Name: methyl-binding dna capture sequencing for patient tissues
Uploaded: 2016-10-31T12:30:00
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...

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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. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>3M sodium acetate pH 5.2</td>
			<td>Sigma</td>
			<td>S7899</td>
			<td>100ml</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 ...

Research

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