for efficient on-target silencing, followed by evaluation of the genome-wide specificity profile of top hits. This allows for reduction of the initial repertoire of candidate gRNAs to a short list of promising ones, whose complexity is suitable for their final&#160;evaluation in the therapeutically relevant setting of interest.

In Vitro Selection of Engineered Transcriptional Repressors for Targeted Epigenetic Silencing

Here, we present a protocol for the in vitro selection of engineered transcriptional repressors (ETRs) with high, long-term, stable, on-target silencing efficiency and low genome-wide, off-target activity. This workflow allows for reducing an initial, complex repertoire of candidate ETRs to a short list, suitable for further evaluation in therapeutically relevant settings.

体外研究 用于靶向表观遗传沉默的工程化转录抑制因子的选择

Gene inactivation is instrumental to study gene function and represents a promising strategy for the treatment of a broad range of diseases. Among ...

生物工程

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 ...

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.

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 ...

为患者组织甲基结合DNA序列捕获

摘要

探索更多视频

此视频中的章节

在慢性淋巴细胞性白血病患者中使用甲基CpG结合域捕获法进行综合DNA甲基化分析

线粒体 DNA 甲基化的精确检测方法

甲基化的DNA免疫沉淀

优化小，解剖定义的大脑区域的DNA甲基化和基因表达分析

胃肠癌DNA甲基化基因组全谱分析

DNA甲基化：亚硫酸氢修改和分析

从基因组DNA选择性捕获的5 - 羟甲基

体外研究用于靶向表观遗传沉默的工程化转录抑制因子的选择

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

有针对性的DNA甲基化分析通过新一代测序

为患者组织甲基结合DNA序列捕获

摘要

探索更多视频

此视频中的章节

在慢性淋巴细胞性白血病患者中使用甲基CpG结合域捕获法进行综合DNA甲基化分析

线粒体 DNA 甲基化的精确检测方法

甲基化的DNA免疫沉淀

优化小，解剖定义的大脑区域的DNA甲基化和基因表达分析

胃肠癌DNA甲基化基因组全谱分析

DNA甲基化：亚硫酸氢修改和分析

从基因组DNA选择性捕获的5 - 羟甲基

体外研究 用于靶向表观遗传沉默的工程化转录抑制因子的选择

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

有针对性的DNA甲基化分析通过新一代测序

体外研究用于靶向表观遗传沉默的工程化转录抑制因子的选择