Name: oncogenic gene fusion detection using anchored multiplex polymerase chain reaction followed by next generation sequencing
Uploaded: 2019-07-05T14:00:00
Description: Watch this Scientific Journal Video about Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing at JoVE.com

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。

1. 图书馆编制和排序
<ol><li>一般测定注意事项和预测定步骤
	<ol>
<li>Assay 运行通常由 7 个临床样本和一个阳性对照组成(尽管可以根据需要调整每个库准备运行的样本数)。使用至少包含多个基因融合(即测定靶点)的正对照,这些基因融合已被制造商确认和/或已被正交方法确认。非模板控制 (NTC) 必须作为附加样本包含在每次检测运行中,但仅通过第二链 cDNA 合成和预测序质量控制(前 Seq QC...

<ol>
	<li>Mitelman, F., Johansson, B., Mertens, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+translocations+and+gene+fusions+on+cancer+causation.">The impact of translocations and gene fusions on cancer causation.</a> Nature Reviews Cancer. 7 (4), 233-245 (2007).</li><li>Daley, G. Q., Van Etten, R. A., Baltimore, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+chronic+myelogenous+leukemia+in+mice+by+the+P210bcr/abl+gene+of+the+Philadelphia+chromosome.">Induction of chronic myelogenous leukemia in mice by the P210bcr/abl gene of the Philadelphia chromosome.</a> Science. 247 (4944), 824-830 (1990).</li><li>Druker, B. J., 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=Activity+of+a+specific+inhibitor+of+the+BCR-ABL+tyrosine+kinase+in+the+blast+crisis+of+chronic+myeloid+leukemia+and+acute+lymphoblastic+leukemia+with+the+Philadelphia+chromosome.">Activity of a specific inhibitor of the BCR-ABL tyrosine kinase in the blast crisis of chronic myeloid leukemia and acute lymphoblastic leukemia with the Philadelphia chromosome.</a> New England Journal of Medicine. 344 (14), 1038-1042 (2001).</li><li>Solomon, B. J., 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=First-line+crizotinib+versus+chemotherapy+in+ALK-positive+lung+cancer.">First-line crizotinib versus chemotherapy in ALK-positive lung cancer.</a> New England Journal of Medicine. 371 (23), 2167-2177 (2014).</li><li>Shaw, A. T., 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=Crizotinib+in+ROS1-rearranged+non-small-cell+lung+cancer.">Crizotinib in ROS1-rearranged non-small-cell lung cancer.</a> New England Journal of Medicine. 371 (21), 1963-1971 (2014).</li><li>Drilon, A., 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=Efficacy+of+Larotrectinib+in+TRK+Fusion-Positive+Cancers+in+Adults+and+Children.">Efficacy of Larotrectinib in TRK Fusion-Positive Cancers in Adults and Children.</a> New England Journal of Medicine. 378 (8), 731-739 (2018).</li><li>Kawai, A., 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=SYT-SSX+gene+fusion+as+a+determinant+of+morphology+and+prognosis+in+synovial+sarcoma.">SYT-SSX gene fusion as a determinant of morphology and prognosis in synovial sarcoma.</a> New England Journal of Medicine. 338 (3), 153-160 (1998).</li><li>de Alava, E., 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=EWS-FLI1+fusion+transcript+structure+is+an+independent+determinant+of+prognosis+in+Ewing's+sarcoma.">EWS-FLI1 fusion transcript structure is an independent determinant of prognosis in Ewing's sarcoma.</a> Journal of Clinical Oncology. 16 (4), 1248-1255 (1998).</li><li>Pierron, G., 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+new+subtype+of+bone+sarcoma+defined+by+BCOR-CCNB3+gene+fusion.">A new subtype of bone sarcoma defined by BCOR-CCNB3 gene fusion.</a> Nature Genetics. 44 (4), 461-466 (2012).</li><li>Papaemmanuil, E., 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=Genomic+Classification+and+Prognosis+in+Acute+Myeloid+Leukemia.">Genomic Classification and Prognosis in Acute Myeloid Leukemia.</a> New England Journal of Medicine. 374 (23), 2209-2221 (2016).</li><li>Arber, D. A., 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=The+2016+revision+to+the+World+Health+Organization+classification+of+myeloid+neoplasms+and+acute.">The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute.</a> Blood. 127 (20), 2391-2405 (2016).</li><li>Sanders, H. 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=Exon+scanning+by+reverse+transcriptase-polymerase+chain+reaction+for+detection+of+known+and+novel+EML4-ALK+fusion+variants+in+non-small+cell+lung+cancer.">Exon scanning by reverse transcriptase-polymerase chain reaction for detection of known and novel EML4-ALK fusion variants in non-small cell lung cancer.</a> Cancer Genetics. 204 (1), 45-52 (2011).</li><li>Camidge, D. 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=Optimizing+the+Detection+of+Lung+Cancer+Patients+Harboring+Anaplastic+Lymphoma+Kinase+(ALK)+Gene+Rearrangements+Potentially+Suitable+for+ALK+Inhibitor+Treatment.">Optimizing the Detection of Lung Cancer Patients Harboring Anaplastic Lymphoma Kinase (ALK) Gene Rearrangements Potentially Suitable for ALK Inhibitor Treatment.</a> Clinical Cancer Research. 16 (22), 5581-5590 (2010).</li><li>Mino-Kenudson, M., 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+novel,+highly+sensitive+antibody+allows+for+the+routine+detection+of+ALK-rearranged+lung+adenocarcinomas+by+standard+immunohistochemistry.">A novel, highly sensitive antibody allows for the routine detection of ALK-rearranged lung adenocarcinomas by standard immunohistochemistry.</a> Clinical Cancer Research. 16 (5), 1561-1571 (2010).</li><li>Suehara, Y., 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=Identification+of+KIF5B-RET+and+GOPC-ROS1+fusions+in+lung+adenocarcinomas+through+a+comprehensive+mRNA-based+screen+for+tyrosine+kinase+fusions.">Identification of KIF5B-RET and GOPC-ROS1 fusions in lung adenocarcinomas through a comprehensive mRNA-based screen for tyrosine kinase fusions.</a> Clinical Cancer Research. 18 (24), 6599-6608 (2012).</li><li>Davies, K. D., 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+Molecular+Testing+Modalities+for+Detection+of+ROS1+Rearrangements+in+a+Cohort+of+Positive+Patient+Samples.">Comparison of Molecular Testing Modalities for Detection of ROS1 Rearrangements in a Cohort of Positive Patient Samples.</a> Journal of Thoracic Oncology. 13 (10), 1474-1482 (2018).</li><li>Reeser, J. W., 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=Validation+of+a+Targeted+RNA+Sequencing+Assay+for+Kinase+Fusion+Detection+in+Solid+Tumors.">Validation of a Targeted RNA Sequencing Assay for Kinase Fusion Detection in Solid Tumors.</a> Journal of Molecular Diagnostics. 19 (5), 682-696 (2017).</li><li>Beadling, 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+Multiplexed+Amplicon+Approach+for+Detecting+Gene+Fusions+by+Next-Generation+Sequencing.">A Multiplexed Amplicon Approach for Detecting Gene Fusions by Next-Generation Sequencing.</a> Journal of Molecular Diagnostics. 18 (2), 165-175 (2016).</li><li>Mamanova, 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=Target-enrichment+strategies+for+next-generation+sequencing.">Target-enrichment strategies for next-generation sequencing.</a> Nature Methods. 7 (2), 111-118 (2010).</li><li>Zheng, Z., 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=Anchored+multiplex+PCR+for+targeted+next-generation+sequencing.">Anchored multiplex PCR for targeted next-generation sequencing.</a> Nature Medicine. 20 (12), 1479-1484 (2014).</li><li>Davies, K. D., 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=Dramatic+Response+to+Crizotinib+in+a+Patient+with+Lung+Cancer+Positive+for+an+HLA-DRB1-MET+Gene+Fusion.">Dramatic Response to Crizotinib in a Patient with Lung Cancer Positive for an HLA-DRB1-MET Gene Fusion.</a> JCO Precision Oncology. 2017 (1), (2017).</li><li>Vendrell, J. A., 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=Detection+of+known+and+novel+ALK+fusion+transcripts+in+lung+cancer+patients+using+next-generation+sequencing+approaches.">Detection of known and novel ALK fusion transcripts in lung cancer patients using next-generation sequencing approaches.</a> Scientific Reports. 7 (1), 12510 (2017).</li><li>Agaram, N. P., Zhang, L., Cotzia, P., Antonescu, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+Spectrum+of+Genetic+Alterations+in+Pseudomyogenic+Hemangioendothelioma+With+Recurrent+Novel+ACTB-FOSB+Gene+Fusions.">Expanding the Spectrum of Genetic Alterations in Pseudomyogenic Hemangioendothelioma With Recurrent Novel ACTB-FOSB Gene Fusions.</a> American Journal of Surgical Pathology. 42 (12), 1653-1661 (2018).</li><li>Skalova, A., 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=Molecular+Profiling+of+Mammary+Analog+Secretory+Carcinoma+Revealed+a+Subset+of+Tumors+Harboring+a+Novel+ETV6-RET+Translocation:+Report+of+10+Cases.">Molecular Profiling of Mammary Analog Secretory Carcinoma Revealed a Subset of Tumors Harboring a Novel ETV6-RET Translocation: Report of 10 Cases.</a> American Journal of Surgical Pathology. 42 (2), 234-246 (2018).</li><li>Guseva, N. V., 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=Anchored+multiplex+PCR+for+targeted+next-generation+sequencing+reveals+recurrent+and+novel+USP6+fusions+and+upregulation+of+USP6+expression+in+aneurysmal+bone+cyst.">Anchored multiplex PCR for targeted next-generation sequencing reveals recurrent and novel USP6 fusions and upregulation of USP6 expression in aneurysmal bone cyst.</a> Genes Chromosomes and Cancer. 56 (4), 266-277 (2017).</li></ol>

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="...

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。

<table>
	<tbody>
		<tr>
			<td>10 mM Tris HCl pH 8.0</td>
			<td>IDT</td>
			<td>11-05-01-13</td>
			<td>Used for TNA dilution</td>
		</tr>
		<tr>
			<td>1M Tris pH 7.0</td>
			<td>Thermo Fisher</td>
			<td>AM9850G</td>
			<td>Used in library pooling</td>
		</tr>
		<tr>
			<td>25 mL Reagent Reservoir with divider</td>
			<td>USA Scientific</td>
			<td>9173-2000</td>
			<td>For use with multi-channel pipetters and large reagent volumes</td>
		</tr>
		<tr>
			<td>96-well TemPlate Semi-Skirt 0.1mL PCR plate-natural</td>
			<td>USA Scientific</td>
			<td>1402-9700</td>
			<td>Plate used for thermocycler steps</td>
		</tr>
		<tr>
			<td>Agencourt AMPure XP Beads</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td>Used in purification after several assay steps</td>
		</tr>
		<tr>
			<td>Agencourt Formapure Kit</td>
			<td>Beckman Coulter</td>
			<td>A33343</td>
			<td>Used in TNA extraction</td>
		</tr>
		<tr>
			<td>Archer FusionPlex Solid Tumor kit</td>
			<td>ArcherDX</td>
			<td>AB0005</td>
			<td>This kit contains most of the reagents necessary to perform library preperation for Illumina sequencing (kits for Ion Torrent sequencing are also available)</td>
		</tr>
		<tr>
			<td>Cold block, 96-well</td>
			<td>Light Labs</td>
			<td>A7079</td>
			<td>Used for keeping samples chilled at various steps</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Decon Labs</td>
			<td>DSP-MD.43</td>
			<td>Used for bead washes</td>
		</tr>
		<tr>
			<td>Library Quantification for Illumina Internal Control Standard</td>
			<td>Kapa Biosystems</td>
			<td>KK4906</td>
			<td>Used for library quantitation</td>
		</tr>
		<tr>
			<td>Library Quantification Primers and ROX Low qPCR mix</td>
			<td>Kapa Biosystems</td>
			<td>KK4973</td>
			<td>Used for library quantitation</td>
		</tr>
		<tr>
			<td>Library Quantification Standards</td>
			<td>Kapa Biosystems</td>
			<td>KK4903</td>
			<td>Used for library quantitation</td>
		</tr>
		<tr>
			<td>Magnet Plate, 96-well (N38 grade)</td>
			<td>Alpaqua</td>
			<td>A32782</td>
			<td>Used in bead purificiation steps</td>
		</tr>
		<tr>
			<td>MBC Adapters Set B</td>
			<td>ArcherDX</td>
			<td>AK0016-48</td>
			<td>Adapters that contain sample-specific indexes to enable multiplex sequencing</td>
		</tr>
		<tr>
			<td>Micro Centrifuge</td>
			<td>USA Scientific</td>
			<td>2641-0016</td>
			<td>Used for spinning down PCR tubes</td>
		</tr>
		<tr>
			<td>MicroAmp EnduraPlate Optical 96 well Plate</td>
			<td>Thermo-Fisher</td>
			<td>4483485</td>
			<td>Used for Pre-Seq QClibrary quantitation</td>
		</tr>
		<tr>
			<td>Microamp Optical Film Compression Pad</td>
			<td>Applied Biosystems</td>
			<td>4312639</td>
			<td>Used for library quantitation</td>
		</tr>
		<tr>
			<td>Mini Plate Spinner</td>
			<td>Labnet</td>
			<td>MPS-1000</td>
			<td>Used for collecing liquid at bottom of plate wells</td>
		</tr>
		<tr>
			<td>MiSeq Reagent Kit v3 (600 cycle)</td>
			<td>Illumina</td>
			<td>MS-102-3003</td>
			<td>Contains components necessary for a MiSeq sequencing run</td>
		</tr>
		<tr>
			<td>MiSeqDx System</td>
			<td>Illumina</td>
			<td></td>
			<td>NGS Sequencing Instrument</td>
		</tr>
		<tr>
			<td>Model 9700 Thermocycler</td>
			<td>Applied Biosystems</td>
			<td></td>
			<td>Used for several steps during assay</td>
		</tr>
		<tr>
			<td>nuclease free water</td>
			<td>Ambion</td>
			<td>9938</td>
			<td>Used as general diluent</td>
		</tr>
		<tr>
			<td>Optical ABI 96-well PCR plate covers</td>
			<td>Thermo-Fisher</td>
			<td>4311971</td>
			<td>Used for Pre-Seq QClibrary quantitation</td>
		</tr>
		<tr>
			<td>PCR Workstation Model 600</td>
			<td>Air Clean Systems</td>
			<td>BZ10119636</td>
			<td>Wet-bench assay steps performed in this &#39;dead air box&#39;</td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Qiagen</td>
			<td>1019499</td>
			<td>Used in TNA extraction</td>
		</tr>
		<tr>
			<td>QuantStudio 5</td>
			<td>Applied Biosystems</td>
			<td>LSA28139</td>
			<td>qPCR instrument used for PreSeq and library quantitation</td>
		</tr>
		<tr>
			<td>Qubit RNA HS Assay Kit</td>
			<td>Life Technologies</td>
			<td>Q32855</td>
			<td>Use for determing RNA concentration in TNA samples</td>
		</tr>
		<tr>
			<td>RNase Away</td>
			<td>Fisher</td>
			<td>12-402-178</td>
			<td>Used for general RNase decontamination of work areas</td>
		</tr>
		<tr>
			<td>Seraseq FFPE Tumor Fusion RNA Reference Material v2</td>
			<td>SeraCare</td>
			<td>0710-0129</td>
			<td>Used as the assay positive control</td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Fisher</td>
			<td>BP359-212</td>
			<td>Used in clean-up steps and for sequencing setup</td>
		</tr>
		<tr>
			<td>SYBR Green Supermix</td>
			<td>Bio Rad</td>
			<td>172-5120</td>
			<td>Component of PreSeq QC Assay</td>
		</tr>
		<tr>
			<td>TempAssure PCR 8-tube Strips</td>
			<td>USA Scientific</td>
			<td>1402-2700</td>
			<td>Used for reagent and sample mixing etc.</td>
		</tr>
		<tr>
			<td>Template RT PCR film</td>
			<td>USA Scientific</td>
			<td>2921-7800</td>
			<td>Used for covering 96-well plates</td>
		</tr>
		<tr>
			<td>U-Bottom 96-well Microplate</td>
			<td>LSP</td>
			<td>MP8117-R</td>
			<td>Used during bead purification</td>
		</tr>
	</tbody>
</table>

oncogenic gene fusion detection using anchored multiplex polymerase chain reaction followed by next generation sequencing

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。

如图3所示,图4和图5是分析用户界面的屏幕截图,展示了肺腺癌样本的结果。在图 3中,示例摘要(上图)显示了所谓的强证据融合,以及 QC 状态(以红色圈)。ADCK4-NUMBL融合(其中列出了 3 个等形)会立即被忽略,因为它是一个持久的转录直读事件(由列表旁边的断开的圆圈图标记录)。图 3的?...

Watch this Scientific Journal Video about Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing at JoVE.com

Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing

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。

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

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples ...

该协议用于检测患者肿瘤样本的信息化基因融合。在一次检测中同时评估数十个基因的融合状态。这项技术的优点是，我可以识别基因融合，而不管融合伙伴的身份如何，这些融合伙伴来自通过配方固定处理的肿瘤样本。临床肿瘤样本中某些基因融合的检测直接为多种癌症类型的诊断、预后和治疗选择提供信息。了解分析算法调用的许多融合都是工件至关重要。分析数据时最困难的任务是区分工件和实际融合调用。首先稀释总核酸，以达到RNA的所需浓度。对于每个样品，将20微升稀释剂转移到预冷却铝块中的随机注注试剂条管中，通过上下移液6至8次混合。简要旋转样品，将整个体积转移到 96 井 PCR 板中，然后用板密封膜密封。将板插入热循环器，用压缩垫盖住，然后合上盖子。在65摄氏度下孵育5分钟。孵育后，将第一链试剂条管中随机注注产品的全部体积转移。通过上下移液和短暂旋转混合。将整个体积转移到 96 井 PCR 板中，并用 RT 薄膜密封。将板插入热循环器，然后根据手稿指示运行反应。执行第二股cDNA合成，并修复，并结合步骤一根据手稿指示，并进入第二结扎步骤。要对分子条形码或 MBC 适配器条管进行编号，请将带铰链的管水平定位到背面，并使用永久标记。将珠子纯化板从第一个结扎步骤中取出，将每个样品的 40 微升转移到 MBC 适配器条管中，注意不要干扰珠子颗粒。通过移液混合试剂，向下旋转管，将整个体积转移到连接步骤两个试剂条管。混合样品，旋转下来，并放在热循环器块中。关闭加热盖，在 22 摄氏度下运行热循环器 5 分钟，然后保持 4 摄氏度。接下来，通过旋转和将50微升到一组新的PCR条管中，准备结扎清理珠子。在磁铁上孵育一分钟，丢弃上一液。从磁铁上取下带状管，通过上下移液将珠子重新插入 50 微升的结扎清洁缓冲液中。将整个样品体积从第二个结扎步骤转移到结扎清理珠条中。将样品混合并留在室温下10分钟。在孵育的中途对样品进行涡旋。之后，旋转样品，旋转下来，并在磁铁上孵育一分钟。丢弃上流液，并添加 200 微升新鲜结扎清理缓冲液。漩涡重新暂停，旋转下来，并放在磁铁上一分钟。两次洗涤后，使用超纯水代替缓冲液进行第三次洗涤。将珠子重新在 20 微升 5 毫米氢氧化钠中，然后将样品转移到 96 井 PCR 板中。将板放入带压缩垫的热循环器中，在 75 摄氏度下运行 10 分钟，然后保持 4 摄氏度。样品冷却至 4 摄氏度后，将板放在磁铁上至少 3 分钟，然后继续使用第一个 PCR。通过将两个微升的 GSP1 底因器添加到第一个 PCR 试剂条的每个井中来准备 PCR 反应。将第二次结扎清理产品的 18 微升转移到第一个 PCR 试剂条管中，然后上下移液混合。旋转样品并转移到 96 井 PCR 板。将它们放入热循环器中，然后根据手稿指示运行 PCR。通过将 PCR 产品的 20 微升添加到每个井填充 24 微升纯化珠的 U 底板中，继续进行珠子纯化。上下移液混合，在室温下离开板五分钟，然后孵育磁铁上的板两分钟。丢弃珠子中的上一分，用200微升70%乙醇洗涤两次。最后洗涤后，取出所有乙醇，让样品干燥两分钟。从磁铁上取下磁石，将珠子重新用 24 微升 10 毫米 Tris-HCl 和 pH 8.0。孵育磁铁三分钟，然后将板放回磁铁上两分钟。在 10 毫米 Tris-HCl 中制备第二个 PCR 产品的 1 到 5 个稀释度，开始库定量。在 10 毫米 Tris-HCl 中连续稀释 1：199、1：199 和 20：80，并添加 05% 聚索酸酯。通过将六微升主混合添加到光学 96 井板的每个井中，然后加入 4 个适当稀释或标准的微升来设置关键 PCR。旋转板并加载到 qPCR 仪器中。根据手稿指示执行 qPCR。库定量完成后，将所有库稀释为两个纳米摩尔，并配有 10 毫米 Tris-HCl。通过将每个规范化库的 10 微升组合成一个 1.5 毫升的微离心管，制作库池。接下来，准备变性安培库，或DAL池，将库池的10微升与10微升的0.2正常氢氧化钠相结合，并在室温下孵育混合物5分钟。孵育后，在pH 7.0下加入10微升200毫米Tris-HCl，然后加入970微升HT1杂交缓冲液。通过组合 300 微升 HT1、25 微升 20 个 picomolar PhiX 和 675 微升的 DAL 池，制作最终负载管。将负载管的整个体积添加到测序器试剂盒的样品井中，并将墨盒加载到测序器中。首先选择阳性对照样本，并确保检测到所有预期的聚变和致癌异构体，并列在强证据选项卡中。对于每个示例，检查每个 GSP2 控制值的平均唯一启动站点，然后通过单击可视化链接来可视化每个潜在融合的支持读取，该链接将用户带至基于 Web 的 JBrowse 视图，该视图包含单个融合支持读取的堆积。确认读取大多没有不匹配，但超过 30 个读取基础与融合伙伴对齐，并且与基因和底像绑定位点之间的断点相邻的序列没有插入或删除。确保每个呼叫融合通常没有错位，并且读取的很大一部分与融合伙伴保持一致至关重要。该协议已用于研究肺腺癌样本中的基因融合状态。摘要显示了强大的证据融合，"读取统计"页显示样本的指标。该样品表现出良好的RNA质量，因此如果没有发现聚变，将报告阴性结果。合法的融合调用具有大量支持读取、支持融合的底像读取率高以及大量启动站点。读取的可视化显示与融合伙伴的大区域良好的对齐方式。相反，在映射到合作伙伴时，工件融合调用具有较低的支持指标和高错误率。分析算法进行的项目融合调用很常见。手动检查每个呼叫，以确保它代表患者样本中真正的基因融合至关重要。

Research

JoVE Journal

Cancer Research

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

新一代测序为可操作的突变在固体和液体瘤检测

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific ...

科研

癌症研究

Detection of Rare Mutations in CtDNA Using Next Generation Sequencing

利用下一代测序检测 CtDNA 中的稀有突变

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. ...

Looking for Driver Pathways of Acquired Resistance to Targeted Therapy: Drug Resistant Subclone Generation and Sensitivity Restoring by Gene Knock-down

寻找获得性抗靶治疗的驱动途径: 耐药亚克隆生成与基因击倒敏感性恢复

The past two decades have seen a shift from cytotoxic drugs to targeted therapy in medical oncology. Although targeted therapeutic agents have shown more ...

Using CRISPR/Cas9 Gene Editing to Investigate the Oncogenic Activity of Mutant Calreticulin in Cytokine Dependent Hematopoietic Cells

利用 CRISPR/Cas9 基因编辑研究细胞因子依赖性造血细胞中突变钙的致癌活性

Clustered regularly interspaced short palindromic repeats (CRISPR) is an adaptive immunity system in prokaryotes that has been repurposed by scientists to ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

数字聚合酶链反应法测定零星家族性腺瘤性息肉病患者的基因变异

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Droplet Digital TRAP (ddTRAP): Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction

Droplet Digital TRAP ddTRAP: Adaptation of the Telomere Repeat Amplification Protocol to Droplet Digital Polymerase Chain Reaction

液滴数字 TRAP (ddTRAP): 对端粒重复放大协议的适应性, 以实现液滴数字聚合酶链反应

The telomere repeat amplification protocol (TRAP) is the most widely used assay to detect telomerase activity within a given a sample. The polymerase ...

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

使用转录组学分析绘制无序致癌转录因子的结构-功能关系

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

人类乳腺上皮细胞致癌转化的体外评价

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Spatial Profiling of Protein and RNA Expression in Tissue: An Approach to Fine-Tune Virtual Microdissection

组织中蛋白质和RNA表达的空间分析：一种微调虚拟显微切割的方法

Multiplexing enables the assessment of several markers on the same tissue while providing spatial context. Spatial Omics technologies allow both protein ...

Transmitochondrial Cybrid Generation From Suspension-Growing Cancer Cells

Transmitochondrial Cybrid Generation Using Cancer Cell Lines

Transmitochondrial Cybrid Generation Using Cancer Cell Lines (Video) | JoVE 

使用癌细胞系的传递软骨Cybrid生成

In recent years, the number of studies dedicated to ascertaining the connection between mitochondria and cancer has significantly risen. However, more ...