Name: comparative lesions analysis through a targeted sequencing approach
Uploaded: 2019-11-05T13:00:00
Description: Watch this Scientific Journal Video about Comparative Lesions Analysis Through a Targeted Sequencing Approach at JoVE.com

这项研究得到了意大利癌症基因组计划的支持。FIRB RBAP10AHJB），意大利协会，意大利皇家空军（AIRC;第12182号给AS，18178号给VC），FP7欧洲共同体赠款（Cam-Pac No 602783到AS）。供资机构在收集、分析和解释数据或编写手稿方面没有作用。

研究中使用的材料是根据当地道德委员会批准的一项特定协议收集的。所有患者均可获得书面知情同意。
1. 组织标本的组织学和免疫本型修正
注：专家病理学家负责下面描述的活动。
<ol><li>根据既定的诊断标准对选定病例进行组织病理学修订。<ol>
<li>使用微体从代表性的FFPE组织块中切割4⁄5μm厚的组织部分，并将部分安装在标准组织学幻灯片上。</li>
	...

<ol>
	<li>Koboldt, D. C., Steinberg, K. M., Larson, D. E., Wilson, R. K., Mardis, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+next-generation+sequencing+revolution+and+its+impact+on+genomics.">The next-generation sequencing revolution and its impact on genomics.</a> Cell. 155 (1), 27-38 (2013).</li><li>McGranahan, N., Swanton, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clonal+Heterogeneity+and+Tumor+Evolution:+Past,+Present,+and+the+Future.">Clonal Heterogeneity and Tumor Evolution: Past, Present, and the Future.</a> Cell. 168 (4), 613-628 (2017).</li><li>Stratton, M. R., Campbell, P. J., Futreal, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cancer+genome.">The cancer genome.</a> Nature. 458 (7239), 719-724 (2009).</li><li>Makohon-Moore, A. P., 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=Limited+heterogeneity+of+known+driver+gene+mutations+among+the+metastases+of+individual+patients+with+pancreatic+cancer.">Limited heterogeneity of known driver gene mutations among the metastases of individual patients with pancreatic cancer.</a> Nature Genetics. 49 (3), 358-366 (2017).</li><li>Seyfried, T. N., Huysentruyt, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+origin+of+cancer+metastasis.">On the origin of cancer metastasis.</a> Critical reviews in oncogenesis. 18 (1-2), 43-73 (2013).</li><li>McLaren, 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=Deriving+the+consequences+of+genomic+variants+with+the+Ensembl+API+and+SNP+Effect+Predictor.">Deriving the consequences of genomic variants with the Ensembl API and SNP Effect Predictor.</a> Bioinformatics. 26 (16), 2069-2070 (2010).</li><li>Robinson, J. 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=Integrative+genomics+viewer.">Integrative genomics viewer.</a> Nature Biotechnology. 29 (1), 24-26 (2011).</li><li>Amato, 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=Molecular+alterations+associated+with+metastases+of+solid+pseudopapillary+neoplasms+of+the+pancreas.">Molecular alterations associated with metastases of solid pseudopapillary neoplasms of the pancreas.</a> The Journal of Pathology. 247 (1), 123-134 (2019).</li><li>Mafficini, 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=Reporting+tumor+molecular+heterogeneity+in+histopathological+diagnosis.">Reporting tumor molecular heterogeneity in histopathological diagnosis.</a> PLoS One. 9 (8), e104979 (2014).</li><li>Shi, 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=Reliability+of+Whole-Exome+Sequencing+for+Assessing+Intratumor+Genetic+Heterogeneity.">Reliability of Whole-Exome Sequencing for Assessing Intratumor Genetic Heterogeneity.</a> Cell Reports. 25 (6), 1446-1457 (2018).</li><li>Simbolo, 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=DNA+qualification+workflow+for+next+generation+sequencing+of+histopathological+samples.">DNA qualification workflow for next generation sequencing of histopathological samples.</a> PLoS One. 8 (6), e62692 (2013).</li><li>Connor, A. 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=Integration+of+Genomic+and+Transcriptional+Features+in+Pancreatic+Cancer+Reveals+Increased+Cell+Cycle+Progression+in+Metastases.">Integration of Genomic and Transcriptional Features in Pancreatic Cancer Reveals Increased Cell Cycle Progression in Metastases.</a> Cancer Cell. 35 (2), 267-282 (2019).</li><li>Priestley, P., 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=Pan-cancer+whole+genome+analyses+of+metastatic+solid+tumors.">Pan-cancer whole genome analyses of metastatic solid tumors.</a> bioRxiv. , (2018).</li></ol>

我们的方法通过整合垂直数据（即形态学、DNA测序和免疫组织化学）与特定患者的不同病变，识别实体肿瘤进展中涉及的分子变化。通过查询409个癌症相关基因8的编码序列，我们证明了我们的方法检测突变沉默肿瘤类型（即胰腺的SPN、固体伪皮毛肿瘤）的克隆和亚克隆事件的能力。此处使用的基于放大素的靶向测序方法的一个优点是覆盖均匀性（90% 的目标碱基覆盖 100 倍，95% ?...

下一代测序（NGS）的出现彻底改变了癌症的诊断和治疗方式。与多区域测序结合的NGS在实体肿瘤2中暴露了高度的肿瘤内异质性（ITH），这在一定程度上解释了靶向治疗的失败，因为存在具有不同药物敏感性的亚克隆2.全基因组测序研究提出的一个重要挑战是必须区分个别癌症的乘客（即中性）和驾驶员突变3。几项研究确实表明，在某些肿瘤中，乘客突变占ITH的大部分，而驾驶员的改变往往在同一个体的病变中保存。同样重要的是要注意，大突变负担（如肺癌和黑色素瘤所示）并不一定意味着一个大的亚克隆突变负担2。因此，在突变性低的肿瘤中可以发现高度的ITH。
转移导致全世界90%以上的癌症相关死亡5;因此，在原发性病变和转移性病变中捕捉驱动基因的突变异质性，对于设计治疗晚期疾病的有效疗法至关重要。临床测序一般对固定组织的核酸进行，由于DNA质量差，使得全基因组的探索变得困难。另一方面，临床测序的目的是识别可操作的突变和/或突变，这些突变可能预测对给定治疗方案的响应/反应能力。就目前情况而言，测序可以限制在基因组的较小部分，以便及时提取临床相关信息。从低通量DNA分析（例如，桑格测序）到NGS的转变使得在高深度下分析数百个与癌症相关的基因成为可能，从而能够检测亚克隆事件。在这里，我们报告一种比较病变分析的方法，该方法允许在同一个体的不同标本中识别克隆和亚克隆种群。此处描述的方法集成了三种成熟的方法（组织分析、高覆盖率多病变测序和免疫表位分析），以预测所识别变异的功能后果。该方法在图1中进行了原理图描述，并应用于胰腺5个固体伪皮毛肿瘤（SPN）转移病例的研究。虽然我们描述正式固定石蜡嵌入 （FFPE） 组织标本的处理和分析，但同样的过程可以应用于来自新鲜冷冻组织的遗传物质。

<table>
	<tbody>
		<tr>
			<td>2100 Bioanalyzer Instrument</td>
			<td>Agilent Technologies</td>
			<td>G2939BA</td>
			<td>&#160;Automated electrophoresis tool</td>
		</tr>
		<tr>
			<td>Agencourt AMPure XP Kit</td>
			<td>Fisher Scientific</td>
			<td>NC9959336</td>
			<td>Beads technology for the purification of PCR products; beads-based purification reagent</td>
		</tr>
		<tr>
			<td>Agilent High Sensitivity DNA Kit</td>
			<td>Agilent Technologies</td>
			<td>5067-4627</td>
			<td>Library quantification</td>
		</tr>
		<tr>
			<td>Anti-BAP1</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-28383</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Anti-GLUT1</td>
			<td>Thermo Scientific</td>
			<td>RB-9052</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Anti-KDM6A</td>
			<td>Cell Signaling</td>
			<td>#33510</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Anti-p53</td>
			<td>Novocastra</td>
			<td>NCL-L-p53-DO7</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Anti-&#946;catenin</td>
			<td>Sigma-Aldrich</td>
			<td>C7207</td>
			<td>Antibody</td>
		</tr>
		<tr>
			<td>Blocking Solution</td>
			<td>home made</td>
			<td>-</td>
			<td>5 % Bovine serum albumin (BSA) in TBST</td>
		</tr>
		<tr>
			<td>Endogenous peroxidases inactivation solution</td>
			<td>home made</td>
			<td>-</td>
			<td>3% H2O2 in Tris-buffered saline (TBS) 1x</td>
		</tr>
		<tr>
			<td>Leica CV ultra</td>
			<td>Leica</td>
			<td>70937891</td>
			<td>Entellan mountin media</td>
		</tr>
		<tr>
			<td>Epitope Retrieval Solution 1</td>
			<td>Leica Biosystems</td>
			<td>AR9961</td>
			<td>Citrate based pH 6.0 epitope retrieval solution</td>
		</tr>
		<tr>
			<td>Epitope Retrieval Solution 2</td>
			<td>Leica Biosystems</td>
			<td>AR9640</td>
			<td>EDTA based pH 9.0 epitope retrieval solution</td>
		</tr>
		<tr>
			<td>Eppendorf 0.2 ml PCR Tubes, clear</td>
			<td>Eppendorf</td>
			<td>951010006</td>
			<td>Tubes</td>
		</tr>
		<tr>
			<td>Eppendorf DNA LoBind Tubes, 1.5 mL</td>
			<td>Eppendorf</td>
			<td>22431021</td>
			<td>Tubes</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>DIAPATH</td>
			<td>A0123</td>
			<td>IHC deparaffinization reagent</td>
		</tr>
		<tr>
			<td>ImmEdge Pen&#160;Hydrophobic&#160;Barrier&#160;Pen</td>
			<td>Vector Laboratories</td>
			<td>H&#173;4000</td>
			<td>Hydrophobic Pen</td>
		</tr>
		<tr>
			<td>ImmPACT DAB&#160;Peroxidase</td>
			<td>Vector Laboratories</td>
			<td>SK&#173;4105</td>
			<td>HRP substrate</td>
		</tr>
		<tr>
			<td>ImmPRESS Anti&#173;Rabbit&#160;Ig&#160;Reagent&#160;Peroxidase</td>
			<td>Vector Laboratories</td>
			<td>MP&#173;7401&#173;50</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>ImmPRESS Anti&#173;Mouse&#160;Ig&#160;Reagent&#160;Peroxidase</td>
			<td>Vector Laboratories</td>
			<td>MP&#173;7402&#173;50</td>
			<td>Secondary antibody</td>
		</tr>
		<tr>
			<td>Integrative Genomics Viewer (IGV)</td>
			<td>Broad Institute</td>
			<td>-</td>
			<td>https://software.broadinstitute.org/software/igv/home</td>
		</tr>
		<tr>
			<td>Ion AmpliSeq Comprehensive Cancer Panel</td>
			<td>Thermofisher Scientific</td>
			<td>4477685</td>
			<td>Multiplexed target selection of 409 cancer related gene. https://assets.thermofisher.com/TFS-Assets/CSD/Reference-Materials/ion-ampliseq-cancer-panel-gene-list.pdf</td>
		</tr>
		<tr>
			<td>Ion AmpliSeq Library Kit 2.0</td>
			<td>Thermofisher Scientific</td>
			<td>4480441</td>
			<td>Preparation of amplicon libraries using Ion AmpliSeq panels</td>
		</tr>
		<tr>
			<td>Ion Chef Instrument</td>
			<td>Thermofisher Scientific</td>
			<td>4484177</td>
			<td>Automated library preparation, template preparation and chip loading</td>
		</tr>
		<tr>
			<td>Ion PI Chip Kit v3 or Ion 540 Chip</td>
			<td>Thermofisher Scientific</td>
			<td>A26771 or A27766</td>
			<td>Barcoded chips for sequencing</td>
		</tr>
		<tr>
			<td>Ion PI Hi-Q Chef Kit or Ion 540 Kit-Chef</td>
			<td>Thermofisher Scientific</td>
			<td>A27198 or A30011</td>
			<td>Template preparation</td>
		</tr>
		<tr>
			<td>Ion PI Hi-Q Sequencing 200 Kit or Ion S5 Sequencing Kit</td>
			<td>Thermofisher Scientific</td>
			<td>A26433 or A30011</td>
			<td>Sequencing</td>
		</tr>
		<tr>
			<td>Ion Proton or Ion GeneStudio S5 System</td>
			<td>Thermofisher Scientific</td>
			<td>4476610 or A38196</td>
			<td>Sequencing system</td>
		</tr>
		<tr>
			<td>Ion Reporter Software - AmpliSeq Comprehensive Cancer Panel tumour-normal pair</td>
			<td>Thermofisher Scientific</td>
			<td>4487118</td>
			<td>Workflow</td>
		</tr>
		<tr>
			<td>Ion Reporter Software - uploader plugin</td>
			<td>Thermofisher Scientific</td>
			<td>4487118</td>
			<td>Data analysis tool</td>
		</tr>
		<tr>
			<td>Ion Torrent Suite Software - Coverege Analysis plugin</td>
			<td>Thermofisher Scientific</td>
			<td>4483643</td>
			<td>Plugin that describe the level of sequance coverage produced</td>
		</tr>
		<tr>
			<td>Ion Torrent Suite Software - Variant Caller plugin</td>
			<td>Thermofisher Scientific</td>
			<td>4483643</td>
			<td>Plugin able to identify single-nucleotide polymorphisms (SNPs), insertions and deletions in a sample across a reference</td>
		</tr>
		<tr>
			<td>Ion Xpress Barcode Adapters 1-96 Kit</td>
			<td>Thermofisher Scientific</td>
			<td>4474517</td>
			<td>Unique barcode adapters</td>
		</tr>
		<tr>
			<td>NanoDrop 2000/2000c Spectrophotometers</td>
			<td>Thermofisher Scientific</td>
			<td>ND-2000</td>
			<td>DNA purity detection</td>
		</tr>
		<tr>
			<td>NCBI reference sequence (RefSeq) database</td>
			<td>NCBI</td>
			<td>-</td>
			<td>https://www.ncbi.nlm.nih.gov/refseq/</td>
		</tr>
		<tr>
			<td>Platinum PCR SuperMix High Fidelity</td>
			<td>Fisher Scientific</td>
			<td>12532-016 or 12532-024</td>
			<td>SuperMix for PCR amplification; high-fidelity PCR mix</td>
		</tr>
		<tr>
			<td>QIAamp DNA Blood Mini Kit</td>
			<td>Quiagen</td>
			<td>51106 0r 51104</td>
			<td>DNA blood extraction kit</td>
		</tr>
		<tr>
			<td>QIAamp DNA FFPE Tissue</td>
			<td>Quiagen</td>
			<td>56404</td>
			<td>DNA FFPE tissue extraction kit</td>
		</tr>
		<tr>
			<td>Qubit 2.0 Fluorometer</td>
			<td>Thermofisher Scientific</td>
			<td>Q32866</td>
			<td>DNA quantification</td>
		</tr>
		<tr>
			<td>Qubit dsDNA BR Assay Kit</td>
			<td>Thermofisher Scientific</td>
			<td>Q32850</td>
			<td>Kit for DNA quantification on Qubit 2.0 Fluorometer</td>
		</tr>
		<tr>
			<td>TBST</td>
			<td>home made</td>
			<td>-</td>
			<td>Tris-buffered saline (TBS) and 0.1% of Tween 20</td>
		</tr>
		<tr>
			<td>Tissue-Tek Prisma Plus &#38; Tissue-Tek Film</td>
			<td>Sakura Europe</td>
			<td>6172</td>
			<td>Automated tissue slide stainer instrument</td>
		</tr>
		<tr>
			<td>Variant Effect Predictor (VEP) software</td>
			<td>EMBI-EBI</td>
			<td>-</td>
			<td>http://grch37.ensembl.org/Homo_sapiens /Tools/VEP</td>
		</tr>
		<tr>
			<td>Xilene, mix of isomeres</td>
			<td>Carlo Erba</td>
			<td>492306</td>
			<td>IHC deparaffinization reagent</td>
		</tr>
	</tbody>
</table>

comparative lesions analysis through a targeted sequencing approach

评估肿瘤内异质性（ITH）对于预测靶向疗法的失败并相应地设计有效的抗肿瘤策略至关重要。尽管由于样品处理和覆盖深度的差异，人们经常引起关注，但下一代实体肿瘤测序已经解开了肿瘤类型中高度可变的ITH程度。通过识别克隆和亚克隆种群来捕捉原发性病变和转移性病变之间的遗传关联性，对于设计早期疾病的治疗方法至关重要。在这里，我们报告一种比较病变分析的方法，该方法允许在同一患者的不同标本中识别克隆和亚克隆种群。本文描述的实验方法集成了三种成熟的方法：组织分析、高覆盖率多病变测序和免疫表位分析。为了尽量减少通过不适当的样品处理对亚克隆事件检测的影响，我们让组织进行仔细的病理检查和肿瘤细胞富集。然后，对肿瘤病变和正常组织进行质量控制的DNA，以409个相关癌症基因的编码区域为目标进行高覆盖率测序。虽然只看有限的基因组空间，我们的方法能够评估体细胞变化（单核苷酸突变和拷贝数变异）与给定患者不同病变的异质程度。通过对测序数据的比较分析，我们可以区分克隆与亚克隆的变化。大多数 ITH 通常归因于乘客突变;因此，我们还使用免疫组化学来预测突变的功能后果。虽然此协议已应用于特定的肿瘤类型，我们预计此处描述的方法广泛适用于其他实体肿瘤类型。

研究工作流如图1所示。针对409个癌症相关基因的编码序列的5个SPN病例的多病变（n = 13）测序共确定了8个基因中的27个体细胞突变（CTNNB1，KDM6A，BAP1，TET1，SMAD4，TP53）， FLT1和FGFR3）。当突变在给定患者的所有病变之间共享时，突变被定义为创始人/克隆，当在给定患者的某些（但不是所有病变）中检测到进展者/子克隆...

Watch this Scientific Journal Video about Comparative Lesions Analysis Through a Targeted Sequencing Approach at JoVE.com

Comparative Lesions Analysis Through a Targeted Sequencing Approach

本文介绍了一种识别特定患者不同标本之间的克隆和亚克隆变化的方法。虽然此处描述的实验侧重于特定的肿瘤类型，但该方法广泛适用于其他实体肿瘤。

通过有针对性的测序方法进行比较病变分析

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective ...

我们的协议允许通过识别与疾病进展有关的分子变化来捕获原发性病变和转移性病变之间的遗传相关性。该方法为探索高灵敏度、高特异性的临床标本遗传相关性提供了机会。这是因为分子和组织病理学分析的整合。癌症研究领域肿瘤内异质性的评价对于设计有效的抗肿瘤策略至关重要，该方法广泛适用于许多实体肿瘤类型。在准备和量化感兴趣的库后，将每个库稀释到无核酸酶水中的 100 个皮摩尔浓度，并将每个稀释库的 10 微升混合在一个管中进行单次运行。然后通过移液混合池库。要启动排序运行，请首先打开连接到测序系统的计算机上的 Torrent 浏览器。使用所选应用程序的通用模板计划新运行，并在套件选项卡下选择离子质子系统或 Ion Gene Studio S5 系统。从芯片类型下拉菜单中选择合适的芯片，然后从库套件下拉列表中选择 Ion AmpliSeq 2.0 库套件。选择模板套件的 Ion Chef 按钮，然后从模板套件下拉列表中选择相应的 Chef 套件。从测序套件下拉菜单中选择相应的测序套件，然后从条形码集下拉列表中选择 Ion Express。在插件选项卡下，选择覆盖率分析插件。在计划选项卡下，从参考库下拉列表中选择 GRCH37HG19 基因组。从目标区域下拉列表中，选择要排序的适当面板。然后设置要排序的条形码数量，并设置每个库的条形码名称和样本名称。对于池库稀释，用无核酸酶水稀释库存库至 25 皮摩尔浓度，用于离子质子芯片，每个稀释库的移液器 50 微升稀释到适当的库样品管的底部。将包含池稀释库的样品管加载到库准备系统中，并开始克隆放大。对于下一代测序，请按照屏幕上的说明初始化仪器。克隆扩增完成后，打开离子 Chef 仪器门，然后打开芯片加载离心机的盖子，然后从库准备系统中卸下两个芯片。将芯片放入单独的芯片存储容器中，将其中一个芯片装入仪器中的芯片室。然后关闭芯片舱盖并开始排序。对于突变分析，打开覆盖分析插件，并使用覆盖分析输出验证覆盖深度和均匀性。使用洪流变体呼叫者插件执行变体调用选择细菌线或躯体工作流。然后下载筛选的变体变体呼叫格式文件，并使用 NCBI RefSeq 数据库中的变体效果预测器软件对变体进行注释。要估计拷贝数变化，使用离子报告器上传器插件将测序数据加载到离子报告器软件中，并使用综合癌面板肿瘤正常对工作流程分析数据。根据软件分配的分数手动筛选突变和拷贝数变异，并使用综合基因组查看器直观地验证变异。然后目视检查对齐方式是否生成异常读取，并检查给定基因上所有安普利子的规范化覆盖，以验证复制数变异。对于每种情况下，从正常组织或血液或原发性肿瘤或转移的测序对突变调用进行索引。标记为细菌线和丢弃调用，这也从细菌线DNA的测序数据中可见。标记为克隆或创始人的突变，共享在给定患者的所有病变。然后标记为亚克隆或进展或突变，检测到在一些，但不是所有给定患者的病变。在这个具有代表性的实验中，针对409个癌症相关基因编码序列的5个固体伪膜肿瘤病例的多病变测序，共确定了8个基因中的27个体细胞突变。突变被定义为在给定患者的所有病变和进展或亚克隆之间共享时，当检测到给定患者的某些（但不是所有）病变时，突变被定义为创始人或克隆。始终如一，β-卡特宁和KMD6A的免疫体化学染色在具有相应基因突变的病例的不同病变中是同质的。KMD6A在突变标本中的适度染色表明，基因改变可能会改变蛋白质的功能，而不是蛋白质的表达。KMD6A在胰腺肿瘤中功能丧失与缺氧标记胶质-1的调节有关。因此，GLUT-1在携带KMD6A突变的情况下过度表达。BAP1和TP53的免疫基础化学证实这些基因的突变是亚克隆的。在这个具有代表性的案例的虚拟 karyotype 中，显示改变基因的位置、接近和拷贝数状态，使用测序数据进行拷贝数变异分析，揭示了所分析的所有样本的变化。与点突变相比，大多数拷贝数变异变化是亚克隆。验证突变和拷贝数变异的验证和索引，并使用肿瘤正常的DNA比较，对于避免生成可能使结果无效的神性调用非常重要。该程序还可用于全基因组测序研究，旨在询问整个基因组，以识别不同实体肿瘤类型病变之间的突变。在癌症研究领域，这些技术是了解具有重要临床意义的疾病生物学的关键工具，旨在设计个性化和有效的靶向疗法。

Research

JoVE Journal

Behavior

Contextual and Cued Fear Conditioning Test Using a Video Analyzing System in Mice

背景和线索恐惧制约试验小鼠中使用视频分析系统

The contextual and cued fear conditioning test is one of the behavioral tests that assesses the ability of mice to learn and remember an association ...

科研

行为学

Measurement of Fronto-limbic Activity Using an Emotional Oddball Task in Children with Familial High Risk for Schizophrenia

的额边缘活度测量在儿童使用情绪穿着或行为古怪的任务与家族性高风险的精神分裂症

Adolescence is a critical developmental period where the early symptoms of schizophrenia frequently emerge. First-degree relatives of people with ...

Experimental Assessment of Mouse Sociability Using an Automated Image Processing Approach

使用自动图像处理方法鼠标社交性的实验评估

Mouse is the preferred model organism for testing drugs designed to increase sociability. We present a method to quantify mouse sociability in which the ...

Pavlovian Conditioned Approach Training in Rats

大鼠条件性巴甫洛夫训练方法

Cues that are contingently paired with unconditioned, rewarding stimuli can acquire rewarding properties themselves through a process known as the ...

Protocol for Data Collection and Analysis Applied to Automated Facial Expression Analysis Technology and Temporal Analysis for Sensory Evaluation

协议进行数据采集和分析应用到自动人脸表情分析技术和时序分析感官评价

We demonstrate a method for capturing emotional response to beverages and liquefied foods in a sensory evaluation laboratory using automated facial ...

Behavioral Disturbances: An Innovative Approach to Monitor the Modulatory Effects of a Nutraceutical Diet

行为障碍：一个创新的方法来监控保健品饮食的调节作用

In dogs, diets are often used to modulate behavioral disturbances related to chronic anxiety and stress caused by intense and restless activity. However, ...

Treatment of Ankle Osteoarthritis with Total Ankle Replacement Through a Lateral Transfibular Approach

外侧 Transfibular 法治疗踝关节骨性关节炎

Total ankle replacement (TAR) is a valid option for the treatment of ankle osteoarthritis. The traditional surgical approach for TAR is the anterior ...

Use of a Wireless Video-EEG System to Monitor Epileptiform Discharges Following Lateral Fluid-Percussion Induced Traumatic Brain Injury

使用无线视频-EEG系统监测侧流冲击后癫痫的放电诱发创伤性脑损伤

The lateral fluid percussion injury (FPI) model is well established and has been used to study TBI and post-traumatic epilepsy (PTE). However, ...

A Machine Learning Approach to Design an Efficient Selective Screening of Mild Cognitive Impairment

设计轻度认知障碍有效选择性筛查的机器学习方法

Mild cognitive impairment (MCI) is the first sign of dementia among elderly populations and its early detection is crucial in our aging societies. Common ...

Evaluating Skilled Prehension in Mice Using an Auto-Trainer

使用自动训练器评估小鼠的熟练前向

We describe a method to introduce na&#239;ve mice to a novel prehension (reach-to-grasp) task. Mice are housed singly in cages with a frontal slot that ...