使用锚定多路聚合酶链反应的致癌基因融合检测,随后进行下一代测序
Summary
本文详细介绍了使用锚定多路聚合酶链式反应基库制备试剂盒,然后进行下一代测序,以评估临床固体肿瘤样本中的致癌基因融合。介绍了湿凳和数据分析步骤。
Abstract
基因融合经常导致许多不同类型的癌症的致癌表型。此外,癌症患者样本中某些融合的存在通常直接影响诊断、预后和/或治疗选择。因此,基因融合的准确检测已成为许多疾病类型临床管理的重要组成部分。直到最近,临床基因融合检测主要通过使用单基因检测来完成。然而,具有临床意义的基因融合的不断增加,已经产生了同时评估多个基因融合状态的需求。基于下一代测序(NGS)的测试通过以大规模平行方式测序核酸的能力满足了这一需求。多种基于NGS的方法,采用不同的策略,基因靶点富集,现在可用于临床分子诊断,每种方法都有自己的优缺点。本文介绍了使用锚定多路PCR(AMP)为基础的靶点浓缩和库制备,然后NGS来评估临床固体肿瘤标本的基因融合。AMP在基于扩基的扩充方法中独树一帜,因为它识别基因融合,而不管融合伙伴的身份如何。这里详细介绍了湿板凳和数据分析步骤,以确保从临床样本中准确检测基因融合。
Introduction
由于大规模染色体变异(包括缺失、重复、插入、反转和易位),两个或两个以上基因融合到单个转录实体中。通过改变转录控制和/或改变表达基因产物的功能特性,这些融合基因可以赋予癌细胞1的致癌特性。在许多情况下,融合基因通过直接激活细胞增殖和生存途径,被认为是原发性致病的驱动因素。
随着费城染色体和相应的BCR-ABL1融合基因在慢性骨髓性白血病(CML)中的发现,基因融合对癌症患者的临床相关性首先显现出来。小分子抑制剂imatinib mesylate被开发专门针对这种融合基因,并在BCR-ABL1阳性CML患者3中表现出显著的疗效。致癌基因融合的治疗靶向在实体肿瘤中也取得了成功,以抑制ALK和ROS1融合基因在非小细胞肺癌中为主要例子4、5。近日,NTRK抑制剂拉罗替尼被FDA批准为NTRK1/2/3融合阳性实体肿瘤,无论疾病部位6。除了治疗选择,基因融合检测在疾病诊断和预后也起着重要作用。这在各种肉瘤和血液恶性肿瘤亚型中尤为普遍,这些亚类型由特定融合的存在和/或存在,直接通知预后7,8,9,10,11.这些只是癌症患者基因融合检测临床应用的几个例子。
由于在临床决策中起着至关重要的作用,从临床样本中准确检测基因融合至关重要。在临床实验室中应用了许多技术进行融合或染色体重排分析,包括:细胞遗传学技术、逆转录聚合酶链反应(RT-PCR)、原位杂交荧光(FISH),免疫组织化学(IHC)和5’/3’表达不平衡分析(除其他外)12,13,14,15 。目前,癌症中可操作基因融合的快速扩大导致需要同时评估多个基因的融合状态。因此,一些一次只能查询一个或几个基因的传统技术正在成为低效的方法,特别是考虑到临床肿瘤样本往往非常有限,不能被分割成几种检测。然而,下一代测序(NGS)是一个分析平台,非常适合多基因检测,基于NGS的检测在临床分子诊断实验室中已经司空见惯。
目前用于融合/重新排列检测的NGS检测在所使用的输入材料、用于库制备和目标扩充的化学成分以及检测中查询的基因数量方面有所不同。NGS检测可以基于从样品中提取的RNA或DNA(或两者)。尽管基于RNA的分析受到临床样本含有高降解RNA的倾向的阻碍,但它规避了对大型且经常重复的内子进行测序的需要,这些子群是基于DNA的聚变测试的目标,但事实证明,这些对NGS来说很困难。数据分析16.基于RNA的NGS测定的目标浓缩策略可以主要分为混合捕获或扩大子介导方法。虽然这两种策略都已成功用于融合检测,但每种策略都比其他17、18具有优势。混合捕获检测通常会导致更复杂的库和减少等位位级落差,而基于安培的检测通常需要较低的输入,导致更少的离目标测序19。然而,传统以扩因为基础的浓缩的主要限制可能是需要对所有已知的聚变伙伴进行引物。这是有问题的,因为许多临床上重要的基因已知与几十个不同的伙伴融合,即使引体设计允许检测所有已知的伙伴,新的融合事件也不会被发现。最近描述的一种称为锚定多路PCR(简称AMP)的技术解决了这一限制20。在 AMP 中,”半功能”NGS 适配器被分割到从输入RNA派生的 cDNA 片段中。目标富集是通过在基因特异性引体和适配器引源之间扩增来实现的。因此,应该检测所有与感兴趣的基因的融合,即使涉及一个新的融合伙伴,也应当检测(见图1)。 本文介绍了 ArcherDx FusionPlex 固体肿瘤试剂盒的使用,这是一种基于 NGS 的检测,采用 AMP 进行靶点浓缩和库制备,用于检测固体肿瘤样本中的致癌基因融合(参见补充表 1完整的基因列表)。湿板协议和数据分析步骤已在临床实验室改进修订 (CLIA) 认证的实验室中经过严格验证。
Protocol
Representative Results
Discussion
锚定多路PCR靶富集和库制备,然后下一代测序非常适合在临床肿瘤样本中的多路复用基因融合评估。通过专注于RNA输入而不是基因组DNA,避免了对大型和重复内子进行测序的需要。此外,由于这种方法放大基因融合,无论融合伙伴的身份,新的融合被检测到。这是临床领域的一个关键优势,在文献21、22、23、24中,通过AMP报告的可操作新基因融合<sup class="…
Declarações
The authors have nothing to disclose.
Acknowledgements
这项工作得到了科罗拉多大学分子病理学共享资源(国家癌症研究所癌症中心支持赠款No.P30-CA046934)和科罗拉多个性化医学中心。
Materials
| 10 mM Tris HCl pH 8.0 | IDT | 11-05-01-13 | Used for TNA dilution |
| 1M Tris pH 7.0 | Thermo Fisher | AM9850G | Used in library pooling |
| 25 mL Reagent Reservoir with divider | USA Scientific | 9173-2000 | For use with multi-channel pipetters and large reagent volumes |
| 96-well TemPlate Semi-Skirt 0.1mL PCR plate-natural | USA Scientific | 1402-9700 | Plate used for thermocycler steps |
| Agencourt AMPure XP Beads | Beckman Coulter | A63881 | Used in purification after several assay steps |
| Agencourt Formapure Kit | Beckman Coulter | A33343 | Used in TNA extraction |
| Archer FusionPlex Solid Tumor kit | ArcherDX | AB0005 | This kit contains most of the reagents necessary to perform library preperation for Illumina sequencing (kits for Ion Torrent sequencing are also available) |
| Cold block, 96-well | Light Labs | A7079 | Used for keeping samples chilled at various steps |
| Ethanol | Decon Labs | DSP-MD.43 | Used for bead washes |
| Library Quantification for Illumina Internal Control Standard | Kapa Biosystems | KK4906 | Used for library quantitation |
| Library Quantification Primers and ROX Low qPCR mix | Kapa Biosystems | KK4973 | Used for library quantitation |
| Library Quantification Standards | Kapa Biosystems | KK4903 | Used for library quantitation |
| Magnet Plate, 96-well (N38 grade) | Alpaqua | A32782 | Used in bead purificiation steps |
| MBC Adapters Set B | ArcherDX | AK0016-48 | Adapters that contain sample-specific indexes to enable multiplex sequencing |
| Micro Centrifuge | USA Scientific | 2641-0016 | Used for spinning down PCR tubes |
| MicroAmp EnduraPlate Optical 96 well Plate | Thermo-Fisher | 4483485 | Used for Pre-Seq QClibrary quantitation |
| Microamp Optical Film Compression Pad | Applied Biosystems | 4312639 | Used for library quantitation |
| Mini Plate Spinner | Labnet | MPS-1000 | Used for collecing liquid at bottom of plate wells |
| MiSeq Reagent Kit v3 (600 cycle) | Illumina | MS-102-3003 | Contains components necessary for a MiSeq sequencing run |
| MiSeqDx System | Illumina | NGS Sequencing Instrument | |
| Model 9700 Thermocycler | Applied Biosystems | Used for several steps during assay | |
| nuclease free water | Ambion | 9938 | Used as general diluent |
| Optical ABI 96-well PCR plate covers | Thermo-Fisher | 4311971 | Used for Pre-Seq QClibrary quantitation |
| PCR Workstation Model 600 | Air Clean Systems | BZ10119636 | Wet-bench assay steps performed in this 'dead air box' |
| Proteinase K | Qiagen | 1019499 | Used in TNA extraction |
| QuantStudio 5 | Applied Biosystems | LSA28139 | qPCR instrument used for PreSeq and library quantitation |
| Qubit RNA HS Assay Kit | Life Technologies | Q32855 | Use for determing RNA concentration in TNA samples |
| RNase Away | Fisher | 12-402-178 | Used for general RNase decontamination of work areas |
| Seraseq FFPE Tumor Fusion RNA Reference Material v2 | SeraCare | 0710-0129 | Used as the assay positive control |
| Sodium Hydroxide | Fisher | BP359-212 | Used in clean-up steps and for sequencing setup |
| SYBR Green Supermix | Bio Rad | 172-5120 | Component of PreSeq QC Assay |
| TempAssure PCR 8-tube Strips | USA Scientific | 1402-2700 | Used for reagent and sample mixing etc. |
| Template RT PCR film | USA Scientific | 2921-7800 | Used for covering 96-well plates |
| U-Bottom 96-well Microplate | LSP | MP8117-R | Used during bead purification |
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