由外显子捕获和大规模平行测序检测体细胞遗传学改变在肿瘤标本
Summary
我们描述条形码DNA文库和随后的杂交为基础的外显子捕获检测的关键癌症相关的基因突变在临床肿瘤标本通过大规模并行的“下一代”测序的准备。靶向外显子的测序提供了高吞吐量，低成本，以及深的序列覆盖率的好处，从而产生较高的灵敏度，用于检测低频率突变。
Abstract
努力发现和调查的关键致癌基因突变已被证明有价值的，以方便对癌症患者进行相应的处理。高通量的建立，大规模并行“下一代”测序已经资助了许多这样的突变的发现。以提高该技术的临床和平移工具，平台必须是高通量，成本效益，并用福尔马林固定，石蜡包埋（FFPE）组织样品可能产生少量的退化或受损的DNA兼容。在这里，我们描述了随后靶向外显子用于癌症相关的突变在新鲜冰冻和石蜡包埋的肿瘤通过大规模平行测序检测的基于杂交的捕获条形码和复用DNA文库的制备。这种方法使序列突变的识别，拷贝数变化，并选择涉及所有目标基因的结构重排。针对外显子测序提供吨他同样拥有高通量，低成本，和深序列覆盖率，从而赋予高灵敏度检测低频率突变。
Introduction
在关键的癌基因和肿瘤抑制基因的“驱动器”肿瘤的遗传事件的识别起着许多癌症1的诊断和治疗的重要作用。利用大规模并行“下一代”测序大规模的研究工作已经启用了很多这样的癌症相关的基因，近年来2的鉴定。然而，这些测序平台通常需要大量的DNA从新鲜冰冻组织中分离，从而构成一个重大的限制在从保存组织，如福尔马林固定，石蜡包埋（FFPE）的肿瘤样品表征和分析DNA突变。改进的努力，高效，可靠地从FFPE肿瘤样本的特征“可操作的”基因组信息将使以前存入银行的标本进行回顾性分析，并进一步鼓励个性化的方法对癌症的管理。
传统上，分子ðiagnostic实验室一直依赖耗时，低通量的方法，如Sanger测序及实时荧光定量PCR进行DNA突变分析。最近，利用复用PCR或质谱基因分型高通量方法已被开发来调查在关键的癌基因3-5复发性体细胞突变。这些方法，但是，只有预先指定的“热点”的突变进行了检测，使得它们不适合用于检测失活突变的肿瘤抑制基因受到限制。大规模并行测序提供了这些策略的几个优点，包括审问整个外显子为双方共同和稀有突变的能力，才能看到其他类基因组改变，如拷贝数收益和损失的能力，异构样品6，在更高的检测灵敏度7 。全基因组测序为代表的突变的发现的最全面的方法，虽然它是相对的LY昂贵，即被用于数据分析和存储大量的计算需求。
对于临床应用，其中的基因组中的仅一小部分可能是临床利益，在测序技术2特别的创新已经变革。首先，通过基于杂交的外显子捕获，可以分离DNA对应键与癌症相关的基因突变有针对性的分析8。第二，通过分子条形码（ 即 DNA序列的6-8个核苷酸长）的结扎，可以有数百个每个测序运行样品并充分利用大规模并行测序仪器10不断增加的容量的优点。合并后，这些创新使肿瘤可为异形更低的成本和更高的吞吐量，更小的计算需求11。进一步，通过重新分配序列覆盖，只有那些基因最关键的特定应用，可以achie已经较大的测序深度为低等位基因频率较高的事件检测灵敏度。
在这里，我们描述了我们的影响分析（中可操作的癌症靶点整合突变图谱），使用自定义的寡核苷酸捕获所有蛋白质编码外显子和选择的279键的癌症相关基因的内含子（它利用外显子捕获的条形码序列库池通过杂交表1 ）。这一战略使基因突变，插入缺失，拷贝数改变的识别，并选择涉及这些279个基因的结构重排。我们的方法是与DNA来自新鲜冷冻和FFPE组织以及细针抽吸等细胞学标本中分离出兼容。
Protocol
Representative Results
Discussion
我们的影响分析产生的高定位速度，较高的目标速度，目标高覆盖，高灵敏度检测基因突变，插入缺失和拷贝数变化。我们已经证明我们的影响分析的能力，同时从新鲜冰冻的DNA序列和归档低的DNA输入FFPE样品。通过执行主要癌症相关的基因有针对性的外显子测序，可以达到非常深的序列覆盖率为这些最关键的基因的外显子从而最大限度地检测低频率突变的能力。使用条形码和复用的，可实现更高…
Divulgations
The authors have nothing to disclose.
Acknowledgements
我们感谢艾格尼丝的Viale博士和MSKCC基因组学核心实验室提供技术援助。此协议是从杰弗里比尼癌症研究中心和农民家庭基金会的支持下开发的。
Materials
| NEBNext End Repair Module | New England Biolabs | E6050L | |
| NEBNext dA-Tailing Module | New England Biolabs | E6053L | |
| NEBNext Quick Ligation Module | New England Biolabs | E6056L | |
| Agencourt AMPure XP | Beckman Coulter Genomics | ||
| NEXTflex PCR-Free Barcodes – 24 | Bioo Scientific | 514103 | |
| HiFi Library Amplification Kit | KAPA Biosystems | KK2612 | |
| COT Human DNA, Fluorometric Grade | Roche Diagnostics | 05 480 647 001 | |
| NimbleGen SeqCap EZ Hybridization and Wash kit | Roche NimbleGen | 05 634 261 001 | |
| SeqCap EZ Library Baits | Roche NimbleGen | ||
| QIAquick PCR Purification Kit | Qiagen | 28104 | |
| Qubit dsDNA Broad Range (BR) Assay Kit | Life Technologies | Q32850 | |
| Qubit dsDNA High Sensitivity (HS) Assay Kit | Life Technologies | Q32851 | |
| Agilent DNA HS Kit | Agilent Technologies | 5067-4626, 4627 | |
| Agilent 2100 Bioanalyzer | Agilent Technologies | ||
| Covaris E220 | Covaris | ||
| Magnetic Stand-96 | Ambion | AM10027 | |
| Illumina Hi-Seq 2000 | Illumina |
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