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>

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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?...

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?...

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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

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 ...

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of intellectual disability and account for 20-40% of drug-resistant epilepsy in childhood. High-resolution brain imaging has facilitated in vivo identification of a large group of MCD phenotypes. Despite the advances in brain imaging, genomic analysis and generation of animal models, a straightforward workflow to systematically prioritize candidate genes and to test functional effects of putative mutations is missing. To overcome this problem, an experimental strategy enabling the identification of novel causative genes for MCD was developed and validated. This strategy is based on identifying candidate genomic regions or genes via array-CGH or whole-exome sequencing and characterizing the effects of their inactivation or of overexpression of specific mutations in developing rodent brains via in utero electroporation. This approach led to the identification of the C6orf70 gene, encoding for a putative vesicular protein, to the pathogenesis of periventricular nodular heterotopia, a MCD caused by defective neuronal migration.

A Novel Strategy Combining Array-CGH, Whole-exome Sequencing and In Utero Electroporation in Rodents to Identify Causative Genes for Brain Malformations

Periventricular nodular heterotopia (PNH) is the most common form of malformation of cortical development (MCD) in adulthood but its genetic basis remains unknown in most sporadic cases. We have recently developed a strategy to identify novel candidate genes for MCDs and to directly confirm their causative role in vivo.

一种新的结合阵列全息、全外测序和宫内电穿孔的方法来识别脑畸形的致病基因

Birth defects that involve the cerebral cortex &#8212; also known as malformations of cortical development (MCD) &#8212; are important causes of ...

神经科学

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 ...

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

由外显子捕获和大规模平行测序检测体细胞遗传学改变在肿瘤标本

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

生物学

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 ...

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

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