Name: vdj-seq: deep sequencing analysis of rearranged immunoglobulin heavy chain gene to reveal clonal evolution patterns of b cell lymphoma
Uploaded: 2015-12-28T13:00:00.000+00:00
Duration: 15 min 7 s
Description: 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

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，扩增的效率在很大程度上取决于样品DNA的质量。特别是，DNA的FFPE样品中提取出低的扩增效率，而且往往只有小FR3片段可以从这些被成功地扩增样品，使它们不适合于随后的分析。由于我们在此描述的研究的目的是要了解的B细胞淋巴瘤，这是一种克隆性疾病与一个或几个主要的克隆支配整个肿瘤的克隆能力，我们只收集和测序的主要的VDJ-PCR产物。对于兴趣编目的整个B细胞克隆群体，例如研究，外周血，包含多个VDJ-PCR产物（多频带或凝胶上的涂抹）凝胶的较大部分可被切下并测序。然而，必须指出，这是不可能的捕获整个重新排列IGH剧目因为可能发生某些SHM在其中引物靶，并损害这些VDJs的扩增是重要的。此外，最近，商业法直接耦合IGH重排扩增和测序MiSeq已经出台。此法简化了样品制备过程。然而，因为不是每个肿瘤样品将能够属TE成功FR1片段，我们仍然建议包括凝胶电泳工序合并样本进行测序之前检查PCR反应的产物。 测序150碱基对的VDJ片段（共300bp的序列信息）的两端具有以下优点:1）300bp的测序可以覆盖多数FR1和整个FR2片段，提供充足的序列信息用于识别的VDJ重排和体细胞超突变; 2）该平台提供快速测序​​周转时间完成下48小时的全部300个循环的VDJ测序的; 3）每次运行可复用多达12个样品，仍然实现了每个样本输出大约一百万配对末端读取。因为克隆携带相同的VDJ重排，特别是在B细胞淋巴瘤样品之间的高序列相似性，是至关重要的尖峰在20-50％PhiX测序运行期间，以允许精确地确定基主叫矩阵。近日，MiSeq软件已经更新来解决这个问题。它建议使用少至5％PhiX尖峰在用于低复杂文库测序。然而，我们的测序设备经历了由制造商，将危及低复杂度的测序结果提供的PhiX尖峰控制的DNA不一致的浓度。因此，在目前的实践中，我们仍然使用10-20％PhiX穗中的VDJ测序。对于整个外周血B-细胞的剧目，通常包含大量的不同的VDJ安排测序，有可能减少PhiX尖峰中的量。尽管确定的每个样品的克隆和亚克隆的数量正具有读测序的数量，一半相关的每个样品的配对末端百万读取足以比较克隆的结构样本之间并推断在不同收集的样品之间克隆演变型态同一患者 12的疾病阶段。它是可能是PE 150 bp的测序可能无法达到那些长期FR1片段（ 即 350-380基点）的足够的覆盖范围。在一些情况下，D段的序列信息可能不被收集或可能没有在V段由SHMS分化亚克隆足够的序列信息。然而，随着的测序技术的进步，已经成为可能的测序时间更长，覆盖整个FR1扩增子。 通过B细胞的IGH剧目的测序是可能的跟踪单个B细胞克隆的膨胀和推断克隆演变过的过程从诊断复发。与使用分析上由SHM型态产生亚克隆的种群，我们能够区分两种模式癌症进展复发通过检查其系统发育。通过多维尺度图和无向图基础上，莱文斯坦距离进一步最新的图形表示异体瓦特我们认识1）各肿瘤的亚克隆重的组合物，2）亚克隆如何个体都与彼此，以及3）如何在疾病进展期间的每个亚克隆演变。另外，统计分析表明，这些系统发育结构显然不同，且该亚克隆种群差异作为关于他们的异质性。最后，通过VDJ测序获得的克隆演变轨迹可以很好的相关性与遗传演变和特点是无论是外显子组或定向重测序12。因此，结合这两种方法都有在淋巴瘤和淋巴瘤复发，以确定司机突变的可能性除了定义B细胞IGH剧目和微量淋巴瘤进展克隆演变，所述的VDJ-SEQ方法可以潜在地用作一个高度敏感的方法，用于跟踪微小残留疾病，监测肿瘤反应疗法，并有可能识别存在的药物抗性克隆。类似的方法也可以应用于给T细胞来映射T细胞受体库，并且可以被用于探测免疫监视响应于癌症。此外，VDJ-SEQ也可使用可得自Hanna等人的引物小鼠模型13 进行 。可以预见，通过在未来几年这种方法收集的信息可导致更好地了解肿瘤发展的动力学以及扩大我们的免疫系统的知识及其在肿瘤侵袭保卫我们的身体的作用。

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><tbody><tr><td>Ethanol</td><td>VWR</td><td>89125-170</td><td>200 proof, for molecular biology</td></tr><tr><td>TE buffer</td><td>Life Technologies</td><td>12090-015</td><td>10 mM Tris&amp;middot;Cl, pH 8.0; 10 mM EDTA</td></tr><tr><td>Xylenes</td><td>VWR</td><td>EM-XX0055-6</td><td>500 ml</td></tr><tr><td>Proteinase K&amp;nbsp;</td><td>Life Technonogies</td><td>25530-015</td><td>100 mg</td></tr><tr><td>Deoxynucleotide triphosphate (dNTP) Solution Mix</td><td>Promega</td><td>U1515</td><td>10 mM each nucleotide</td></tr><tr><td>10x PCR Buffer&amp;nbsp;</td><td>Roche</td><td>11699105001</td><td>Without MgCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>AmpliTaq Gold DNA Polymerase with Gold Buffer and MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Life Technonogies</td><td>4311806</td><td>50 &amp;micro;l at 5 U/&amp;micro;l</td></tr><tr><td>Specimen Control Size Ladder</td><td>Invivoscribe Technologies&amp;nbsp;</td><td>2-096-0020</td><td>33 reactions</td></tr><tr><td>IGH Somatic Hypermutation Assay v2.0 - Gel Detection</td><td>Invivoscribe Technologies&amp;nbsp;</td><td>5-101-0030</td><td>33 reactions, Mix 2 for IGVHFR1 detection</td></tr><tr><td>IGH Gene Clonality Assay - Gel Detection</td><td>Invivoscribe Technologies&amp;nbsp;</td><td>1-101-0020</td><td>33 reactions, Tube B for IGVHFR2 detection</td></tr><tr><td>GoTaq Flexi DNA Polymerase</td><td>Promega</td><td>M8291</td><td>20 &amp;micro;l at 5 U/&amp;micro;l</td></tr><tr><td>10X Tris-Borate-EDTA (TBE) buffer</td><td>Corning (cellgro)</td><td>46-011-CM</td><td>6x1 L</td></tr><tr><td>50x TAE BUFFER&amp;nbsp;&amp;nbsp;</td><td>VWR</td><td>101414-298</td><td>1 L</td></tr><tr><td>Ethidium bromide solution</td><td>Sigma-Aldrich</td><td>E1510</td><td>10 mg/ml</td></tr><tr><td>25 bp DNA Ladder</td><td>Life Technologies</td><td>10597011</td><td>1 &amp;micro;g/&amp;micro;l</td></tr><tr><td>100 bp DNA Ladder</td><td>Life Technologies</td><td>15628-019</td><td>1 &amp;micro;g/&amp;micro;l</td></tr><tr><td>UltraPure Agarose</td><td>Life Technologies</td><td>16500-500</td><td>500 g</td></tr><tr><td>40% Acrylamide/Bis Solution</td><td>Bio-Rad Laboratories</td><td>1610144</td><td>500 ml</td></tr><tr><td>QIAquick Gel Extraction Kit</td><td>Qiagen</td><td>28704</td><td>50 columns</td></tr><tr><td>Agencourt AMPure XP</td><td>Beckman Coulter</td><td>A63881</td><td></td></tr><tr><td>Qubit dsDNA High Sensitivity Assay Kit</td><td>Life Technologies</td><td>Q32854</td><td></td></tr><tr><td>High Sensitivity DNA Kit</td><td>Agilent Technologies</td><td>5067-4626</td><td></td></tr><tr><td>2100 Bioanalyzer</td><td>Agilent Technologies</td><td></td><td></td></tr><tr><td>PhiX Control v3</td><td>Illumina</td><td>FC-110-3001</td><td></td></tr><tr><td>MiSeq</td><td>Illumina</td><td></td><td></td></tr><tr><td>Qubit 2.0 Fluorometer</td><td>Life Technologies</td><td>Q32872</td><td></td></tr><tr><td>Resuspension buffer (Illumina TruSeq DNA Sample Preparation Kit v2)</td><td>Illumina</td><td>FC-121-2001</td><td></td></tr><tr><td>End repair mix (Illumina TruSeq DNA Sample Preparation Kit v2)</td><td>Illumina</td><td>FC-121-2001</td><td></td></tr><tr><td>A-tailing mix (Illumina TruSeq DNA Sample Preparation Kit v2)</td><td>Illumina</td><td>FC-121-2001</td><td></td></tr><tr><td>Ligation mix (Illumina TruSeq DNA Sample Preparation Kit v2)</td><td>Illumina</td><td>FC-121-2001</td><td></td></tr><tr><td>DNA adaptor index (Illumina TruSeq DNA Sample Preparation Kit v2)</td><td>Illumina</td><td>FC-121-2001</td><td></td></tr><tr><td>Stop ligation buffer (Illumina TruSeq DNA Sample Preparation Kit v2)</td><td>Illumina</td><td>FC-121-2001</td><td></td></tr><tr><td>PCR primer cocktail (Illumina TruSeq DNA Sample Preparation Kit v2)</td><td>Illumina</td><td>FC-121-2001</td><td></td></tr><tr><td>PCR master mix (Illumina TruSeq DNA Sample Preparation Kit v2)</td><td>Illumina</td><td>FC-121-2001</td><td></td></tr><tr><td>Magnetic stand</td><td>Life Technologies</td><td>4457858</td><td></td></tr><tr><td>Gel imaging system</td><td>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碱基对），和FR3（70-120基点）。从其中仅FR3片段可从VDJ PCR获得的样品排除在剩余研究，由于短产品不用于下游数据分析目的提供的序列信息足够量。只有试样的一个主要FR1或FR2 PCR产物可在th被可视化Ë琼脂糖凝胶进行处理，以产生测序文库。主要PCR产物凝胶纯化后的产率范围为10〜50纳克在总和。 整个纯化的PCR产物用于随后的文库构建用于测序文库。适配器连接后，每个样品获得其自己的唯一索引。在文库扩增，10个循环的PCR进行可能产生超过30纳克作为最终​​库产品。该库的质量是由生物分析仪进行访问。 如图3，没有与〜125碱基的尺寸移位库样品中的一种单一产品形成原始的VDJ PCR扩增子产物表示附加适配器的。对于每个测序运行，5-10库均在7μM汇集起来。此外，由于VDJ PCR产物之间的高序列相似性，该库池用50％PhiX混合尖峰中，以确保在运行和正确识别碱的复杂性每个测序循环后更多。文库的DNA片段进行测序的150bp的两端，其允许的序列信息跨越性病和DJ路口为FR1片段足够量的检索，和SHM突变模式的系统发育分析。 先前，我们进行对在诊断和复发从14名患者收集32 DLBCL样品的VDJ测序（几个患者有多个样品收集在任诊断或复发阶段） 使用 12个上述的条件。通过执行的配对样本，一个"早-发散"和"晚-发散"模式DLBCL复发相关克隆演变之间的主要VħDJħ重排的SHM型材的系统发生分析鉴定12。这里，相同的方法被施加到一个新收集的DLBCL诊断和复发对。 P后两个样品在FR1区得到CR扩增。一个主要的PCR产物与周围344碱基对大小（由生物分析仪测得的）中的同时从诊断和复发样品获得。测序后，总共0.70亿配对末端读得到用于诊断样品和0.73百万配对末端读为复发样品，这是在以前的研究中，每个样品读取得到的数的范围内（0.38- 142万，平均0.75±0.26亿美元）12。后映射配对末端读针对IMGT数据库，读出不具有在所有THREE V命中，D和J区被丢弃。 V ^ h DJ ^ h安排被分配给每个配对末端读。总共有064万和067万VħDJħ路口进行诊断和复发样品中分别确定的，代表大于90％的取向率。通过计数有多少次第VħDJ H的每组合被发现个体样品中，808不同Vħ&lt;/ SUB&gt; DJ ^ h路口发现诊断样品和复发样本中581独特的V ^ h DJ ^ h路口研究。这两个样本中占主导地位的V ^ h DJ ^ h交界处IGHV4-34 * 2 IGHD5-5 * 01 IGHJ2 * 01，确认它们是克隆相关。此主导VħDJħ结占整个映射VħDJħ结读取在两个诊断样品和复发样品的97％。 要追溯这种DLBCL复发情况下，克隆演变，进行了这对诊断和复发的样本之间的主要V ^ h DJ ^ h重排的SHM型材系统发育分析。我们观察到，该一对的克隆演变图案继"晚发散"路径（图4），即从上所述系统发育树的同一分支聚在一起的诊断和复发肿瘤，显性亚克隆诊断亚克隆和共享类似的SHM模式与几个其他突变占主导地位的复发亚克隆发生复发亚克隆。这些结果表明，有可能有一个在诊断肿瘤的亚克隆，这是任化疗耐药或从处理逃出，然后最终发展成复发肿瘤。 为了进一步表征和可视化过程中复发过程中的每个样本，克隆演变模式的克隆构型，两个新的分析已经被开发出来。在这些分析中，整个组中的每个样本中的主要VJ重排的亚克隆被用来计算通过SHM图案所限定的所有亚克隆之间的成对串距离。然后，通过使用多维标度（图5A，5B）或者通过建立在数据分析中描述的无向图（图5C，5D），创建表示亚克隆的分布和异质性图。如图 4所示，对MDS情节表现出晚期发散例（ 图5A）相比，在早期发散例（ 图5B）主要亚克隆之间少得多的序列多样性。此外，诊断样品的子克隆和复发样品的子克隆在初期发散壳体彼此完全分离，指示缺乏这些肿瘤之间的SHM图案相似的，即使它们携带相同的VDJ重排。同样，在（图5C）和（图5D）的无向图表明亚克隆计数的后期发散情况下（图5C）的分布不同于早期发散（图5D）。后者具有更突出的，但遗传多样性主要克隆从诊断到复发。此外，缺少边和较小亚克隆连接在主要诊断和复发克隆之间早发散的情况下（图5D），表明早期发散复发的情况下的序列多样性性质。 <img alt="图1" src="/files/ftp_upload/53215/53215fig1.jpg" /> 图1:一步的VDJ-SEQ方法步骤流程图 VDJ序列的整体流程如图<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53215/53215fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53215/53215fig2.jpg" /> 图2:VDJ PCR扩增子产物代表性图像 。凝胶图像，显示代表IgVH基因-FR1（左图）和IgVH基因-FR2（右图）从DNA的淋巴瘤患者样本中提取PCR产物。 PCR产物不能在样品5可能是由于无论是低得在那受损的PCR反应引物位点的DNA样品质量或SHM。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53215/53215fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53215/53215fig3.jpg" /> 图3:的VDJ PCR扩增子和随后的测序文库由生物分析仪的QC代表性生物分析仪跟踪之前文库构建（上图）和文库构建（底部面板）之后的相同的VDJ扩增子。请注意，从334基点的大小转移到459 bp的指示添加适配器。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53215/53215fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53215/53215fig4.jpg" /> 图4: </strong&gt; 一对DLBCL诊断和复发样品的克隆演变的分析，诊断，复发DLBCL样本对显示"晚分歧"克隆演变模式的系统发育分析。颜色代号表示突变状态。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53215/53215fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53215/53215fig5.jpg" /> 图5: 新的图形化方法来可视化克隆演变模式 （A，B）多维尺度地块整合SHM配置文件和样本对A显示了后期发散复发模式 （A）和样品对B的亚克隆计数显示早期发散复发模式 。 （B）中。圆的半径表示subcl的计那些对应于特定SHM轮廓和对应于该诊断（蓝色）样品或复发（红色）样品的颜色。 X轴和Y轴代表MDA的顶部2组成。 （C，D）样品对A显示了后期发散复发模式（C）和样品对B显示早期发散复发模式（D）的无向图。边缘对应于具有一个字母分离字符串距离的两个SHM型材及毕业底纹（彩色标尺条）指示的序列映射到对应于诊断（红色）样品或复发的特定SHM轮廓的比例（黄色）样品。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53215/53215fig5large.jpg" target="_blank">请这里查看该图的放大版本。</a>

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

Universidad San Sebastian

Research

JoVE Journal

Medicine

26.6K 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

Flow-sorting and Exome Sequencing of the Reed-Sternberg Cells of Classical Hodgkin Lymphoma

The Hodgkin Reed-Sternberg cells of classical Hodgkin lymphoma are sparsely distributed within a background of inflammatory lymphocytes and typically comprise less than 1% of the tumor mass. Material derived from bulk tumor contains tumor content at a concentration insufficient for characterization. Therefore, fluorescence activated cell sorting using eight antibodies, as well as side- and forward-scatter, is described here as a method of rapidly separating and concentrating with high purity thousands of HRS cells from the tumor for subsequent study. At the same time, because standard protocols for exome sequencing typically require 100-1,000 ng of input DNA, which is often too high, even with flow sorting, we also provide an optimized, low-input library construction protocol capable of producing high-quality data from as little as 10 ng of input DNA. This combination is capable of producing next-generation libraries suitable for hybridization capture of whole-exome baits or more focused targeted panels, as desired. Exome sequencing of the HRS cells, when compared against healthy intratumor T or B cells, can identify somatic alterations, including mutations, insertions and deletions, and copy number alterations. These findings elucidate the molecular biology of HRS cells and may reveal avenues for targeted drug treatments.

Here, we describe a combined flow cytometric cell sorting and low-input, next-generation library construction protocol designed to produce high-quality, whole-exome data from the Hodgkin Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (CHL).

经典霍奇金淋巴瘤的芦苇细胞的流分选和外显子测序

The Hodgkin Reed-Sternberg cells of classical Hodgkin lymphoma are sparsely distributed within a background of inflammatory lymphocytes and typically ...

科研

遗传学

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 ...

医学

Immunoglobulin Gene Sequence Analysis In Chronic Lymphocytic Leukemia: From Patient Material To Sequence Interpretation

During B cell maturation, the complex process of immunoglobulin (IG) gene V(D)J recombination coupled with somatic hypermutation (SHM) gives rise to a unique DNA sequence within each individual B cell. Since B cell malignancies result from the clonal expansion of a single cell, IG genes represent a unique molecular signature common to all the malignant cells within an individual patient; thus, IG gene rearrangements can be used as clonal markers. In addition to serving as an important clonal identifier, the IG gene sequence can act as a &#39;molecular timeline&#39; since it is associated with specific developmental stages and hence reflects the history of the B cell involved in the neoplastic transformation. Moreover, for certain malignancies, in particular chronic lymphocytic leukemia (CLL), the IG gene sequence holds prognostic and potentially predictive capabilities. That said, extrapolating meaningful conclusions from IG gene sequence analysis would be impossible if robust methods and tools were not available to aid in their analysis. This article, drawing on the vast experience of the European Research Initiative on CLL (ERIC), details the technical aspects and essential requirements necessary to ensure reliable and reproducible IG gene sequence analysis in CLL, a test that is now recommended for all CLL patients prior to treatment. More specifically, the various analytical stages are described ranging from the identification of the clonotypic IG gene rearrangement and the determination of the nucleotide sequence to the accurate clinical interpretation of the IG gene sequence data.

Herein, we present a protocol that details the technical aspects and essential requirements to ensure robust IG gene sequence analysis in patients with chronic lymphocytic leukemia (CLL), based on the accumulated experience of the European Research initiative on CLL (ERIC).

慢性淋巴细胞白血病免疫球蛋白基因序列分析: 从患者材料到序列解释

During B cell maturation, the complex process of immunoglobulin (IG) gene V(D)J recombination coupled with somatic hypermutation (SHM) gives rise to a ...

癌症研究

Characterizing Mutational Load and Clonal Composition of Human Blood

Hematopoietic stem and progenitor cells (HSPCs) gradually accumulate DNA mutations during a lifespan, which can contribute to age-associated diseases such as leukemia. Characterizing mutation accumulation can improve understanding of the etiology of age-associated diseases. Presented here is a method to catalogue somatic mutations in individual HSPCs, which is based on whole-genome sequencing (WGS) of clonal primary cell cultures. Mutations that are present in the original cell are shared by all cells in the clonal culture, whereas mutations acquired in vitro after cell sorting are present in a subset of cells. Therefore, this method allows for accurate detection of somatic mutations present in the genomes of individual HSPCs, which accumulate during life. These catalogues of somatic mutations can provide valuable insights into mutational processes active in the hematopoietic tissue and how these processes contribute to leukemogenesis. In addition, by assessing somatic mutations that are shared between multiple HSPCs of the same individual, clonal lineage relationships and population dynamics of blood populations can be determined. As this approach relies on in vitro expansion of single cells, the method is limited to hematopoietic cells with sufficient replicative potential.

Somatic mutation patterns in cells reflect previous mutagenic exposure and can reveal developmental lineage relationships. Presented here is a methodology to catalogue and analyze somatic mutations in individual hematopoietic stem and progenitor cells.

人体血液的突变载荷和克隆成分的特征

Hematopoietic stem and progenitor cells (HSPCs) gradually accumulate DNA mutations during a lifespan, which can contribute to age-associated diseases such ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

从排序的永生B细胞产生的重组人IgG单克隆抗体

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

免疫与感染

Generation of Discriminative Human Monoclonal Antibodies from Rare Antigen-specific B Cells Circulating in Blood

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the antibody needs to distinguish between highly related structures (e.g., a normal protein and a mutated version thereof). The current way of generating such discriminative mAbs involves extensive screening of multiple Ab-producing B cells, which is both costly and time consuming. We propose here a rapid and cost-effective method for the generation of discriminative, fully human mAbs starting from human blood circulating B lymphocytes. The originality of this strategy is due to the selection of specific antigen binding B cells combined with the counter-selection of all other cells, using readily available Peripheral Blood Mononuclear Cells (PBMC). Once specific B cells are isolated, cDNA (complementary deoxyribonucleic acid) sequences coding for the corresponding mAb are obtained using single cell Reverse Transcription-Polymerase Chain Reaction (RT-PCR) technology and subsequently expressed in human cells. Within as little as 1 month, it is possible to produce milligrams of highly discriminative human mAbs directed against virtually any desired antigen naturally detected by the B cell repertoire.

We describe a method for the production of human antibodies specific for an antigen of interest, starting from rare B cells circulating in human blood. Generation of these natural antibodies is efficient and rapid, and the antibodies obtained can discriminate between highly related antigens.

血液循环中稀有抗原特异 B 细胞的鉴别人单克隆抗体的生成

Monoclonal antibodies (mAbs) are powerful tools useful for both fundamental research and in biomedicine. Their high specificity is indispensable when the ...

Identification of Mouse and Human Antibody Repertoires by Next-Generation Sequencing

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular mechanisms underlying the production of the vast repertoire of antibodies by the acquired immune systems, it has not yet been possible to arrive at a global view of a complete antibody repertoire. In particular, B cell repertoires have been regarded as a black box because of their astronomical number of antibody clones. However, next-generation sequencing technologies are enabling breakthroughs to increase our understanding of the B cell repertoire. In this report, we describe a simple and efficient method to visualize and analyze whole individual mouse and human antibody repertoires. From the immune organs, representatively from spleen in mice and peripheral blood mononuclear cells in humans, total RNA was prepared, reverse transcribed, and amplified using the 5&#39;-RACE method. Using a universal forward primer and antisense primers for the antibody class-specific constant domains, antibody mRNAs were uniformly amplified in proportions reflecting their frequencies in the antibody populations. The amplicons were sequenced by next-generation sequencing (NGS), yielding more than 105 antibody sequences per immunological sample. We describe the protocols for antibody sequence analyses including V(D)J-gene-segment annotation, a bird&#39;s-eye view of the antibody repertoire, and our computational methods.

Here, we describe protocols for the analysis and visualization of the structure and constitution of whole antibody repertoires. This involves the acquisition of vast sequences of antibody RNA using next-generation sequencing.

近一代测序鉴定小鼠与人体抗体的分泌

The immense adaptability of antigen recognition by antibodies is the basis of the acquired immune system. Despite our understanding of the molecular ...

Single-cell Screening Method for the Selection and Recovery of Antibodies with Desired Specificities from Enriched Human Memory B Cell Populations

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational methods, such as next generation sequencing (NGS), yield enormous sets of data on the antibody repertoire at the sequence level, functional data is required to identify which sequences are relevant for a particular antigen or set of antigens. Here, we describe a method to identify and recover individual antigen-specific antibodies from peripheral blood mononuclear cells (PBMCs) from a human blood donor. This method utilizes an initial enrichment of mature B cells and requires a combination of phenotypic cell markers and fluorescently-labeled protein to isolate IgG memory B cells via flow cytometry. The heavy and light chain variable regions are then cloned and re-screened. Although limited to the memory B cell compartment, this method takes advantage of flow cytometry to interrogate millions of B cells and returns paired heavy and light chain sequences from a single cell in a format ready for expression and confirmation of specificity. Antibodies recovered with this method can be considered for therapeutic potential, but can also link specificity and function with bioinformatic approaches to assess the B cell repertoire within individuals.

The BSelex method to identify and recover individual antigen-specific antibodies from human peripheral blood mononuclear cells combines flow cytometry with single cell PCR and cloning.

从富足人类记忆B细胞群中选择和恢复具有所需特异性的抗体的单细胞筛选方法

The human antibody repertoire represents a largely untapped source of potential therapeutic antibodies and useful biomarkers. While current computational ...

Analysis of Somatic Hypermutation in the JH4 intron of Germinal Center B cells from Mouse Peyer's Patches

Within the germinal centers of lymphoid organs, mature B cells alter their expressed immunoglobulin (Ig) by introducing untemplated mutations into the variable coding exons of the Ig heavy and light chain gene loci. This process of somatic hypermutation (SHM) requires the enzyme activation-induced cytidine deaminase (AID), which converts deoxycytidines (C), into deoxyuridines (U). Processing the AID-generated U:G mismatches into mutations by the base excision and mismatch repair pathways introduces new Ig coding sequences that may produce a higher affinity Ig. Mutations in AID or DNA repair genes can block or significantly alter the types of mutations observed in the Ig loci. We describe a protocol to quantify JH4 intron mutations that uses fluorescence activated cell sorting (FACS), PCR, and Sanger sequencing. Although this assay does not directly measure Ig affinity maturation, it is indicative of mutations in Ig variable coding sequences. Additionally, these methods utilize common molecular biology techniques which&#160;analyze&#160;mutations in Ig sequences of multiple B cell clones. Thus, this assay is an invaluable tool in the study of SHM and Ig diversification.

Presented here is an assay to quantify somatic hypermutation within the immunoglobulin heavy chain gene locus using germinal center B cells from mouse Peyer&#8217;s patches.

从小鼠佩耶的补丁中对Germinal中心B细胞JH4因特龙的体细胞突变分析

Within the germinal centers of lymphoid organs, mature B cells alter their expressed immunoglobulin (Ig) by introducing untemplated mutations into the ...

T and B Cell Receptor Immune Repertoire Analysis using Next-generation Sequencing

Immunological memory, the hallmark of adaptive immunity, is orchestrated by T and B lymphocytes. In circulation and different organs, there are billions of unique T and B cell clones, and each one can bind a specific antigen, leading to proliferation, differentiation and/or cytokine secretion. The vast heterogeneity in T and B cells is generated by random recombination of different genetic segments. Next-generation sequencing (NGS) technologies, developed in the last decade, enable an unprecedented in-depth view of the T and B cell receptor immune repertoire. Studies in various inflammatory conditions, immunodeficiencies, infections and malignancies demonstrated marked changes in clonality, gene usage, and biophysical properties of immune repertoire, providing important insights about the role of adaptive immune responses in different disorders.
Here, we provide a detailed protocol for NGS of immune repertoire of T and B cells from blood and tissue. We present a pipeline starting from DNA isolation through library preparation, sequencing on NGS sequencer and ending with basic analyses. This method enables exploration of specific T and B cells at the nucleotide or amino-acid level, and thus can identify dynamic changes in lymphocyte populations and diversity parameters in different diseases. This technique is slowly entering clinical practice and has the potential for identification of novel biomarkers, risk stratification and precision medicine.

The current protocol describes a method for DNA isolation from blood samples and intestinal biopsies, generation of TCR&#946; and IGH PCR libraries for next-generation sequencing, performance of a NGS run and basic data analysis.

使用下一代测序的 T 和 B 细胞受体免疫再剧目分析

Immunological memory, the hallmark of adaptive immunity, is orchestrated by T and B lymphocytes. In circulation and different organs, there are billions ...