The authors would like to thank Dr Rita Shaknovich and members of Elemento lab, Melnick lab, and Tam lab for thoughtful discussions. We would also like to thank the Genomics Resources Core Facility at Weill Cornell Medical College for performing the VDJ-sequencing. YJ was supported by ASH Scholar Award. WT and OE are supported by Weill Cornell Cancer Center Pilot Grant. OE is supported by the NSF CAREER award, the Starr Cancer Consortium and the Hirschl Trust. We would also like to thank Katherine Benesch, JD, MPH for her generous support to this project.

1. VDJ放大 1.1）的肿瘤样本DNA提取<ol><li>提取冻结十月嵌入式正常或恶性组织的薄片（10-20微米）的DNA。 <ol><li>消化十月三十零日薄于4毫升核酸裂解缓冲液（0.0075摩尔Tris盐酸，pH值8.2; 0.3M NaCl的; 0.002，1M ​​的Na 2 EDTA）中削减了一个低温恒温器切片包埋组织切片用蛋白酶K（0.5毫克/毫升，终浓度）和0.625％SDS中在37℃水浴中过夜15ml离心管中。 </li><li>加入1毫升饱和NaCl（5米）的消化混合物，剧烈摇晃15秒。 </li><li>离心在1100×g离心15分钟，在室温下。 </li><li>上清转移到一个新的15ml离心管中，加两卷100％的乙醇。 </li><li>通过倒转试管6-8次混匀。离心机在5000×g离心60分钟，在4℃下以收集沉淀的DNA。 </li><li>用70％乙醇洗两次DNA沉淀。 CENTRifuge在5000 XG为每次收集沉淀15分钟。 </li><li>溶于100-400微升TE缓冲液中的DNA在室温下在振荡器上过夜。的DNA产量是5至200微克（50-500纳克/微升之间最终浓度）取决于组织的大小</li></ol></li><li>提取福尔马林固定，石蜡包埋（FFPE）正常或恶性组织薄切片（10-20微米）的DNA。 <ol><li>孵化石蜡切片在1毫升二甲苯在1.5ml微量离心管中室温下每次10分钟两次，以解除paraffinize。通过在13000×g离心纺丝5分钟在室温下收集的组织切片。 </li><li>孵育在1ml 100％乙醇部分两次在室温下，每次以除去残余的二甲苯10分钟。通过在13000×g离心纺丝5分钟在室温下收集的组织切片。石蜡是完全在这一点上溶解并只将组织部分的剩余量。 </li><li>空气干燥，在室温TE的章节mperature 10-15分钟。 </li><li>使0.5毫克/毫升蛋白酶K溶液用1×PCR缓冲液（10×从PCR缓冲液稀释无核酸酶的水）。添加蛋白酶K溶液的样品中的50-100微升最终体积孵育过夜，在37℃。 </li><li>加热在95℃下对样品进行10分钟以灭活蛋白酶K. 注意：在这个步骤中，样本DNA溶解在PCR缓冲液，并且可以在步骤直接1.2和1.3被使用。的DNA产量是0.5至20微克（10-200纳克/微升之间最终浓度）取决于组织的大小。 </li></ol></li></ol> 1.2）DNA的质量评价<ol><li>混合0.25微升Taq DNA聚合酶用45微升主混合物的从在一个PCR管商业梯试剂盒。 </li><li>添加从1.1.1.7或1.1.2.5制备5微升DNA导入PCR管并通过上下抽吸拌匀至少5倍。 </li><li>使用以下条件来扩增该DNA：95℃7分钟;接着45秒的35个循环的95℃，45秒60℃，和90秒72℃;然后72℃10分钟，并保持在15℃。 </li><li>准备在TBE液（Tris /硼酸盐/ EDTA）在2％琼脂糖凝胶。 </li><li>混合20微升与4μl的6×上样染料PCR反应，并装载到2％琼脂糖凝胶。 </li><li>染色用0.5微克/毫升溴化乙锭溶液中的琼脂糖凝胶和检测PCR产物用凝胶想象系统。注意：得到5- PCR产物，在100，200，300，400，和600碱基对大小的样品将被继续产生的VDJ扩增子。 小心：溴化乙锭是一种强效的诱变剂。戴防护装备， 如手套非常谨慎，请处理和处置成每个机构的指导方针特定的容器。 </li></ol> 1.3）VDJ PCR 1.3.1）的框架区1中扩增重组的IgH VDJ段（IgVHFR1） <ol><li>从管标签混合45μl反应混合液编为“混合2”商业体细胞超变异IGH检测方法的凝胶检测试剂盒的，0.25微升Taq DNA聚合酶，和从1.1.1.7或1.1.2.5在PCR管制备5μl的样品DNA。 </li><li>使用以下条件来扩增该DNA：95℃7分钟;接着45秒的35个循环的95℃，45秒60℃，和90秒72℃;然后72℃10分钟，并保持在15℃。 </li><li>解决整个PCR产物通过电泳在2％琼脂糖凝胶。 </li><li>染色用0.5微克/毫升溴化乙锭溶液中的琼脂糖凝胶和检测PCR产物用凝胶成像系统。单克隆扩增，预计用310-380基点的大小范围。 </li><li>切下含有310-380碱基之间的单克隆扩增的凝胶部分。 </li><li>使用根据制造商的协议标准的凝胶提取试剂盒纯化从切下的凝胶中的DNA。注意：对于FR1片段可以得到的样品，没有必要充分IgV结构HFR2和IgVHFR3。 </li></ol> 1.3.2）的框架区2中扩增重组的IgH VDJ段（IgVHFR2） <ol><li>从标记为从1.1.1.7或1.1.2.5在PCR管准备凝胶检测试剂盒“B管”商业IgH基因克隆测定的，0.25微升Taq DNA聚合酶，和5μl样本DNA的管混合45微升主混合物。 </li><li>使用以下条件来扩增该DNA：95℃7分钟;接着45秒的35个循环的95℃，45秒60℃，和90秒72℃;然后72℃10分钟，并保留在15℃。 </li><li>解决整个PCR产物通过电泳在2％琼脂糖凝胶。 </li><li> 0.5微克/毫升溴化乙锭染色进行可视化的PCR产物（多个）。单克隆扩增子可以由250-295基点的尺寸范围内观察到。 </li><li>切下含有250-295碱基之间的单克隆扩增的凝胶部分。 </li><li>使用标准的凝胶中提取纯化从凝胶切除DNA根据制造商的协议离子试剂盒。 </li></ol> 1.4）优化VDJ PCR <ol><li>使用下面的修饰的PCR条件IgVHFR1和IgVHFR2使用次优的DNA扩增：95℃7分钟，40个循环的95℃60秒，60℃60秒，72℃90秒，在最后的延伸72℃下进行10分钟。 </li></ol> 2. VDJ扩增子文库制备和测序 2.1）文库制备 2.1.1）末端修复<ol><li>从1.3传输的VDJ PCR产物成PCR管并从DNA样品制备试剂盒中添加悬浮缓冲液，使体积达60微升。 </li><li>加入40微升末端修复混合的DNA样品制备试剂盒调匀。 </li><li>孵育在30℃下进行30分钟的反应中预热的热循环仪（30℃）与预先加热盖在100℃下。 </li><li>混合136微升磁珠和24微升的PCR克RADE水首先在1.5ml管中，然后传送整个最终修复反应从2.1.1.3至1.5毫升管，并与小珠溶液充分混合。 </li><li>在磁性支架2分钟地方管，以允许从溶液中珠的分离。吸出上清液，并用80％的新鲜制备的乙醇的珠两次，而该管上的磁性支架上。 </li><li>完全吸出乙醇溶液，并允许珠粒空气干燥15分钟，在室温下搅拌15分钟。 </li><li>以管从磁力架，重悬珠在17.5微升再悬浮缓冲液。 </li><li>地方管重新安装到磁静置2分钟，以从所述重悬缓冲液单独的珠。除去重悬浮缓冲液（现在最终修复产物再悬浮于重悬浮缓冲液）到一个干净的PCR管。 </li></ol> 2.1.2）A尾<ol><li>添加12.5微升拖尾混合到含有末端修复产品中的PCR管调匀。 </LI&gt; <li>孵育将该PCR管在37℃下30分钟在预先加热的热循环仪（37℃）与预先加热盖在100℃下。 </li></ol> 2.1.3）接头连接<ol><li>加入2.5微升再悬浮缓冲液，2.5微升式反应，以及2.5微升DNA适配器指数进入尾反应。 </li><li>孵育在30℃进行30 MINL反应在预加热的热循环仪（30℃）与预先加热盖在100℃下。 </li><li>添加5微升站连接缓冲液到每个反应调匀。 </li><li>拌42.5微升充分混合磁珠清洁以下步骤2.1.1.5至2.1.1.8反应。加入50微升重悬浮缓冲液以洗脱衔接子 - 连接产物。 </li><li>通过使用50微升充分混合AMPure XP珠粒清洗衔接子连接的产物进行第二次，和洗脱在25μl的重悬缓冲液的产品到一个干净的PCR管。 </li></ol> 2.1.4）扩增DNA片段<ol><li>添加5微升的PCR引物鸡尾酒和25微升的PCR主混合物含有衔接子连接的产物，将该PCR管并充分混合。 </li><li>在一个预编程热循环具有以下条件进行扩增：98℃30秒; 10个循环的98℃，10秒，60℃30秒和72℃30秒;然后72℃5分钟，并保持在10℃。 </li><li>清理反应用50μl的充分混合的磁珠，并且洗脱最终产品在30微升缓冲液重悬。 </li></ol> 2.1.5）库验证<ol><li>量化，使用制造商的协议荧光计的最终产品。最终库浓度为2.5〜20纳克/微升之间。 </li><li>通过使用具有根据制造商的协议高灵敏度的DNA芯片的分析仪器评价最终产品的质量。期望与任一IGVHFR1，IGVHFR2或IGVHFR3预期大小的单条带。电子库产物的xpected大小等于原始的VDJ扩增子的大小加上适配器（〜125 bp）的大小。 </li></ol> 2.2）的VDJ-PCR库池和测序<ol><li>使用下列公式计算每个文库级分的摩尔浓度：纳米= [（毫微克/ 1,000）/ bp的* 660]×10 9，其中n是在步骤2.1.5.1测定的VDJ-PCR文库的浓度和基点是峰在VDJ-PCR库的大小测定步骤2.1.5.2。 </li><li>稀释库为2nM（10微升总）与无DNA酶的水。 </li><li>结合将10μl稀释为2nM库一起放入1.5ml试管。 </li><li>添加的PhiX尖峰控制等体积为1.5 ml管。 </li><li>加载游泳池晚上7点集中到流动池。 </li><li>测序使用根据制造商的协议的配对末端150循环音序器的库。 </li></ol> 3.数据分析注意：SUMM在本节中使用的生物信息学脚本进制可以发现作为辅助代码文件。 3.1）校准和QC <ol><li>地图配对末端测序读针对人IGH V，D和J区数据库使用基于“BLASTN&#39;可得自NCBI与缺口开放罚分2，1缺口延伸罚一个适于核苷酸BLAST算法从IMGT网站11下载的， 7字长，和10 -4 e值的阈值。丢弃所读一对不映射到既是IGH V及J区。 </li><li>算剩余的读对具有IGH V和J的映射，以获得匹配的VJ组合在所有读取从样品上的测序运行得到的对的频率。计数读一对，如果它被映射到一个IGH V和J基因，然后将其等位基因增加相应的计用于特定VJ组合。 </li><li>排名从3.1.2高得到的每个重组VDJ区的数美国东部时间到最低。该VDJ地区具有最高的读取次数被定义为主要的重排组合。 </li><li>丢弃，覆盖在3.1.3中标识的域大的重排的小于35％比对的序列。 </li></ol> 3.2）SHM档案鉴定<ol><li>计算所有SHM模式在整个读取步骤3.1.3。定义每个SHM图案作为亚克隆。 </li><li>计数分别对应于不同的唯一SHM模式，将亚克隆的数量，和对读取映射到各个亚克隆该号码。 </li></ol>结果3.3）图形表示<ol><li>执行使用基于邻域加入与R包“猿”的方法，根据制造商的协议及其对应的SHM模式的亚克隆系统发育分析。计算从各不同路线的距离矩阵来重新创建亚克隆系统发育植根于V的种系序列Ĵ组合为每个配对样本集的问题。 </li><li>使用每个亚克隆作为字符载体的核苷酸序列，并计算在主要的VJ重排为诊断和使用Levenshtein距离量度相应复发样品，其中数学2路线之间的距离为d 的x，y给出的所有亚克隆之间的近似串距离（I，J）其中d 的x，y（I，J）= 1，如果I≠j和否则为0，然后求和这个函数在字符串的长度相对应的核苷酸序列i和j。 </li><li>图形方式显示所产生的亚克隆的距离及其相应的亚克隆计数以下列两种方式中的矩阵： <ol><li>使用ř包“。MASS”根据制造商的协议来多维尺度应用于亚克隆距离矩阵和生成的二维坐标原理图，然后将其与对数作图沿的亚克隆计为圆的半径。 </li><li>构造基于所述Levenshtein距离，其中该顶点对应于从主要VJ重排所产生的每一个不同的亚克隆无向图。 注意：如果两个不同的克隆之间的距离等于一个则存在这两个顶点之间的边。如果距离大于一，那么就没有边缘连接它们。 </li><li>使用tkplot函数在“IGRAPH”R包具有根据制造商的协议的镰河合布局的绘制3.3.3.2的曲线图。顶点的半径成正比的克隆映射到它的总数量的立方根和遮光使用红色和蓝色的范围来表示的克隆的，要么属于诊断（蓝色）或复发（红色）的样品的比例。 </li></ol></li></ol> 3.4）异质性（熵）测量<ol><li>检查的数量和亚克隆计数体细胞突变的主要VJ重排的每个个体出现的图案配对设置的样品。 </li><li>计算经验熵和残余经验性熵调整不同的克隆在每个样品中的使用标准直方图基于熵估计的数量。 </li></ol>

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The authors have no conflicts of interest to disclose.

因为几乎无限数量的通过的VDJ重排和SHM在人B细胞的IGH轨迹编码序列信息迭代的，检查整个IGH剧目由高通量深测序证明是划定克隆一种有效和全面的方式和子克隆B细胞群。此外，该策略可用于通过比较沿该疾病的不同阶段收集的患者样本的克隆和子克隆构研究B细胞肿瘤的发展，缓解，复发的克隆演变路径。虽然很容易在实验室环境中使用可商购的引物进行从初级样品DNA重排的VDJ序列的扩增，扩增?...

癌症是一种克隆性疾病。自从三十年前，当彼得C.诺维尔提出了癌症克隆演化模型1，许多研究都试图肿瘤样本中剖析克隆群体和重建克隆扩增和发展模式，背后的肿瘤发生过程中2。最近，全基因组测序，使调查人员深吸看克隆异质性和演化3,4。然而，由于缺乏在许多细胞类型易于处理的标记物，它是很难推断精确的克隆构和进化路径。幸运的是在成熟B细胞，许多淋巴组织的恶性肿瘤，包括DLBCL，源于自然的克隆性标志。响应于抗原刺激，每个B细胞可以从这些区段的大池通过加入为V H（可变的），一个D（多样性）形成单个生产性的IgH VDJ序列和歼轰（接合）片段一起。在这个公关ocess，原始序列的小部分可以被删除和附加非模板的核苷酸可以添加到创建一个唯一的VDJ重排。这个特定的VDJ重排可在该B细胞的所有子代继承，因此标注个体成熟的B细胞和其后代5。此外，SHM发生在随后的生发中心（GC）的反应重组的VDJ序列引入另外的突变的抗体池的膨胀和抗体亲和6的提高。因此，通过比较和对比已经经过这些过程淋巴瘤样品的VDJ和SHM图案，肿瘤内的异质性，可以划定及疾病的克隆演变路径可以被推断出来。 先前，VDJ重排和SHM可以通过PCR鉴定扩增重组的区域，克隆PCR产物，并随后Sanger测序来获得序列信息。这种方法我的低通量和低产量，检索整个重组的VDJ剧目只有一个非常小的部分，并阻碍给定样品中的克隆群的整体表现的表征。一种改进的方法是由来自VDJ PCR产物生成索引NGS测序文库并进行PE 2x150碱基序列，获得半数以上一万元左右的样品重组VDJ序列创建。此外，定制的管道的开发是为了进行质量控制（QC），对齐，滤波器的VDJ测序读数，以确定每次读取的重排和SHMS，以及执行系统发育分析每个样品的克隆构。此外，一种新的方法已经建立，以进一步表征克隆演变模式在不同疾病阶段收集的样本。 我们应用这种技术来DLBCL患者样本。 DLBCL是频繁复发的非霍奇金淋巴瘤的最多一个第一种侵袭性IRD的患者7。 DLBCL复发通常发生早，在2至3年的初步诊断，但也有一些确实发生5年8之后。愈后复发的患者差，只有10％实现3年无进展生存期，由于有限的治疗选择。这是基础，迫切需要新的方法来治疗DLBCL复发9,10。然而，DLBCL复发相关的分子机制仍知之甚少。具体地，克隆的异质性，在诊断和克隆演变的DLBCL复发发育过程中的作用是当前未表征，因此很难确定准确的和有用的生物标志物来预测复发。为了解决这些问题，我们运用我们的VDJ测序的方法对多对匹配的初步诊断复发DLBCL样本对。复发的两个不同的克隆的进化方案从克隆构的诊断和复发SAMP之间的比较出现莱这表明多个分子机制可能参与了DLBCL复发。

<table border="0" cellpadding="0" cellspacing="0" width="841">
	<tbody>
		<tr height="60">
			<td height="60" style="height:60px;width:359px;">Ethanol</td>
			<td style="width:163px;">VWR</td>
			<td style="width:123px;">89125-170</td>
			<td style="width:197px;">200 proof, for molecular biology</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:359px;">TE buffer</td>
			<td>Life Technologies</td>
			<td>12090-015</td>
			<td style="width:197px;">10 mM Tris&#183;Cl, pH 8.0; 10mM EDTA</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Xylenes</td>
			<td>VWR</td>
			<td>EM-XX0055-6</td>
			<td>500 ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Proteinase K&#160;</td>
			<td style="width:163px;">Life Technonogies</td>
			<td>25530-015</td>
			<td>100 mg</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Deoxynucleotide triphosphate (dNTP) Solution Mix</td>
			<td>Promega</td>
			<td style="width:123px;">U1515</td>
			<td style="width:197px;">10 mM each nucleotide</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10x PCR Buffer&#160;</td>
			<td>Roche</td>
			<td>11699105001</td>
			<td>Without MgCl2</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:359px;">AmpliTaq Gold DNA Polymerase with Gold Buffer and MgCl2</td>
			<td style="width:163px;">Life Technonogies</td>
			<td style="width:123px;">4311806</td>
			<td>50 &#181;l at 5 U/&#181;l</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">Specimen Control Size Ladder</td>
			<td style="width:163px;">Invivoscribe Technologies&#160;</td>
			<td>2-096-0020</td>
			<td>33 reactions</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:359px;">IGH Somatic Hypermutation Assay v2.0 - Gel Detection</td>
			<td style="width:163px;">Invivoscribe Technologies&#160;</td>
			<td style="width:123px;">5-101-0030</td>
			<td style="width:197px;">33 reactions, Mix 2 for IGVHFR1 detection</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;">IGH Gene Clonality Assay - Gel Detection</td>
			<td style="width:163px;">Invivoscribe Technologies&#160;</td>
			<td>1-101-0020</td>
			<td style="width:197px;">33 reactions, Tube B for IGVHFR2 detection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GoTaq Flexi DNA Polymerase</td>
			<td>Promega</td>
			<td style="width:123px;">M8291</td>
			<td>20 &#181;l at 5 U/&#181;l</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">10X Tris-Borate-EDTA (TBE) buffer</td>
			<td>Corning (cellgro)</td>
			<td style="width:123px;">46-011-CM</td>
			<td style="width:197px;">6x1 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50X TAE BUFFER&#160;&#160;</td>
			<td>VWR</td>
			<td>101414-298</td>
			<td style="width:197px;">1 L</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethidium bromide solution</td>
			<td>Sigma-Aldrich</td>
			<td>E1510</td>
			<td style="width:197px;">10 mg/ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">25 bp DNA Ladder</td>
			<td>Life Technologies</td>
			<td>10597011</td>
			<td style="width:197px;">1 &#181;g/&#181;l</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">100 bp DNA Ladder</td>
			<td>Life Technologies</td>
			<td>15628-019</td>
			<td style="width:197px;">1 &#181;g/&#181;l</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">UltraPure Agarose</td>
			<td>Life Technologies</td>
			<td>16500-500</td>
			<td style="width:197px;">500 g</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">40% Acrylamide/Bis Solution</td>
			<td>Bio-Rad Laboratories</td>
			<td>1610144</td>
			<td style="width:197px;">500 ml</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">QIAquick Gel Extraction Kit</td>
			<td>Qiagen</td>
			<td>28704</td>
			<td style="width:197px;">50 columns</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:359px;">Agencourt AMPure XP</td>
			<td style="width:163px;">Beckman Coulter</td>
			<td style="width:123px;">A63881</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Qubit dsDNA High Sensitivity Assay Kit</td>
			<td style="width:163px;">Life Technologies</td>
			<td style="width:123px;">Q32854</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">High Sensitivity DNA Kit</td>
			<td style="width:163px;">Agilent Technologies</td>
			<td style="width:123px;">5067-4626</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:359px;">2100 Bioanalyzer</td>
			<td style="width:163px;">Agilent Technologies</td>
			<td></td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">PhiX Control v3</td>
			<td style="width:163px;">Illumina</td>
			<td>FC-110-3001</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">MiSeq</td>
			<td style="width:163px;">Illumina</td>
			<td></td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Qubit 2.0 Fluorometer</td>
			<td>Life Technologies</td>
			<td>Q32872</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:359px;">Resuspension buffer (Illumina TruSeq DNA Sample Preparation Kit v2)</td>
			<td style="width:163px;">Illumina</td>
			<td>FC-121-2001</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:359px;">End repair mix (Illumina TruSeq DNA Sample Preparation Kit v2)</td>
			<td style="width:163px;">Illumina</td>
			<td>FC-121-2001</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:359px;">A-tailing mix (Illumina TruSeq DNA Sample Preparation Kit v2)</td>
			<td style="width:163px;">Illumina</td>
			<td>FC-121-2001</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:359px;">Ligation mix (Illumina TruSeq DNA Sample Preparation Kit v2)</td>
			<td style="width:163px;">Illumina</td>
			<td>FC-121-2001</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:359px;">DNA adaptor index (Illumina TruSeq DNA Sample Preparation Kit v2)</td>
			<td style="width:163px;">Illumina</td>
			<td>FC-121-2001</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:359px;">Stop ligation buffer (Illumina TruSeq DNA Sample Preparation Kit v2)</td>
			<td style="width:163px;">Illumina</td>
			<td>FC-121-2001</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:359px;">PCR primer cocktail (Illumina TruSeq DNA Sample Preparation Kit v2)</td>
			<td style="width:163px;">Illumina</td>
			<td>FC-121-2001</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:359px;">PCR master mix (Illumina TruSeq DNA Sample Preparation Kit v2)</td>
			<td style="width:163px;">Illumina</td>
			<td>FC-121-2001</td>
			<td style="width:197px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:359px;">Magnetic stand</td>
			<td style="width:163px;">Life Technologies</td>
			<td align="right">4457858</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:359px;">Gel imaging system</td>
			<td style="width:163px;">Bio-Rad Laboratories</td>
			<td>170-8370</td>
			<td></td>
		</tr>
	</tbody>
</table>

vdj-seq: deep sequencing analysis of rearranged immunoglobulin heavy chain gene to reveal clonal evolution patterns of b cell lymphoma

了解肿瘤的克隆是了解参与肿瘤的发生和疾病进展的机制是至关重要的。此外，理解内肿瘤发生响应于特定微环境或治疗方法，所述组合物克隆变化可能会导致更复杂的和有效的方法的设计消除肿瘤细胞。然而，跟踪肿瘤克隆亚种群已具有挑战性，由于缺乏可区分的标记。为了解决这个问题，一个VDJ-SEQ协议创建通过利用VDJ重组和体细胞突变（SHM），B细胞淋巴瘤两个独特的功能，以跟踪弥漫性大B细胞淋巴瘤（DLBCL）复发的克隆演变图案。 在这个协议中，新一代测序（NGS）库，索引潜在的扩增重排的免疫球蛋白重链（IgH重）VDJ区，构建由对初步诊断和复发DLBCL SAMP的LES。每个样品平均超过五十万的VDJ序列测序，同时含有的VDJ重排和SHM信息后获得。此外，自定义生物信息学管道开发，以充分利用序列信息FR3区​​域，VDJ剧目这些样品中的表征。此外，该管道允许重建和个体肿瘤的克隆构，这使得肿瘤的诊断和演绎的诊断和复发性肿瘤对之间克隆演变模式内的克隆异质性的检查的比较。当应用这一分析的几个诊断复发对，我们发现关键证据多个独特的肿瘤进化模式可能会导致DLBCL复发。此外，这种方法可以扩展为其它临床方面，如识别微小残留病，监测复发进展和治疗反应和免疫repertoir的调查ES在非淋巴瘤上下文。

VDJ测序（VDJ-SEQ），包括DNA提取的整体过程，重组的VDJ区域扩增和纯化，测序文库构建，读取处理，和系统发育分析，表示在图1中。按常规5-200微克的DNA可以从被检索冷冻固体组织切片或从福尔马林固定的石蜡包埋组织切片0.5-20微克的DNA。视质量，重排的模式，和个体样本SHM程度，可以得到各种的VDJ PCR产物来自不同样品（图2），包括IGVHFR1（310-380碱基），FR2（250-295碱基?...

Watch this Scientific Journal Video about VDJ-Seq: Deep Sequencing Analysis of Rearranged Immunoglobulin Heavy Chain Gene to Reveal Clonal Evolution Patterns of B Cell Lymphoma at JoVE.com

VDJ-Seq: Deep Sequencing Analysis of Rearranged Immunoglobulin Heavy Chain Gene to Reveal Clonal Evolution Patterns of B Cell Lymphoma

This protocol describes an approach to interrogate the recombined immunoglobulin heavy chain VDJ regions of lymphomas by deep-sequencing and retrieve VDJ rearrangement and somatic hypermutation status to delineate clonal architecture of individual tumor. Comparing clonal architecture between paired diagnosis and relapse samples reveals lymphoma relapse clonal evolution modes.

VDJ-SEQ：重排免疫球蛋白重链基因的深度测序分析，以揭示的B细胞淋巴瘤克隆演变模式

Understanding tumor clonality is critical to understanding the mechanisms involved in tumorigenesis and disease progression. In addition, understanding ...

vdj-seq-deep-sequencing-analysis-rearranged-immunoglobulin-heavy

Research

JoVE Journal

Medicine

26.5K 次观看.  Weill Cornell Medical College. This protocol describes an approach to interrogate the recombined immunoglobulin heavy chain VDJ regions of lymphomas by deep-sequencing and retrieve VDJ rearrangement and somatic hypermutation status to delineate clonal architecture of individual tumor. Comparing clonal architecture between paired diagnosis and relapse samples reveals lymphoma relapse clonal evolution modes. 

视频: VDJ-Seq: Deep Sequencing Analysis of Rearranged Immunoglobulin Heavy Chain Gene to Reveal Clonal Evolution Patterns of B Cell Lymphoma

Dissection of Human Vitreous Body Elements for Proteomic Analysis

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions between vitreous substructures and the retina underlie several vitreoretinal diseases, including retinal tear and detachment, macular pucker, macular hole, age-related macular degeneration, vitreomacular traction, proliferative vitreoretinopathy, proliferative diabetic retinopathy, and inherited vitreoretinopathies. 1,2 The molecular composition of the vitreous substructures is not known. Since the vitreous body is transparent with limited surgical access, it has been difficult to study its substructures at the molecular level. We developed a method to separate and preserve these tissues for proteomic and biochemical analysis. The dissection technique in this experimental video shows how to isolate vitreous base, anterior hyaloid, vitreous core, and vitreous cortex from postmortem human eyes. One-dimensional SDS-PAGE analyses of each vitreous component showed that our dissection technique resulted in four unique protein profiles corresponding to each substructure of the human vitreous body. Identification of differentially compartmentalized proteins will reveal candidate molecules underlying various vitreoretinal diseases.

This video shows an effective technique for differentiating and dissecting the various semi-transparent structures of the human vitreous body in post mortem eyes.

人类蛋白质组分析玻璃体元素剖析

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions ...

科研

医学

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

在免疫缺陷小鼠的建立和传播人类视网膜母细胞瘤肿瘤

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Production and Detection of Reactive Oxygen Species (ROS) in Cancers

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive molecules contain an oxygen and include H2O2 (hydrogen peroxide), NO (nitric oxide), O2- (oxide anion), peroxynitrite (ONOO-), hydrochlorous acid (HOCl), and hydroxyl radical (OH-).
Oxidative species are produced not only under pathological situations (cancers, ischemic/reperfusion, neurologic and cardiovascular pathologies, infectious diseases, inflammatory diseases 1, autoimmune diseases 2, etc&hellip;) but also during physiological (non-pathological) situations such as cellular metabolism 3, 4. Indeed, ROS play important roles in many cellular signaling pathways (proliferation, cell activation 5, 6, migration 7 etc..). ROS can be detrimental (it is then referred to as "oxidative and nitrosative stress") when produced in high amounts in the intracellular compartments and cells generally respond to ROS by upregulating antioxidants such as superoxide dismutase (SOD) and catalase (CAT), glutathione peroxidase (GPx) and glutathione (GSH) that protects them by converting dangerous free radicals to harmless molecules (i.e. water). Vitamins C and E have also been described as ROS scavengers (antioxidants).
Free radicals are beneficial in low amounts 3. Macrophage and neutrophils-mediated immune responses involve the production and release of NO, which inhibits viruses, pathogens and tumor proliferation 8. NO also reacts with other ROS and thus, also has a role as a detoxifier (ROS scavenger). Finally NO acts on vessels to regulate blood flow which is important for the adaptation of muscle to prolonged exercise 9, 10. Several publications have also demonstrated that ROS are involved in insulin sensitivity 11, 12.
Numerous methods to evaluate ROS production are available. In this article we propose several simple, fast, and affordable assays; these assays have been validated by many publications and are routinely used to detect ROS or its effects in mammalian cells. While some of these assays detect multiple ROS, others detect only a single ROS.

Production and Detection of Reactive Oxygen Species ROS in Cancers

Here we propose simple methods to test and evaluate the presence of reactive oxygen species in cells.

在癌症的生产和检测活性氧（ROS）的

Reactive oxygen species include a number of molecules that damage DNA and RNA and oxidize proteins and lipids (lipid peroxydation). These reactive ...

DNA Vector-based RNA Interference to Study Gene Function in Cancer

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing1, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible2,3. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression. 
We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. 
Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer4,5, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution.

RNA interference (RNAi) possesses many advantages over gene knockout and has been broadly used as a tool in gene functional studies. The invention of DNA vector-based RNAi technology has made long term and inducible gene knockdown possible, and also increased the feasibility of gene silencing in vivo.

基于DNA载体的RNA干扰在癌症研究基因功能

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA ...

Deep Neuromuscular Blockade Leads to a Larger Intraabdominal Volume During Laparoscopy

Shoulder pain is a commonly reported symptom following laparoscopic procedures such as myomectomy or hysterectomy, and recent studies have shown that lowering the insufflation pressure during surgery may reduce the risk of post-operative pain. In this pilot study, a method is presented for measuring the intra-abdominal space available to the surgeon during laproscopy, in order to examine whether the relaxation produced by deep neuromuscular blockade can increase the working surgical space sufficiently to permit a reduction in the CO2 insufflation pressure. Using the laproscopic grasper, the distance from the promontory to the skin is measured at two different insufflation pressures: 8 mm Hg and 12 mm Hg. After the initial measurements, a neuromuscular blocking agent (rocuronium) is administered to the patient and the intra-abdominal volume is measured again. Pilot data collected from 15 patients shows that the intra-abdominal space at 8 mm Hg with blockade is comparable to the intra-abdominal space measured at 12 mm Hg without blockade. The impact of neuromuscular blockade was not correlated with patient height, weight, BMI, and age. Thus, using neuromuscular blockade to maintain a steady volume while reducing insufflation pressure may produce improved patient outcomes.

This article presents a method for measuring the working abdominal volume during laproscopic myomectomy using the surgical grasper, and applies this technique to obtain pilot data on whether deep neuromuscular blockade (NMB) can enable the use of lower insufflation pressure. Reduced insufflation pressure during laproscopic surgery has been shown to reduce post-operative pain, and thus the use of NMB during surgery may enable improved patient outcomes.

深神经肌肉阻滞信息到更大的在腹腔镜腹腔容积

Shoulder pain is a commonly reported symptom following laparoscopic procedures such as myomectomy or hysterectomy, and recent studies have shown that ...

Detection of Architectural Distortion in Prior Mammograms via Analysis of Oriented Patterns

We demonstrate methods for the detection of architectural distortion in prior mammograms of interval-cancer cases based on analysis of the orientation of breast tissue patterns in mammograms. We hypothesize that architectural distortion modifies the normal orientation of breast tissue patterns in mammographic images before the formation of masses or tumors. In the initial steps of our methods, the oriented structures in a given mammogram are analyzed using Gabor filters and phase portraits to detect node-like sites of radiating or intersecting tissue patterns. Each detected site is then characterized using the node value, fractal dimension, and a measure of angular dispersion specifically designed to represent spiculating patterns associated with architectural distortion.
Our methods were tested with a database of 106 prior mammograms of 56 interval-cancer cases and 52 mammograms of 13 normal cases using the features developed for the characterization of architectural distortion, pattern classification via quadratic discriminant analysis, and validation with the leave-one-patient out procedure. According to the results of free-response receiver operating characteristic analysis, our methods have demonstrated the capability to detect architectural distortion in prior mammograms, taken 15 months (on the average) before clinical diagnosis of breast cancer, with a sensitivity of 80% at about five false positives per patient.

Detection of Architectural Distortion in Prior Mammograms via Analysis of Oriented Patterns

We demonstrate methods for the detection of architectural distortion in prior mammograms. Oriented structures are analyzed using Gabor filters and phase portraits to detect sites of radiating tissue patterns. Each site is characterized and classified using measures to represent spiculating patterns. The methods should assist in the detection of breast cancer.

在检测之前乳房X光检查建筑失真<em&gt;通过</em面向模式的&gt;分析

We demonstrate methods for the detection of architectural distortion in prior mammograms of interval-cancer cases based on analysis of the orientation of ...

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson&#039;s Disease

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

基因-环境相互作用模型揭开帕金森病的隐形易感机制

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

注重形成：一个基于Cell的检测，以确定一个基因的致癌性

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

Unbiased Deep Sequencing of RNA Viruses from Clinical Samples

Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from clinical and biological sources. The method is unbiased and universal; it uses random primers for cDNA synthesis and requires no prior knowledge of the viral sequence content. Before library construction, selective RNase H-based digestion is used to deplete unwanted RNA &#8212; including poly(rA) carrier and ribosomal RNA &#8212; from the viral RNA sample. Selective depletion improves both the data quality and the number of unique reads in viral RNA sequencing libraries. Moreover, a transposase-based &#39;tagmentation&#39; step is used in the protocol as it reduces overall library construction time. The protocol has enabled rapid deep sequencing of over 600 Lassa and Ebola virus samples-including collections from both blood and tissue isolates-and is broadly applicable to other microbial genomics studies.

This protocol describes a rapid and broadly applicable method for unbiased RNA-sequencing of viral samples from human clinical isolates.

临床样品RNA病毒的无偏深度测序

Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from ...

Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E (sIgE)

Allergic disease is common in both adults and children. Identification of the causative allergens is significant in disease management and prevention. However, a specific immunoglobulin E (IgE) measurement system with a high price-performance ratio is lacking in mainland China, especially in the primary care hospitals. This paper describes the principle and operation procedures of using a microfluidic cartridge-based chemiluminescence system to detect allergen-specific IgE in serum. The results were compared with those from ImmunoCAP (System 1), the industrial standard, and the reproducibility of the system to detect patients sensitized to common allergens is evaluated. The results showed that in comparison with ImmunoCAP (System 1), the BioIC System (System 2) has good precision and sensitivity in detecting serum-specific IgE against various inhalant and food allergens but with a significantly lower cost. It can serve as a good alternative to System 1 in primary care hospitals in mainland China who have lower financial affordability.

Application of Biochip Microfluidic Technology to Detect Serum Allergen-specific Immunoglobulin E sIgE

This paper presents a protocol for detecting serum-specific immunoglobulin E with a microfluidic cartridge-based chemiluminescence system and the evaluation of its usefulness in allergy diagnoses.

生物芯片微流控技术在血清过敏源特异性免疫球蛋白 E (sIgE) 检测中的应用

Allergic disease is common in both adults and children. Identification of the causative allergens is significant in disease management and prevention. ...