Name: gdna enrichment by a transposase-based technology for ngs analysis of the whole sequence of brca1, brca2, and 9 genes involved in dna damage repair
Uploaded: 2014-10-06T13:00:00.000+00:00
Duration: 8 min 15 s
Description: Watch this Scientific Journal Video about gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair at JoVE.co

We thank the Ligue contre le Cancer de C&#244;te d&#8217;Or and the Centre Georges-Fran&#231;ois Leclerc for their financial support. We thank Philip Bastable for the editing of the manuscript.

基因组DNA 1。评估（基因组DNA）产量<ol><li>量化新鲜提取基因组DNA的制备图书馆前。使用荧光估产方法量化完整的基因组DNA（避免使用分光光度计进行基因组DNA的产量评估）。测量（二百八十零分之二百六十nm）的比率，并确保它是在1.8和2注:50纳克基因组DNA的需要进行实验。 </li></ol> 2，基因组DNA富集:第1天，上午<ol><li>开始之前<ol><li>从DNA富集试剂盒（见表材料/设备），解冻DNA缓冲液（TD）和冰的DNA酶（TDE1）解决方案。至少30分钟，在使用前，使纯化的磁珠和停止目标（ST）的缓冲液至室温（RT）。每个样品中添加20微升基因组DNA样品在2-2.5纳克/微升直接放入96孔板中，1个井。重要提示:使用阳性对照，以验证协议（与已知序列变异样本）。 </li></ol></li><li> Tagmentation <ol><li> M九，所有试剂彻底颠倒5倍，后短暂离心。在室温，加入25微升的TD缓冲液到每个孔中含有50的gDNA纳克（20微升），然后取5μl的TDE1缓冲器。将吸管50微升，轻轻吸取整个音量上下10倍。 </li><li>覆盖板用粘接膜和离心机在280 XG 1分钟，在20℃。然后，将板在热循环仪（盖在100℃加热），然后运行下面的程序:55℃5分钟，10℃，无时间限制。 </li><li>在此培养后，制备与实验管理器软件的样品片材，以定义将要使用的索引。 </li></ol></li><li> Tagmentation净化 注意:该步骤是关键的。要非常小心。 <ol><li>检查无沉淀净化磁珠和ST缓冲器。如果沉淀物存在，热身手中的解决方案。解冻再悬浮缓冲（RSB）冰上。 </li><li>在RT下，涡街溶液和添加15μl到每个孔中含有的样品。将吸管65微升，轻轻吸取整个音量上下10倍。孵育5分钟，在室温。覆盖板和离心机在280 XG 1分钟，在20℃。 </li><li>加入52微升的预先混合纯化的磁珠在每个孔中。将吸管117微升，轻轻吸取整个音量上下10倍。下室温温育10分钟。覆盖板和离心机在280 XG 1分钟，在20℃。 </li><li>除去粘合膜。放置在96孔板上的磁性支架在室温2分钟。确保上清出现彻底清除。 </li><li>制备新鲜的80％乙醇溶液（24个样品，加入2毫升蒸馏水至8毫升乙醇中）。取出并弃去上清液，注意不要打扰珠。 </li><li>同时使板在磁性支架，加入200微升新鲜制备的80％乙醇中，以每孔，而不会干扰珠孵育该板为至少30秒在室温下进行。弃上清，重复此洗了第二次。确保乙醇已被删除。让干燥，在室温下搅拌10分钟或直到乙醇完全蒸发。 </li><li>从磁底座上卸下板并添加22.5微升RSB缓冲。移液器设置为22.5微升，轻轻吸取整个体积上下10倍。放置在96孔板上的磁性支架并孵育2分钟。确保上清出现彻底清除。轻轻的转移在一个新的96孔板20μL上清液。 注意:任选地，通过使用一个片段分析器确定tagmented样本的大小。而不是上面提到的步骤，使用高分辨率的丙烯酰胺凝胶电泳。片段应为150个基点至1000个基点之间。 </li></ol></li><li>第1次PCR扩增<ol><li>解冻PCR扩增预混含有聚合酶（LP-PMM），索引1引，索引2引物溶液在冰上。匹配组合Ø˚F指标与实验管理器示例表。对于多路，准备从索引1（I7，N7xx）对每个样品不同I7。 </li><li>在每孔加入20微升的LP-PMM，5微升指数1和5微升指数2将吸管50微升，轻轻吸取整个音量上下10倍。覆盖板用粘合剂微密封膜。离心机在280 XG 1分钟，在20℃。 </li><li>将板在热循环仪（盖在100℃加热），然后运行程序1（ 表1）。 注:该过程可以停止在这一点上与该反应过夜储存在热循环或2至8℃下2天。 </li></ol></li></ol> 3，基因组DNA富集:第1天，下午<ol><li>开始之前<ol><li>融寡聚物（CSO），捕获目标缓冲器1（NCT1），并在冰上RSB的解决方案。至少30分钟，在使用前，使纯化的磁珠至室温。 </li></ol></li><li> PCR纯化<ol><li>从步骤2.4离心盘，持续1分钟，在280 XG在20℃。 </li><li>在RT下，在各孔中加入45微升混合纯化的磁珠。移液器设置为95微升，轻轻吸取整个体积上下10倍。下室温温育10分钟。放置在96孔板上的磁性支架在室温2分钟。 </li><li>确保上清出现彻底清除。制备新鲜的80％乙醇溶液（24个样品，加入2毫升蒸馏水至8毫升乙醇中）。弃去上清液，注意不要打扰颗粒。 </li><li>同时使板在磁性支架，加入200微升新鲜制备的80％乙醇中，以每孔，而不会干扰珠孵育该板进行30秒，在室温下进行。弃上清，重复此洗了第二次。确保乙醇已被删除。让干燥，在室温下搅拌15分钟。 </li><li>从磁底座上卸下板加入40μl的RSB缓冲。设置管TTE至40微升，轻轻吸取整个音量上下10倍。 </li><li>放置在96孔板上的磁性支架并孵育2分钟。上清应该出现彻底清除。轻轻转移38微升上清液到新的96孔板中。 </li><li>测定各样品的通过荧光法的产率。如果所评估的产率是30至50纳克/微升之间的协议的第一部分将被验证。 </li></ol></li><li> 1号杂交<ol><li>普尔高达每池12个样本在这个阶段通过将500ng的每种样品中一种新的96孔板中。确保每个池的最终体积不超过40微升。 注:如果需要的话，减少池的体积通过使用真空浓缩装置​​在RT（注意:DNA的浓度而不能加热进行修改，即使在30℃下加热使DNA降解）。通过添加RSB溶液调整最终体积至40微升。 </li><li>充分混匀NCT1解决方案。对于每个孔含有40微升合并的DNA样品中，加50微升NCT1的，和10μl的CSO。移液器设置为100微升，轻轻吸取整个体积上下10倍。密封该板，并离心1分钟，在280 XG在20℃。 </li><li>将板在热循环仪（盖在100℃加热），然后运行程序2（ 表1）。 </li></ol></li></ol> 4，基因组DNA富集:2日，上午<ol><li>开始之前<ol><li>从DNA富集试剂盒（见表材料/设备）解冻2 N氢氧化钠（HP3），目标洗脱缓冲液1（ET1），目标洗脱缓冲液1 ET2，链亲和素磁珠（SMB），洗涤液1（WS1），洗解决方案2（WS2），洗涤液3（WS3），CSO和NCT1解决方案，在室温。 </li></ol></li><li> 1号杂交洗<ol><li>从该热循环和离心分离1分钟，在280 XG取出96孔板在20℃。取下粘合盖很carefuLLY。 </li><li>从平板转移100μl的反应混合物到新的MIDI 96孔板中并加入250微升以及涡旋SMB溶液。移液器设置为350微升，轻轻吸取整个体积上下10倍。密封该板，并在室温下孵育30分钟。 </li><li>离心机在280 XG 1分钟，在20℃下，除去了粘合剂密封，并放置在盘上的磁性支架，持续2分钟。确保上清出现彻底清除。弃去上清液，并从磁性底座上卸下盘。 </li><li>加入200μl的良好混合WS1溶液进入井含珠。移液器设置为200微升，轻轻吸取的整个体积上下15X避免气泡/泡沫形成。 </li><li>将板在磁性支架，持续2分钟。确保上清出现彻底清除。弃去上清液，并从磁性底座上卸下盘。 </li><li>加入200μl的良好混合WS2溶液进入井其所含进不去珠。移液器设置为200微升，轻轻吸取的整个体积上下15倍，避免形成气泡/泡沫。 </li><li>将板在磁性支架，持续2分钟。确保上清出现彻底清除。弃去上清液，并从磁性底座上卸下盘。 </li><li>加入200μl的良好混合WS2溶液到含有珠的孔中。移液器设置为200微升，轻轻吸取的整个体积上下15x连接。 </li><li>传送在新的96孔板的整个体积。密封该板，并将该板在热循环仪（盖加热至100℃）。在42℃下启动热循环30分钟。 </li><li> Imediately放置盘上的磁性支架，持续2分钟。确保上清出现彻底清除。取下密封并立即丢弃所有上清液。然后，从磁底座上卸下盘。 </li><li>加入200μlWS 2成含珠孔。将吸管200微升，轻轻吸取的整个体积上下15倍，避免形成气泡/泡沫。密封该板，并将该板在热循环仪（盖加热至100℃）。在42℃下启动热循环30分钟。 </li><li>立即将板在磁性支架，持续2分钟。确保上清出现彻底清除。取下密封并立即丢弃所有上清液。然后，从磁底座上卸下盘。 </li><li>加入200μl的良好混合WS3溶液到含有珠的孔中。移液器设置为200微升，轻轻吸取的整个体积上下15x连接。 </li><li>将板在磁性支架，持续2分钟。确保上清出现彻底清除。丢弃所有上清液，并从磁性底座上卸下盘。 </li><li>重复此WS3洗了第二次。 </li><li>除去上清液后，密封板，并短暂离心​​拉下残留上清。在放置板磁性支架，持续2分钟。丢弃剩余的上清液。 </li><li>在0.2 ml管中，混匀28.5微升ET1和1.5微升HP3解决方案。这样的搭配是1池，若再池准备，以备池的数量乘以这些卷。 </li><li>从磁底座上卸下板。加入23微升上述制备的混合物。移液器设置为23微升，轻轻吸取的整个体积上下15x连接。密封该板，并留下5分钟，在室温。离心机在280 XG 1分钟，在20℃。 </li><li>将板在磁性支架，持续2分钟。确保上清出现彻底清除。除去粘合膜和转移21微升上清液到新的96孔板中。 </li><li>加入4微升ET2溶液到每个孔中。移液器设置为25微升，轻轻吸取的整个体积的上下10倍和密封板。 注:该过程可以停止在这一点上与反应贮存7天，在-15℃下。如果板被冻结，启动第二杂交之前完全解冻。 </li></ol></li><li> 第二杂交<ol><li>离心盘，持续1分钟，在280 XG在20℃。除去粘合膜，并添加50μl的NCT1，10微升的CSO，与15微升的PCR级水至25μl的库。移液器设置为100微升，轻轻吸取的整个体积上下10倍。 </li><li>密封该板，并离心1分钟，在280 XG在20℃。 </li><li>将板在热循环仪（盖在100℃加热），然后运行程序2（ 表1）。 </li></ol></li></ol> 5，基因组DNA富集:2日，下午<ol><li>开始之前<ol><li>解冻ET1，ET2，SMB，WS1，WS2，WS3和HP3溶液在RT杂交洗涤。从DNA富集试剂盒，解冻PCR扩增预混含有聚合酶（TC-PMM），PCR引物鸡尾酒（PPC），和RSB的解决方案，在冰上进行PCR扩增。 </li></ol></li><li> 第二杂交洗<ol><li>按照完全相同的协议为4.2 - 1号杂交洗。 </li></ol></li><li> PCR扩增<ol><li>在新的96孔板中，混合20微升洗脱的来自步骤5.2中，将25μl的TC-PMM的，和5μl的PPC。移液器设置为50微升，轻轻吸取的整个体积上下10倍。密封该板，并离心1分钟，在280 XG在20℃。 </li><li>将板在热循环仪（盖在100℃加热），然后运行程序3（ 表1）。 </li></ol></li></ol> 6，基因组DNA富集:3天<ol><li>开始之前<ol><li>冰解冻RSB的解决方案。至少30分钟，在使用前，使纯化的磁珠至室温。 </li></ol></li><li> PCR纯化<ol><li>离心反应板从步骤5.3，持续1分钟，在280 XG在20℃，并除去粘合剂覆盖。 </li><li>在室温下，加入90微升的previously混合纯化的磁珠在每个孔中。移液器设置为140微升，轻轻吸取整个体积上下10倍。孵育15分钟，在室温。放置在96孔板上的磁性底座5分钟，在室温。确保上清出现彻底清除。 </li><li>制备新鲜的80％乙醇溶液（2样品中，加入200微升的蒸馏水，以800微升乙醇）。弃去上清液，注意不要打扰颗粒。 </li><li>同时使板在磁性支架，加入200微升新鲜制备的80％乙醇中，以每孔，而不会干扰珠孵育该板进行30秒，在室温。弃上清，重复此清洗一次。确保乙醇已被删除。让干燥在室温进行15分钟或直到乙醇完全蒸发，而该板是在磁性支架上。 </li><li>从磁底座上卸下板，加入30微升的RSB缓冲。移液器设置为30微升，并轻轻吸取吨他整个音量上下10倍。将培养板在室温下2分钟。 </li><li>放置在96孔板上的磁性支架并孵育5分钟，或直至上清液出现完全清楚。轻轻转移28微升上清液到新的96孔板（板类型:TCY）。 注:该过程可以停止在这个阶段，并存储在-15℃下进行长达7天的反应。 </li></ol></li><li>库验证和定量<ol><li>通过使用片段分析仪确定库的质量。高分辨率的聚丙烯酰胺凝胶电泳可以用来代替上述的步骤，但不推荐使用。片段应为150个基点至1000个基点之间。 注:推荐:通过qPCR定量库测序过程中获得集群的最佳数量。 </li><li>作为参考，使用经过验证库（2米）。如果第一个库正在准备中，使用规定的sequenc校准噬菌体PhiX的DNA（10纳米）荷兰国际集团的设备。 </li><li>制备阳性对照的系列稀释液，得到7个点，20，16，8,6，4,2，和下午1点在EBT（EB洗脱缓冲液+0.1％吐温20）。淡化利益图书馆在1/1000〜1/2000。每一点必须以一式三份进行分析。 </li><li>制备下列混合物（乘法卷所使用的孔的数目）:对于1个孔中，添加10微升的SYBR Green含有定量PCR主混合物（2×），0.2微升正向引物（10μM，AATGATACGGCGACCACCGAGAT），0.2微升反向引物的（10μM，CAAGCAGAAGACGGCATACGA），和7.6微升的PCR级水。 </li><li>混合后，废除在96孔板中，18微升混合液到每个孔中，并加入2μl的阳性对照和文库稀释。密封该板，并离心1分钟，在280 XG在20℃。然后，把平板的定量PCR热循环和运行程序4（ 表1）。 </li><li>分析结果如常规绝对量化，得到每个升的产率ibrary。定量PCR的优点是，它仅量化的DNA，将与流动池进行交互。 注:请不要使用的DNA荧光定量由于DNA分子模拟在试剂中的一个存在。这种量化方法将高估图书馆产量，从而低估了集群得分。 </li></ol></li><li>排序下水<ol><li>稀释每个库在4纳米的30微升的洗脱缓冲液（EB）的最终体积，并集中所有的图书馆，拌匀。 </li><li>准备在EB缓冲液新鲜的0.2N的NaOH溶液，搅拌10微升该NaOH溶液与10微升汇集库。搅拌均匀，准确地孵育5分钟在室温。 </li><li> 5分钟后，立即加入980μL杂交液（从测序盒）拌匀。然后，混合400μL与600μL杂交液这种混合物。拌匀并注入了8转600微升到300周期盒（2×150）PM。启动顺序。 </li></ol></li></ol> 7，数据分析<ol><li>一旦设备处理的数据中，.bam和.vcf文件转移到新电脑上。 注:转座子碎片结合的脚印。因此，软件会自动修剪这些数据。 </li><li>收集与所述设备分析所获得的遗传变异。 </li><li>通过按照制造商的指示进行分析导出的文件（.bam，的.vcf）与另一软件（阿拉穆特）。然后，比较用两种分析中得到的遗传变异。唯一的变化检测两次都被认为存在。 </li></ol> 表1 PCR条件 <table><tbody><tr><td> 节目 </td><td> 温度 </td><td> 时间 </td><td> 民。重复 </td></tr><tr><td rowspan="7"> 1 </td><td> 72℃ </td><td> 3分钟</td><td> 1 </td></tR&gt; <tr><td> 98℃ </td><td> 30秒</td><td> 1 </td></tr><tr><td> 98℃ </td><td> 10秒</td><td rowspan="3"> 10 </td></tr><tr><td> 60℃ </td><td> 30秒</td></tr><tr><td> 72℃ </td><td> 30秒</td></tr><tr><td> 72℃ </td><td> 5分钟</td><td> 1 </td></tr><tr><td> 10℃ </td><td>无限</td><td></td></tr><tr><td colspan="4"></td></tr><tr><td rowspan="19"> 2 </td><td> 95℃ </td><td> 10分钟</td><td> 1 </td></tr><tr><td> 93℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 91℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 89℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 87℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 85°C </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 83℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 81℃ </tD&gt; <td> 1分钟</td><td> 1 </td></tr><tr><td> 79℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 77℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 75℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 71℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 69℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 67℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 65℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 63℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 61℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 59℃ </td><td> 1分钟</td><td> 1 </td></tr><tr><td> 58℃ </td><td> 1分钟</td><td> 16-18小时</td></tr><tr><td colspan="4"></td></tr><tr><td rowspan="6"> 3 </td><td> 98℃ </td><td> 30秒</td><td> 1 </td></tr><tr><td> 98℃ </td><td> 10秒</td><td rowspan="3"> 10 </td></tr><tr><td> 60℃ </td><td> 30秒</td></tr><tr><td> 72℃ </td><td> 30秒</td></tr><tr><td> 72℃ </td><td> 5分钟</td><td> 1 </td></tr><tr><td> 10℃ </td><td>无限</td><td></td></tr><tr><td colspan="4"></td></tr><tr><td rowspan="3"> 4 </td><td> 95℃ </td><td> 10分钟</td><td> 1 </td></tr><tr><td> 95℃ </td><td> 15秒</td><td rowspan="2"> 40 </td></tr><tr><td> 60℃ </td><td> 1分钟</td></tr></tbody></table>

<ol>
	<li>Cornelisse, C. J., Cornelis, R. S., Devilee, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+responsible+for+familial+breast+cancer.">Genes responsible for familial breast cancer.</a> Pathol. Res. Pract. 192 (7), 684-693 (1996).</li><li>Culver, J., Lowstuter, K., Bowling, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+breast+cancer+risk+and+BRCA1/2+carrier+probability.">Assessing breast cancer risk and BRCA1/2 carrier probability.</a> Breast Dis. 27, 5-20 (2007).</li><li>Cox, D. 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=Common+variants+of+the+BRCA1+wild-type+allele+modify+the+risk+of+breast+cancer+in+BRCA1+mutation+carriers.">Common variants of the BRCA1 wild-type allele modify the risk of breast cancer in BRCA1 mutation carriers.</a> Hum. Mol. Genet. 20 (23), 4732-4747 (2011).</li><li>Maia, 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=Effects+of+BRCA2+cis-regulation+in+normal+breast+and+cancer+risk+amongst+BRCA2+mutation+carriers.">Effects of BRCA2 cis-regulation in normal breast and cancer risk amongst BRCA2 mutation carriers.</a> Breast Cancer Res. 14 (2), 63 (2012).</li><li>Anczuk&#243;w, O., 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=BRCA2+deep+intronic+mutation+causing+activation+of+a+cryptic+exon:+opening+toward+a+new+preventive+therapeutic+strategy.">BRCA2 deep intronic mutation causing activation of a cryptic exon: opening toward a new preventive therapeutic strategy.</a> Clin. Cancer Res. 18 (18), 4903-4909 (2012).</li><li>Brewster, B. 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=Identification+of+fifteen+novel+germline+variants+in+the+BRCA1+3'UTR+reveals+a+variant+in+a+breast+cancer+case+that+introduces+a+functional+miR-103+target+site.">Identification of fifteen novel germline variants in the BRCA1 3'UTR reveals a variant in a breast cancer case that introduces a functional miR-103 target site.</a> Hum. Mutat. 33 (12), 1665-1675 (2012).</li><li>Roy, R., Chun, J., Powell, S. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BRCA1+and+BRCA2:+different+roles+in+a+common+pathway+of+genome+protection.">BRCA1 and BRCA2: different roles in a common pathway of genome protection.</a> Nat Rev Cancer. 12 (1), 68-78 (2012).</li><li>Ma, X. 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=First+evidence+for+the+contribution+of+the+genetic+variations+of+BRCA1-interacting+protein+1+(BRIP1)+to+the+genetic+susceptibility+of+cervical+cancer.">First evidence for the contribution of the genetic variations of BRCA1-interacting protein 1 (BRIP1) to the genetic susceptibility of cervical cancer.</a> Gene. 524 (2), 208-213 (2013).</li><li>Haanp&#228;&#228;, M., Pylk&#228;s, K., Moilanen, J. S., Winqvist, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+need+for+routine+clinical+testing+of+PALB2+c.1592delT+mutation+in+BRCA+negative+Northern+Finnish+breast+cancer+families.">Evaluation of the need for routine clinical testing of PALB2 c.1592delT mutation in BRCA negative Northern Finnish breast cancer families.</a> BMC Med. Genet. 14, 82 (2013).</li><li>Southey, M. C., Teo, Z. L., Winship, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PALB2+and+breast+cancer:+ready+for+clinical+translation.">PALB2 and breast cancer: ready for clinical translation.</a> Appl. Clin. Genet. 6, 43-52 (2013).</li><li>Ulahannan, D., Kovac, M. B., Mulholland, P. J., Cazier, J. B., Tomlinson, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+and+implementation+issues+in+using+next-generation+sequencing+of+cancers+in+clinical+practice.">Technical and implementation issues in using next-generation sequencing of cancers in clinical practice.</a> Br. J. Cancer. 109, 827-835 (2013).</li><li>Hernan, I., Borr&#224;s, E., de Sousa Dias, M., Gamundi, M. J., Ma&#241;&#233;, B., Llort, G., Ag&#250;ndez, J. A., Blanca, M., Carballo, M. <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+genomic+variations+in+BRCA1+and+BRCA2+genes+by+long-range+PCR+and+next-generation+sequencing.">Detection of genomic variations in BRCA1 and BRCA2 genes by long-range PCR and next-generation sequencing.</a> J. Mol. Diagn. 14, 286-293 (2012).</li><li>Knierim, E., Lucke, B., Schwarz, J. M., Schuelke, M., Seelow, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+comparison+of+three+methods+for+fragmentation+of+long-range+PCR+products+for+next+generation+sequencing.">Systematic comparison of three methods for fragmentation of long-range PCR products for next generation sequencing.</a> Plos One. 6, e28240 (2011).</li><li>de Sousa Dias, M., Hernan, I., Pascual, B., Borr&#224;s, E., Ma&#241;&#233;, B., Gamundi, M. J., Carballo, M. <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+novel+mutations+that+cause+autosomal+dominant+retinitis+pigmentosa+in+candidate+genes+by+long-range+PCR+amplification+and+next-generation+sequencing.">Detection of novel mutations that cause autosomal dominant retinitis pigmentosa in candidate genes by long-range PCR amplification and next-generation sequencing.</a> Mol. Vis. 19, 654-664 (2013).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Breast+Cancer+Linkage+Consortium.+Cancer+Risks+in+BRCA2+Mutation+Carriers.">The Breast Cancer Linkage Consortium. Cancer Risks in BRCA2 Mutation Carriers.</a> J. Natl. Cancer Inst. 91, 1310-1316 (1999).</li><li>Levy-Lahad, E., Friedman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+risks+among+BRCA1+and+BRCA2+mutation+carriers.">Cancer risks among BRCA1 and BRCA2 mutation carriers.</a> Br. J. Cancer. 96 (1), 11-15 (2007).</li><li>Risch, H. 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=Population+BRCA1+and+BRCA2+mutation+frequencies+and+cancer+penetrances:+a+kin-cohort+study+in+Ontario,+Canada.">Population BRCA1 and BRCA2 mutation frequencies and cancer penetrances: a kin-cohort study in Ontario, Canada.</a> J. Natl. Cancer Inst. 98 (23), 1694-1706 (2006).</li><li>Slater, E. 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=PALB2+mutations+in+European+familial+pancreatic+cancer+families.">PALB2 mutations in European familial pancreatic cancer families.</a> Clin. Genet. 78, 490-494 (2010).</li><li>Brennan, G. T., Relias, V., Saif, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BRCA+and+pancreatic+cancer.">BRCA and pancreatic cancer.</a> J.O.P. 14 (4), 325-328 (2013).</li></ol>

The authors have no conflicts of interest to declare.

广泛使用的NGS的设备和技术已经在癌症和遗传疾病的研究提供新的机会。除了全基因组测序和RNA测序，产生大量的无数的患者选择的基因组DNA序列的分析同时提供了诊断的前景十分广阔。在这里，我们开发了使用Nextera技术，同时研究11完整的基因在24例中等通量测序装置（表材料/设备）的特定设计（随叫随到）。该协议允许快速生成的数据，使患者的错误的低风险的担忧快速反应。如示于图6中 ，用Sanger测序检测到的所有遗传变异，通过使用转座子为基础的制备试剂盒，还检测到。该方法是可靠和容易理解，尤其适用于直接分析复杂的插入缺失的改变。然而，重要的是要注意，对于基因位于负链，它是必要的补充（不可逆）的核苷酸。目前的工作进行了设计的完整的11个基因的分析，但该协议是任何设计的选择是一样的。转座为基础的技术也可以用于从远距离的PCR产物12的文库制备。实际上，该算法（由生产商开发的）用于设计（制造商网站工具，请参阅表的材料/设备）特别地专用于该协议。除了转座为基础的技术，DNA片段化的另外两个机制图书馆前的准备工作还有:机械破碎和酶的碎片。机械DNA片段是可重现的，但需要一个超声发生器，以及库制剂是更昂贵和更耗时。酶的DNA片段往往导致因限制性酶切位点的位置问题进行DNA采集，导致在缺乏靶序列覆盖。然而，每一个策略的DNA片段都有优点和缺点，但似乎得到相当类似的结果，至少在长范围PCR产物的碎片13。使用一个新的酶鸡尾酒的似乎提供了相同的结果的那些机械DNA片段14获得。转座为基础的技术需要高质量的DNA（不适用于从DNA FFPE组织中提取）。此外，转座活性的需要超过300 bp的片段，表明感兴趣的片段必须比该长度更长。这种特殊性说明需要为高质量的，未片段化DNA。 到现在为止，BRCA1和BRCA2的遗传变异进 ​​行了研究，在家族性乳腺癌和卵巢癌。然而，BRCA基因，特别是BRCA2的介入，现在怀疑在其他癌症，特别是在胰腺15，前列腺16，和睾丸癌17。 PALB2，BRCA基因的合作伙伴，对基因的改变也与胰腺癌相关的18。此外，最近出现患者窝藏BRCA基因突变，以及在较小程度上PALB2的突变，表现出他们的胰腺癌到PARP抑制剂19更好的反应。由于BRCA1，BRCA2和PALB2与癌症的家族性风险，并可能与易患具体的治疗有关，它探索了BRCA1和BRCA2伙伴筛查家族性癌症的风险，并在筛查癌症治疗的反应似乎很重要。 从这些数据中，DNA断裂修复相关基因的分析似乎不仅对敏感性的筛选，而且对治疗应答的推测的指示器是很重要的。而且，随着该协议提出完整的基因分析，可以COV呃所有改变，可能是先天本（以及存在于癌细胞），如突变的启动子，剪接位点，或位于内含子调控剪接位点。迄今为止，在非编码基因序列的改变还没有深入研究，但全基因组关联研究的发展能够突出这些序列的重要功能。 总之，本文研制的转座为基础的设计是一个有趣的方式来探索基因异常在参与DNA损伤修复的癌症易感基因。测序11完整基因的24名患者的可能性同时是一个重要的优点与Sanger测序方法，这是一个相当困难的技术进行筛选比较。

2010年，有近150万人（主要是女性）开发的全球乳腺癌。据估计，5％至10％的这些情况是遗传的。大约20年前，BRCA1和BRCA2被认定为参与遗传性乳腺癌和卵巢癌1。自从大约15年前，BRCA1和BRCA2基因的编码区已被测序，以确定遗传倾向的乳腺癌和卵巢癌。在BRCA1和BRCA2的变化在10％至20％选家庭2表明这些区域的分析是不够有效的筛查检测。近日，BRCA1和BRCA2基因的非编码序列（启动子，内含子，3&#39;UTR）的分析，强调了新的基因突变/变异可能与乳腺癌3-6的风险较高。 BRCA1和BRCA2蛋白参与同源重组修复（HHR），它是由众多的合作伙伴7完成。而在BRCA1或BRCA2基因的改变诱发的DNA修复缺陷，其他伙伴也可能会影响到乳腺癌的危险性。这个假设似乎因为BRIP1 8已被验证，PALB2 9对宫颈癌和乳腺癌的一个行之有效的影响，分别为。此外，还有两个其他的"中度风险"的乳腺癌易感基因，ATM和CHEK2，也可以定期研究10。 从这些研究之后，我们决定开发一个协议分析11个基因（ 自动取款机，BARD1，BRCA1，BRCA2，BRIP1，CHEK2，PALB2，RAD50，RAD51C，RAP80和TP53），24例同时使用一个非常简单且相对基于转座技术快速的协议，以充实和测序中等吞吐量的设备上。谢谢该技术，我们测序完成基因的启动子的3&#39;-UTR的端部的起动，除了RAP80，对于其中的2500 bp的内含子区域没有被覆盖的（CHR 5:176,381,588-176,390,180）。这代表共约1000300 BP研究与2,734探头。通常，BRCA1和BRCA2的外显子序列由Sanger测序，这需要1.5个月时间少于20名患者进行分析。本协议（ 图1），在相同的时间，11个完整的基因为大于75的病人可以进行分析。

<table><tbody><tr><td>MiSeq</td><td>Illumina</td><td>SY-410-1001</td><td>Sequencing/medium throughput device</td></tr><tr><td>Nextera Enrichment kit</td><td>Illumina</td><td>FC-123-1208</td><td>Transposase based technology</td></tr><tr><td>300 cycle cartridge</td><td>Illumina</td><td>15033624</td><td></td></tr><tr><td>AMPure beads</td><td>Beckman Coulter</td><td>A63881</td><td>Magnetic purification beads</td></tr><tr><td>Magnetic stand</td><td>Alpaqua</td><td>A32782</td><td></td></tr><tr><td>96-well Plates</td><td>Life Technologies</td><td>4306737</td><td></td></tr><tr><td>MIDI 96-well plates</td><td>Biorad</td><td>AB0859</td><td></td></tr><tr><td>Microseal A</td><td>Biorad</td><td>MSA-5001</td><td>This seal is necessary only for PCR amplification. Other standard seals can be used throughout the experiment</td></tr><tr><td>MiSeq</td><td>Illumina</td><td></td><td>Provided with the sequencing device&lt;br/&gt; Experiment Manager software&lt;br/&gt; Internet adress: http://designstudio.illumina.com/NexteraRc/project/new&lt;br/&gt; Manufacturer website tool</td></tr></tbody></table>

gdna enrichment by a transposase-based technology for ngs analysis of the whole sequence of brca1, brca2, and 9 genes involved in dna damage repair

广泛使用的新一代测序开辟了为癌症研究和诊断的新途径。农工商会带来癌症大量的新数据，尤其是癌症遗传学。目前的知识和未来发现将使得有必要研究一种巨大的基因可能涉及遗传因素对癌症的数目。在这方面，我们开发了一个Nextera设计研究11参与DNA损伤修复完整的基因。该协议的开发是为了安全地同时学习11个基因（ATM，BARD1，BRCA1，BRCA2，BRIP1，CHEK2，PALB2，RAD50，RAD51C，RAD80和TP53）从启动到3&#39;-UTR 24例。这个协议中，根据转座技术和基因组DNA富集，给出了一个很大的优点的时间为基因诊断由于采样复用条件。这个协议可以安全地使用与血液的gDNA。

样品QC成果这种方法来确定靶基因的序列的能力是基于基因组DNA（ 图2A）的质量和tagmentation步骤的质量。如果tagmentation是不够（ 图2B，上图），测序将不会令人满意。如上面所提到的，tagmentation纯化后，将基因组DNA的应tagmented成片段从150 100bp到1000bp的多数大约300 bp的片段（ 图2B，下图）。 在文库制备结束，库质量是通过使用生物分析器检查。所获得的信息应该是大致相同tagmentation后但具有更高数目的片段（ 图2C）。如果文库质量不等同于图1C中 ，测序不应该执行。 一旦测序发动的测序装置，该聚类的得分是在9个周期可用的和应该是80万和110万簇/平方毫米（ 图3A）之间。如果这个数较低，则排序将不令人满意，尤其是在阅读深度。如果它是高时，图像会模糊（ 图3B），并没有分析将由于不可能区分集群完成。 在运行结束时，重要的是要检查的质量得分。按照上面详述的方案，该运行的质量得分将对应于图4。测序的质量是由Q-得分（Q-30）来表示。以验证实验中，至少约75％的序列（群）应具有的Q分数大于或等于30。 测序结果这个实验的目的是研究宪法的遗传异常。在这种情况下，基因异常为prESENT在基因组的50％。正因为如此，在测序深度不是很重要。在此，我们准备同时进行测序2库12​​例为11完整的基因。以获得高的测序深度，多路复用患者数目必须减少。作为基因组DNA富集是基于捕获由探针中，富集不均质的（ 图5）。与我们的协议，我们产生了大约6千兆读取，诱导150的平均覆盖率为读取每个基地，以最小20，最大为330读取。这些读出的深度是根据在临床实践11使用NGS的。尽管读取深处，这可能是由于基因组DNA富集捕获和基因不是主要的重排的非均质性，值得注意的是，在Q-得分总是优于30（ 图5），从而验证所述序列是很重要的。 然而，通过比较的结果来验证技术是很重要的■对于那些与Sanger测序获得的所得。在17例由双方Sanger测序法测序（编码BRCA1和BRCA2基因的序列）和转座基础的技术，我们发现同样的330遗传变异（SNP和突变）。作为一个例子，在BRCA1基因的点突变（ 图6左）和缺失中的BRCA2基因4个碱基（ 图6右）由两个桑格和转座为基础的方法进行检测。如BRCA1是13号染色体上的反向链，值得注意的是，它是必要的，以补充（但不反转）中获得的序列，而对于BRCA2（正链），将获得的序列并不需要进行补充是重要。如示于图5中 ，NGS技术给出了遗传变异的估计频率，而桑格方法没有。此外，特别是对于插入缺失变异的解释是，吴更容易的技术。 如图6右边，插入缺失变异的序列显示为加扰的电泳图谱，需要正向和反向测序破译插入或删除序列。与NGS分析，插入或删除的序列被直接确定，从而减少误解的危险。最后，转座基础的技术使我们能够分析更多的靶基因，我们发现并没有涵盖的Sanger测序许多新的遗传变异序列。这些结果将另行公布。 <img alt="图1" fo:content-width="6in" src="/files/ftp_upload/51902/51902fig1highres.jpg" width="600" /> 图1的程序流程示意图。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51902/51902fig1large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> 图2:质量控制之前和图书馆准备了。 A.光度法信息的gDNA，可以安全地用于tagmentation。该DNA产率必须高于5纳克/微升，二百八十○分之二百六和二百三十〇分之二百六十零比率必须高于1.8和2。tagmented的gDNA B.片段分析器配置文件。上图表示不够tagmented基因组DNA。下图显示了一个完美的tagmented样本。备库的三片段分析仪配置文件只是顺序启动之前。片段的大小是一样的tagmented基因组DNA（B，下图），但放大的量。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51902/51902fig2large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> &gt; <img alt="图3" fo:content-width="6in" src="/files/ftp_upload/51902/51902fig3highres.jpg" width="600" /> 图3:检查集群生成。在运行过程中的测序设备的答屏幕截图。集群密度应介于800至1,000 K /平方毫米。B.不同的图像对应低密度（上图），高密度（下图）和完善的密度（中图）。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51902/51902fig3large.jpg" target="_blank">请点击这里查看该图的放大版本。 。</a> <img alt="图4" fo:content-width="6in" src="/files/ftp_upload/51902/51902fig4highres.jpg" width="600" /> 图4检查的Q分数。在运行结束时，验证序列应具有生成的群中的至少75％具有Q得分高于30。JPG"目标="_ blank将"&gt;请点击这里查看该图的放大版本。 <img alt="图5" fo:content-width="6in" src="/files/ftp_upload/51902/51902fig5highres.jpg" width="600" /> 图5代表按覆盖数据和其对应的Q分数的阅读深度是异质所有沿覆盖基因。然而，Q分数覆盖区域总是优于Q-30， <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51902/51902fig5large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图6" fo:content-width="6in" src="/files/ftp_upload/51902/51902fig6highres.jpg" width="600" /> 与Sanger测序和转座为基础的技术得到图6。代表性的成果。与Sanger测序发现所有的遗传变异进 ​​行了Ðetected与转座为基础的技术。而点突变很容易理解，插入缺失的改变有时是相当困难的桑格方法来研究。与中等通量设备相关的转座为基础的技术，插入缺失的改变是简单的发现。不过，需要注意的是，有必要补充（而不是反转）获得序列时，靶基因位于负链（这里的BRCA1基因）是非常重要的。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/51902/51902fig6large.jpg" target="_blank">请点击这里查看该图的放大版本。</a>

Watch this Scientific Journal Video about gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair at JoVE.co

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

基因组DNA富集转座为基础的技术的整个序列的农工商分析<em&gt; BRCA1基因</em&gt;<em&gt; BRCA2</em&gt;和9个基因参与DNA损伤修复

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

gdna-enrichment-transposase-based-technology-for-ngs-analysis-whole

Universidad San Sebastian

Research

JoVE Journal

Biology

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

12.2K 次观看.  Centre Georges-François Leclerc. gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair. 

视频: gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

Detection of Rare Mutations in CtDNA Using Next Generation Sequencing

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. However, the application of this method is challenging due to its low sensitivity in analyzing the trace amount of ctDNA in the blood. Furthermore, the method may generate false positive and negative results from this sequencing and subsequent analysis. To improve the feasibility and reliability of ctDNA detection in the clinic, here we present a technique which enriches rare mutations for sequencing, Enrich Rare Mutation Sequencing (ER-Seq). ER-Seq can distinguish a single mutation out of 1 x 107 wild-type nucleotides, which makes it a promising tool to detect extremely low frequency genetic alterations and thus will be very useful in studying disease heterogenicity. By virtue of the unique sequencing adapter&#39;s ligation, this method enables an efficient recovery of ctDNA molecules, while at the same time correcting for errors bidirectionally (sense and antisense). Our selection of 1021 kb probes enriches the measurement of target regions that cover over 95% of the tumor-related driver mutations in 12 tumors. This cost-effective and universal method enables a uniquely successful accumulation of genetic data. After efficiently filtering out background error, ER-seq can precisely detect rare mutations. Using a case study, we present a detailed protocol demonstrating probe design, library construction, and target DNA capture methodologies, while also including the data analysis workflow. The process to carry out this method typically takes 1-2 days.

This manuscript describes a technique for detecting mutations of low frequency in ctDNA, ER-Seq. This method is differentiated by its unique use of two-directional error correction, a special background filter, and efficient molecular acquirement.

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. ...

科研

癌症研究

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

CRISPR/Cas9 和 SsODN 在人细胞中催化点突变修复功能的标准方法研究

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

遗传学

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

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples from cancer patients often directly influences diagnosis, prognosis, and/or therapy selection. As a result, the accurate detection of gene fusions has become a critical component of clinical management for many disease types. Until recently, clinical gene fusion detection was predominantly accomplished through the use of single-gene assays. However, the ever-growing list of gene fusions with clinical significance has created a need for assessing fusion status of multiple genes simultaneously. Next generation sequencing (NGS)-based testing has met this demand through the ability to sequence nucleic acid in massively parallel fashion. Multiple NGS-based approaches that employ different strategies for gene target enrichment are now available for use in clinical molecular diagnostics, each with its own strengths and weaknesses. This article describes the use of anchored multiplex PCR (AMP)-based target enrichment and library preparation followed by NGS to assess for gene fusions in clinical solid tumor specimens. AMP is unique among amplicon-based enrichment approaches in that it identifies gene fusions regardless of the identity of the fusion partner. Detailed here are both the wet-bench and data analysis steps that ensure accurate gene fusion detection from clinical samples.

This article details the use of an anchored multiplex polymerase chain reaction-based library preparation kit followed by next-generation sequencing to assess for oncogenic gene fusions in clinical solid tumor samples. Both wet-bench and data analysis steps are described.

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 ...

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome compared to matched normal tissue. Based on these analyses it is evident that there are many undiscovered genetic drivers of cancer1. Unfortunately these drivers are hidden within a much larger number of passenger anomalies in the genome that do not directly contribute to tumor formation. Another aspect of the cancer genome is that there is considerable genetic heterogeneity within similar tumor types. Each tumor can harbor different mutations that provide a selective advantage for tumor formation2. Performing an unbiased forward genetic screen in mice provides the tools to generate tumors and analyze their genetic composition, while reducing the background of passenger mutations. The Sleeping Beauty (SB) transposon system is one such method3. The SB system utilizes mobile vectors (transposons) that can be inserted throughout the genome by the transposase enzyme. Mutations are limited to a specific cell type through the use of a conditional transposase allele that is activated by Cre Recombinase. Many mouse lines exist that express Cre Recombinase in specific tissues. By crossing one of these lines to the conditional transposase allele (e.g. Lox-stop-Lox-SB11), the SB system is activated only in cells that express Cre Recombinase. The Cre Recombinase will excise a stop cassette that blocks expression of the transposase allele, thereby activating transposon mutagenesis within the designated cell type. An SB screen is initiated by breeding three strains of transgenic mice so that the experimental mice carry a conditional transposase allele, a concatamer of transposons, and a tissue-specific Cre Recombinase allele. These mice are allowed to age until tumors form and they become moribund. The mice are then necropsied and genomic DNA is isolated from the tumors. Next, the genomic DNA is subjected to linker-mediated-PCR (LM-PCR) that results in amplification of genomic loci containing an SB transposon. LM-PCR performed on a single tumor will result in hundreds of distinct amplicons representing the hundreds of genomic loci containing transposon insertions in a single tumor4. The transposon insertions in all tumors are analyzed and common insertion sites (CISs) are identified using an appropriate statistical method5. Genes within the CIS are highly likely to be oncogenes or tumor suppressor genes, and are considered candidate cancer genes. The advantages of using the SB system to identify candidate cancer genes are: 1) the transposon can easily be located in the genome because its sequence is known, 2) transposition can be directed to almost any cell type and 3) the transposon is capable of introducing both gain- and loss-of-function mutations6. The following protocol describes how to devise and execute a forward genetic screen using the SB transposon system to identify candidate cancer genes (Figure 1).

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

A method of identifying unknown drivers of carcinogenesis using an unbiased approach is described. The method uses the Sleeping Beauty transposon as a random mutagen directed to specific tissues. Genomic mapping of transposon insertions that drive tumor formation identifies novel oncogenes and tumor suppressor genes

鉴定<em&gt;睡美人</em&gt;转座子插入连接器介导的PCR实体瘤

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome ...

医学

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter assays, embryonic stem cell viability assays, and therapeutic drug-based sensitivity assays. Although they have clarified a lot of BRCA1 variants, these assays involving the use of exogenously expressed BRCA1 variants are associated with overexpression issues and cannot be applied to post-transcriptional regulation. To resolve these limitations, we previously reported a method for functional analysis of BRCA1 variants via CRISPR-mediated cytosine base editor that induce targeted nucleotide substitution in living cells. Using this method, we identified variants whose functions remain ambiguous, including c.-97C&#62;T, c.154C&#62;T, c.3847C&#62;T, c.5056C&#62;T, and c.4986+5G&#62;A, and confirmed that CRISPR-mediated base editors are useful tools for reclassifying the variants of uncertain significance in BRCA1. Here, we describe a protocol for functional analysis of BRCA1 variants using CRISPR-based cytosine base editor. This protocol provides guidelines for the selection of target sites, functional analysis and evaluation of BRCA1 variants.

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

People with BRCA1 mutations have a higher risk of developing cancer, which warrants accurate evaluation of the function of BRCA1 variants. Herein, we described a protocol for functional assessment of BRCA1 variants using CRISPR-mediated cytosine base editors that enable targeted C:G to T:A conversion in living cells.

使用 CRISPR 介质基础编辑器对 BRCA1 变体进行功能评估

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter ...

Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter

Double strand breaks (DSBs) in DNA are the most cytotoxic type of DNA damage. Because a myriad of insults can result in these lesions (e.g., replication stress, ionizing radiation, unrepaired UV damage), DSBs occur in most cells each day. In addition to cell death, unrepaired DSBs reduce genome integrity and the resulting mutations can drive tumorigenesis. These risks and the prevalence of DSBs motivate investigations into the mechanisms by which cells repair these lesions. Next generation sequencing can be paired with the induction of DSBs by ionizing radiation to provide a powerful tool to precisely define the mutations associated with DSB repair defects. However, this approach requires computationally challenging and cost prohibitive whole genome sequencing to detect the repair of the randomly occurring DSBs associated with ionizing radiation. Rare cutting endonucleases, such as I-Sce1, provide the ability to generate a single DSB, but their recognition sites must be inserted into the genome of interest. As a result, the site of repair is inherently artificial. Recent advances allow guide RNA (sgRNA) to direct a Cas9 endonuclease to any genome locus of interest. This could be applied to the study of DSB repair making next generation sequencing more cost effective by allowing it to be focused on the DNA flanking the Cas9-induced DSB. The goal of the manuscript is to demonstrate the feasibility of this approach by presenting a protocol that can define mutations that stem from the repair of a DSB upstream of the CD4 gene. The protocol can be adapted to determine changes in the mutagenic potential of DSB associated with exogenous factors, such as repair inhibitors, viral protein expression, mutations, and environmental exposures with relatively limited computation requirements. Once an organism's genome has been sequenced, this method can be theoretically employed at any genomic locus and in any cell culture model of that organism that can be transfected. Similar adaptations of the approach could allow comparisons of repair fidelity between different loci in the same genetic background.

Presented here is sgRNA/CAS9 endonuclease and next-generation sequencing protocol that can be used to identify the mutations associated with double strand break repair near the CD4 promoter.

使用下一代测序来鉴定与CD4启动子附近CAS9诱导的双链断裂修复相关的突变

Double strand breaks (DSBs) in DNA are the most cytotoxic type of DNA damage. Because a myriad of insults can result in these lesions (e.g., replication ...

Analysis of DNA Double-strand Break (DSB) Repair in Mammalian Cells

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death. Cells repair DSBs using two major pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Perturbations of NHEJ and HR are often associated with premature aging and tumorigenesis, hence it is important to have a quantitative way of measuring each DSB repair pathway. Our laboratory has developed fluorescent reporter constructs that allow sensitive and quantitative measurement of NHEJ and HR. The constructs are based on an engineered GFP gene containing recognition sites for a rare-cutting I-SceI endonuclease for induction of DSBs. The starting constructs are GFP negative as the GFP gene is inactivated by an additional exon, or by mutations. Successful repair of the I-SceI-induced breaks by NHEJ or HR restores the functional GFP gene. The number of GFP positive cells counted by flow cytometry provides quantitative measure of NHEJ or HR efficiency.

Analysis of DNA Double-strand Break DSB Repair in Mammalian Cells

This article describes GFP-based fluorescence in vivo assays that separately quantify homologous recombination and nonhomologous end joining in mammalian cells.

在哺乳动物细胞中的DNA双链断裂（DSB）的维修分析

DNA double-strand breaks are the most dangerous DNA lesions that may lead to massive loss of genetic information and cell death.  Cells repair DSBs using ...

生物学

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using bioinformatics pipelines (&#34;dry bench&#34;). Both elements are essential to produce accurate and reliable results, which are particularly critical for clinical laboratories. Targeted NGS technologies have increasingly found favor in oncology applications to help advance precision medicine objectives, yet the methods often involve disconnected and variable wet and dry bench workflows and uncoordinated reagent sets. In this report, we describe a method for sequencing challenging cancer specimens with a 21-gene panel as an example of a comprehensive targeted NGS system. The system integrates functional DNA quantification and qualification, single-tube multiplexed PCR enrichment, and library purification and normalization using analytically-verified, single-source reagents with a standalone bioinformatics suite. As a result, accurate variant calls from low-quality and low-quantity formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration (FNA) tumor biopsies can be achieved. The method can routinely assess cancer-associated variants from an input of 400 amplifiable DNA copies, and is modular in design to accommodate new gene content. Two different types of analytically-defined controls provide quality assurance and help safeguard call accuracy with clinically-relevant samples. A flexible &#34;tag&#34; PCR step embeds platform-specific adaptors and index codes to allow sample barcoding and compatibility with common benchtop NGS instruments. Importantly, the protocol is streamlined and can produce 24 sequence-ready libraries in a single day. Finally, the approach links wet and dry bench processes by incorporating pre-analytical sample quality control results directly into the variant calling algorithms to improve mutation detection accuracy and differentiate false-negative and indeterminate calls. This targeted NGS method uses advances in both wetware and software to achieve high-depth, multiplexed sequencing and sensitive analysis of heterogeneous cancer samples for diagnostic applications.

An integrated system for targeted next-generation sequencing of oncology specimens is described. This cross-platform system is optimized for low-quality and low-quantity tumor biopsies, accommodates low DNA inputs, includes well-characterized multi-variant controls, and features a novel variant caller that is informed by quantitative pre-analytical quality control measures.

低质量和低数量肿瘤活检丰枯台过程的集成优化了有针对性的下一代测序

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using ...

Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is essential in revealing cellular events associated with the recruitment and assembly of DNA repair complexes at the site of DNA damage. In the last few years, several powerful tools have been developed to induce site-specific DNA damage. Moreover, novel seminal techniques allow us to study these processes at the single-cell resolution level using both fixed and living cells. Although these techniques have been used to study various biological processes, herein we present the most widely used protocols in the field of DNA repair, Fluorescence Immunostaining (IF) and Chromatin Immunoprecipitation (ChIP), which in combination with endonuclease-based site-specific DNA damage make it possible to visualize and quantify the genomic occupancy of DNA repair factors in a directed and regulated fashion, respectively. These techniques provide powerful tools for the researchers to identify novel proteins bound to the damaged genomic locus as well as their post-translational modifications necessary for their fine-tune regulation during DNA repair.

This article introduces essential steps of immunostaining and chromatin immunoprecipitation. These protocols are commonly used to study DNA damage-related cellular processes and to visualize and quantify the recruitment of proteins implicated in DNA repair.

可视化和量化基于内分泌酶的站点特定DNA损伤

Cells are continuously exposed to various DNA damaging agents, inducing different cellular responses. Applying biochemical and genetic approaches is ...

对GFP和mCherry报告细胞进行FACS分析，以评估NHEJ和HR效率

Analysis of Nonhomologous End Joining and Homologous Recombination Efficiency in HEK-293T Cells Using GFP-Based Reporter Systems

DNA double-strand breaks (DSBs) represent the most perilous DNA lesions, capable of inducing substantial genetic information loss and cellular demise. In response, cells employ two primary mechanisms for DSB repair: nonhomologous end joining (NHEJ) and homologous recombination (HR). Quantifying the efficiency of NHEJ and HR separately is crucial for exploring the relevant mechanisms and factors associated with each. The NHEJ assay and HR assay are established methods used to measure the efficiency of their respective repair pathways. These methods rely on meticulously designed plasmids containing a disrupted green fluorescent protein (GFP) gene with recognition sites for endonuclease I-SceI, which induces DSBs. Here, we describe the extrachromosomal NHEJ assay and HR assay, which involve co-transfecting HEK-293T cells with EJ5-GFP/DR-GFP plasmids, an I-SceI expressing plasmid, and an mCherry expressing plasmid. Quantitative results of NHEJ and HR efficiency are obtained by calculating the ratio of GFP-positive cells to mCherry-positive cells, as counted by flow cytometry. In contrast to chromosomally integrated assays, these extrachromosomal assays are more suitable for conducting comparative investigations involving multiple established stable cell lines.

作者聚焦:通过染色体外 NHEJ 检测和 HR 检测解码 DNA 修复

This protocol describes an extrachromosomal nonhomologous end joining (NHEJ) assay and homologous recombination (HR) assay to quantify the efficiency of NHEJ and HR in HEK-293T cells.

使用基于 GFP 的报告系统分析 HEK-293T 细胞中的非同源末端连接和同源重组效率

DNA double-strand breaks (DSBs) represent the most perilous DNA lesions, capable of inducing substantial genetic information loss and cellular demise. In ...

基因组DNA富集转座为基础的技术的整个序列的农工商分析

摘要

探索更多视频

此视频中的章节