The authors have no acknowledgements.

伦理声明:获得机构审查委员会（IRB）的批准后进行人体样品所有测试。 1. DNA的提取FFPE组织，外周血和骨髓吸<ol><li>对于DNA FFPE提取试剂盒骨髓FFPE组织<ol><li>与来自未染色的载玻片FFPE组织开始（5 - 5 10部分 - 10微米厚）。 注意:如果开始与组织屑，使用3 - 6个切片在5 - 10微米厚，并跳到步骤1.1.6。 </li><li>放置在滑动的滑动篮和制备四种洗涤储层（两个用于二甲苯和两个100％酒精）。添加600毫升的溶液，以每笼最小体积。 </li><li>通过执行在第一托盘上的5分钟洗涤二甲苯Deparaffinize幻灯片。转移滑动到第二二甲苯洗蓄水池额外的5分钟。 </li><li>脱石蜡后，洗净，用100％乙醇将载玻片5分钟。转移的幻灯片第二醇洗蓄水池额外的5分钟。 </li><li>允许的幻灯片用刀片刮他们入微量离心管中之前，罩下完全干燥。 注意:如果与肿瘤区域的H＆E滑动指示是可用的，对准的幻灯片并刮去仅肿瘤区域。 </li><li>从制造商讲义包括在试剂盒说明书进行。 </li></ol></li><li>对于BM吸出物和外周血<ol><li>根据制造商的说明施舍这些规范提取。 注:有许多市售试剂盒，并从FFPE组织和细胞中的DNA提取方法。实际上，所有的都可以使用。使用是建立在实验室的方法。 <ol><li>使用200μL的外周血（PB）或100μL的BM + 100μLPBS。 </li><li>使用4μL核糖核酸酶A原液。 </li><li>用100微升洗脱缓冲液洗脱。 </li></ol></li></ol></li><li> DNA定量<ol><li>测量使用分光光度计确保约1.8的260 / 280nm光比（纯DNA）的DNA浓度。如果该比率低明显，它可以指示蛋白质，苯酚或其他污染物的存在可与下游应用干扰。 </li><li>调节DNA浓度至约50 - 100纳克/μL与适当的洗脱缓冲液。 </li></ol></li></ol> 2.野生型阻断PCR <ol><li>引物设计<ol><li>对于根据以前感兴趣的基因设计引物，获得描述PCR引物设计13的一般准则。包括M13正向和反向PCR引物通用测序引物。 注:MYD88测定的开发是为了扩增MYD88的外显子5（G259 - N291）和110个核苷酸位于5&#39; 内含子区域，以覆盖内含子4向前的剪接位点和部分和反向引物被设计具有5&#39; -M13序列（M13正向:TGT AAA ACG ACG GCC AGT; M13反向:CAG GAA ACA GCT ATG ACC）以允许互补的测序引物退火（见材料表 ）。 </li></ol></li><li>锁核酸寡核苷酸设计<ol><li>设计阻断寡核苷酸为大约10 - 的长度为15个碱基并互补于其中突变体富集所需WT模板。 注:较短的寡核苷酸将提高不匹配歧视。为了实现高目标专一，不要使用过多的阻挡核苷酸，因为这可能会导致一个非常"粘"寡核苷酸是很重要的。的内容和长度以及阻断核苷酸应该根据需要被阻挡的特定核苷酸进行优化。实现高结合特异性不影响二次结构和冒着自身互补性是很重要的。 </li><li>设计阻断寡核苷酸具有的Tm 10 - 15℃的延伸TEMPERAT上述热循环过程中URE。阻断G或C碱基的- （4个碱基3）通过添加或移除阻塞碱或通过用于DNA代阻断碱基同时避免长段调整的Tm。 <ol><li>导航到寡工具网站（见材料表 ），然后选择"阻断寡核苷酸TM预测"的工具。 </li><li>粘贴期望被阻止进入"寡序列"框中的WT模板的序列。添加"+"在基地前，表示拦截基地。 </li><li>选择"计算"按钮，以确定DNA阻滞剂混合的近似的Tm。 </li></ol></li><li>设计阻断寡核苷酸，以避免二级结构的形成或自二聚体。 <ol><li>导航到寡工具网站（见材料表 ），然后选择"阻断寡核苷酸优化"工具。 </li><li>粘贴期望被阻止进入所述箱WT模板的序列。添加"+"在基地前，表示阻止基地。 </li><li>选择"二级结构"和"自我，只有"两个盒子。按分析按钮审查潜在麻烦的二级结构或自二聚体寡。 注:分数代表在T M"的二级结构和自二聚体第非常粗略的估计。得分较低，因此最佳的，并且可以通过限制阻断受体阻滞剂配对来实现。对于MYD88 [MYD88blocker（TCAGA + AG + C + G + A + C + T + G + A + T + CC / invdT /）]被设计为覆盖氨基酸Q262-I266，并设有一个3&#39;-反转的阻断寡核苷酸的dT同时抑制由延伸DNA聚合酶和降解3&#39;-核酸外切酶。这个特定的阻断寡核苷酸是与被表示为"+ N" 10个阻断碱基的17聚体。剩余的7个碱基是普通DNA核苷酸。 </li></ol></li></ol></li><li> WTB-PCR设置和热循环<ol><li>准备WTB-PCR主设备MiX（MMX）使用2.5μLPCR反应缓冲液10倍重量/ 20毫摩尔MgCl 2，250倍μM的dNTP，0.2μM的正向和反向引物，1.2μMMYD88blocker，和DNA酶，不含RNA酶的，超纯H 2 O操作产生最终溶液体积的每个反应21.75μL。 注:工作浓度为25μL的最终反应体积（加入DNA模板和聚合酶之后）。常规PCR MMX是通过简单地省略除了阻断剂制备。所有协议步骤保持两者WTB和常规PCR相同。这可以验证使用，并确定富集加入阻滞剂的实现。 <ol><li>当计算MMX的量，以制备确保考虑到3个额外的反应（阳性和阴性对照和非模板对照检查污染）和移液误差至少1个另外的反应。 </li></ol></li><li>涡MMX彻底。的MMX可以储存在-80℃下长达着y耳。 </li><li>添加每反应0.25μLTaq DNA聚合酶到MMX和反转混合。一旦聚合酶被添加到MMX，保持它在冰上。 </li><li>把一个新的96孔PCR板上的冷板和移液管的MMX 22μL用聚合酶到每个反应孔。 </li><li>加入3微升基因组DNA（50 - 100毫微克/微升到各自包含MMX聚合酶孔中。 </li><li>将板密封并短暂离心以确保溶液达到各孔的底部。 </li><li>加载热循环仪上的PCR板。 <ol><li>在95℃下进行6分钟初始变性;:设置WTB-PCR反应的热循环条件如下在95℃下变性30秒，引物退火在56℃下进行30秒，和延伸72℃1分钟20秒的40个循环;在72℃下最终延伸10分钟。 注:最佳实践包括完成在物理上分离的区域中的所述协议的其余部分，以避免扩增子污染我ñ未来设置。 </li></ol></li></ol></li><li>根据先前描述的方案14以确定是否WTB-PCR成功，并确认在非模板对照缺乏扩增进行凝胶电泳。 </li><li>通过磁珠PCR产物的纯化<ol><li>从4℃除去磁珠并使其达到室温。 </li><li>转移到新的PCR板10μL的PCR产物。 </li><li>涡磁珠大力完全悬浮的磁性粒子。 </li><li>加入18微升磁珠，每孔上的新盘。移液器混合10倍。 </li><li>在室温下孵育5分钟。 </li><li>放置PCR板到侧裙边磁铁板2分钟，以从溶液中分离磁珠。 </li><li>吸出上清液用多道移液器，避免了珠粒料。 </li><li>分配150μL70％乙醇到每个孔中并在室温下孵育FOř至少30秒。吸用多道移液器乙醇和丢弃的提示。重复这一清洗过程一次。 </li><li>使用20μL的多道移液器吸从每个剩余的乙醇井并丢弃提示。 </li><li>一旦井干燥（〜10分钟），从磁铁板除去和无核酸酶的水加入40μL到各孔中，移液管混合15倍或轻轻涡旋。 </li><li>在室温下孵育2分钟。 </li><li>放置PCR板到磁铁板1分钟以从溶液中分离磁珠。 </li><li>转移35微升纯化的产物，以一个新的PCR板。这是现在纯化的PCR产物准备与双向测序继续或直到需要可以在-20℃保存。 注意:当开发一种新的测定法中，可能需要纯化的PCR产物的定量。 1 - 3纳克/μLDNA扩增子是最优的双向测序。如果浓度一直很低，增加PCR产物和磁珠比例onally（1:1.8）。如果浓度是一致的高，洗脱用的水更大的体积。 </li></ol></li></ol> 3. WTB-PCR产物的测序<ol><li>双向测序 注意:下面的协议是已经过优化以使用较少的试剂制造商的说明书的修饰形式。 <ol><li>准备正向和反向测序反应，用0.25μL现成反应混合物，1.88μL测序缓冲液，1.78μMM13-F或M13-R测序引物和混合添加和DNA酶，不含RNA酶的，超纯H 2 O操作创建最终的溶液每反应9μL体积。这种测序反应混合物可以储存在-20℃下长达一年。 </li><li>吸管9μL正向测序反应混合物成用于前向测序反应新的96孔PCR板的每个孔中的。重复上用于反向测序反应的单独板。 </li><li>添加纯化的WTB-PCR产物吨的1μLÒ每个孔上的正向和反向板两者。 </li><li>将板密封并短暂离心以确保溶液达到各孔的底部。 </li><li>加载热循环仪上的PCR板。 <ol><li>设置了测序反应的热循环条件如下:96℃1分钟;的96℃30个循环保持10秒，50℃5秒，60℃4分钟;保持在4℃下，直到准备用于纯化。 </li></ol></li></ol></li><li>净化测序产品<ol><li>制备的3M乙酸钠（pH 5.2）和100％乙醇中的新鲜1:25溶液。 </li><li>制备新鲜的70％乙醇溶液。 </li><li>添加乙酸钠30μL/ 100％乙醇溶液以正向和反向测序板和移液管混合5倍的各孔中。 </li><li>重新密封板并在室温下在黑暗中孵育20分钟。 </li><li>离心板在2250×g离心15分钟。 </li><li>移开板盖和倒置在浪费容器。 注:反转只有一次或颗粒可以从孔底部松动。 </li><li>放置在干净的纸巾和离心机反相板在150×g离心1分钟。 </li><li> 150μL的70％乙醇添加到每个孔中。 </li><li>重新密封板和旋转在2250×g离心5分钟。 </li><li>重复步骤3.2.6和3.2.7。 </li><li>如果井没有完全干透，让他们风干在室温下。确保样品从光，而干燥的保护。 </li><li>加入10微升甲酰胺，每孔和吸管搭配10倍。重新密封板。 </li><li>变性上热循环仪在95℃下进行3分钟，随后4℃5分钟。 </li><li>变性后，根据制造商的说明15替换测序平台与隔膜和顺序板盖。 </li></ol></li></ol> 4.测序结果分析<ol><li>使用测序DAT可视化测序踪迹根据制造商的说明16和对准相应的参考序列的分析软件。对齐MYD88到NCBI参考序列"NM_002468"。 </li></ol>

<ol>
	<li>Vogelstein, B., Kinzler, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+PCR.">Digital PCR.</a> Proc Natl Acad of Sci U S A. 96 (16), 9236-9241 (1999).</li><li>Milbury, C. A., Li, J., Makrigiorgos, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PCR-based+methods+for+the+enrichment+of+minority+alleles+and+mutations.">PCR-based methods for the enrichment of minority alleles and mutations.</a> Clin Chem. 55 (4), 632-640 (2009).</li><li>Albitar, A., Ma, W., DeDios, I., Estella, J., Agersborg, S., Albitar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+selection+and+high+sensitivity+test+for+MYD88+mutations+using+locked+nucleic+acid.">Positive selection and high sensitivity test for MYD88 mutations using locked nucleic acid.</a> Int J Lab Hematol. 38 (2), 133-140 (2016).</li><li>Dominguez, P. L., Kolodney, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wild-type+blocking+polymerase+chain+reaction+for+detection+of+single+nucleotide+minority+mutations+from+clinical+specimens.">Wild-type blocking polymerase chain reaction for detection of single nucleotide minority mutations from clinical specimens.</a> Oncogene. 24 (45), 6830-6834 (2005).</li><li>Gray, P. N., Dunlop, C. L. M., Elliott, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Not+All+Next+Generation+Sequencing+Diagnostics+are+Created+Equal:+Understanding+the+Nuances+of+Solid+Tumor+Assay+Design+for+Somatic+Mutation+Detection.">Not All Next Generation Sequencing Diagnostics are Created Equal: Understanding the Nuances of Solid Tumor Assay Design for Somatic Mutation Detection.</a> Cancers. 7 (3), 1313-1332 (2015).</li><li>Smith, E. N., 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=Biased+estimates+of+clonal+evolution+and+subclonal+heterogeneity+can+arise+from+PCR+duplicates+in+deep+sequencing+experiments.">Biased estimates of clonal evolution and subclonal heterogeneity can arise from PCR duplicates in deep sequencing experiments.</a> Genome Biol. 15 (8), 420 (2014).</li><li>Varettoni, 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=Prevalence+and+clinical+significance+of+the+MYD88+(L265P)+somatic+mutation+in+Waldenstr&#246;m's+macroglobulinemia+and+related+lymphoid+neoplasms.">Prevalence and clinical significance of the MYD88 (L265P) somatic mutation in Waldenstr&#246;m's macroglobulinemia and related lymphoid neoplasms.</a> Blood. 121 (13), 2522-2528 (2013).</li><li>Xu, 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=MYD88+L265P+in+Waldenstr&#246;m+macroglobulinemia,+immunoglobulin+M+monoclonal+gammopathy,+and+other+B-cell+lymphoproliferative+disorders+using+conventional+and+quantitative+allele-specific+polymerase+chain+reaction.">MYD88 L265P in Waldenstr&#246;m macroglobulinemia, immunoglobulin M monoclonal gammopathy, and other B-cell lymphoproliferative disorders using conventional and quantitative allele-specific polymerase chain reaction.</a> Blood. 121 (11), 2051-2058 (2013).</li><li>Gertz, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Waldenstr&#246;m+macroglobulinemia:+2012+update+on+diagnosis,+risk+stratification,+and+management.">Waldenstr&#246;m macroglobulinemia: 2012 update on diagnosis, risk stratification, and management.</a> Amer J Hematol. 87 (5), 503-510 (2012).</li><li>Salar, 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=MYD88+(L265P)+Mutation+Confers+Very+Poor+Response+and+Outcome+after+Second-Line+Therapy+in+Patients+with+Diffuse+Large+B-Cell+Lymphoma+(DLBCL).">MYD88 (L265P) Mutation Confers Very Poor Response and Outcome after Second-Line Therapy in Patients with Diffuse Large B-Cell Lymphoma (DLBCL).</a> Blood. 124 (21), 1690-1690 (2014).</li><li>Ngo, V. N., 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=Oncogenically+active+MYD88+mutations+in+human+lymphoma.">Oncogenically active MYD88 mutations in human lymphoma.</a> Nature. 470 (7332), 115-119 (2011).</li><li>Pasqualucci, 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=Analysis+of+the+coding+genome+of+diffuse+large+B-cell+lymphoma.">Analysis of the coding genome of diffuse large B-cell lymphoma.</a> Nat Genet. 43 (9), 830-837 (2011).</li><li>Dieffenbach, C., Lowe, T., Dveksler, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=General+concepts+for+PCR+primer+design.">General concepts for PCR primer design.</a> PCR Methods Appl. 3 (3), S30-S37 (1993).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+Gel+Electrophoresis+for+the+Separation+of+DNA+Fragments.">Agarose Gel Electrophoresis for the Separation of DNA Fragments.</a> J Vis Exp. (62), e3923 (2012).</li><li>Applied Biosystems. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> User Guide for Applied Biosystems 3730/3730xl DNA Analyzer. , (2014).</li><li>Applied Biosystems. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> User Guide for SeqScape Software 3. , (2012).</li><li>Mouritzen, P., Nielsen, A. T., Pfundheller, H. M., Choleva, Y., Kongsbak, L., M&#248;ller, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single+nucleotide+polymorphism+genotyping+using+locked+nucleic+acid+(LNA).">Single nucleotide polymorphism genotyping using locked nucleic acid (LNA).</a> Expert Rev Mol Diagn. 3 (1), 27-38 (2003).</li><li>Quach, N., Goodman, M. F., Shibata, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+mutation+artifacts+after+formalin+fixation+and+error+prone+translesion+synthesis+during+PCR.">In vitro mutation artifacts after formalin fixation and error prone translesion synthesis during PCR.</a> BMC Clin Pathol. 4 (1), (2004).</li><li>Gallegos Ruiz, M. I., Floor, K., Rijmen, F., Gr&#252;nberg, K., Rodriguez, J. A., Giaccone, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EGFR+and+K-ras+mutation+analysis+in+non-small+cell+lung+cancer:+comparison+of+paraffin+embedded+versus+frozen+specimens.">EGFR and K-ras mutation analysis in non-small cell lung cancer: comparison of paraffin embedded versus frozen specimens.</a> Anal Cell Pathol. 29 (3), 257-264 (2007).</li><li>Solassol, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KRAS+mutation+detection+in+paired+frozen+and+formalin-fixed+paraffin-embedded+(FFPE)+colorectal+cancer+tissues.">KRAS mutation detection in paired frozen and formalin-fixed paraffin-embedded (FFPE) colorectal cancer tissues.</a> Int J Mol Sci. 12 (5), 3191-3204 (2011).</li><li>Yost, S. 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=Identification+of+high-confidence+somatic+mutations+in+whole+genome+sequence+of+formalin-fixed+breast+cancer+specimens.">Identification of high-confidence somatic mutations in whole genome sequence of formalin-fixed breast cancer specimens.</a> Nucleic Acids Res. 40 (14), e107-e107 (2012).</li><li>Do, H., Wong, S. Q., Li, J., Dobrovic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+sequence+artifacts+in+amplicon-based+massively+parallel+sequencing+of+formalin-fixed+paraffin-embedded+DNA+by+enzymatic+depletion+of+uracil-containing+templates.">Reducing sequence artifacts in amplicon-based massively parallel sequencing of formalin-fixed paraffin-embedded DNA by enzymatic depletion of uracil-containing templates.</a> Clin Chem. 59 (9), 1376-1383 (2013).</li><li>Oldenburg, R. P., Liu, M. S., Kolodney, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+amplification+of+rare+mutations+using+locked+nucleic+acid+oligonucleotides+that+competitively+inhibit+primer+binding+to+wild-type+DNA.">Selective amplification of rare mutations using locked nucleic acid oligonucleotides that competitively inhibit primer binding to wild-type DNA.</a> J Invest Dermatol. 128 (2), 398-402 (2008).</li><li>Mancini, 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=Two+novel+methods+for+rapid+detection+and+quantification+of+DNMT3A+R882+mutations+in+acute+myeloid+leukemia.">Two novel methods for rapid detection and quantification of DNMT3A R882 mutations in acute myeloid leukemia.</a> J Mol Diagn. 17 (2), 179-184 (2015).</li><li>Parsons, B. L., Heflich, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotypic+selection+methods+for+the+direct+analysis+of+point+mutations.">Genotypic selection methods for the direct analysis of point mutations.</a> Mutat Res Rev Muta Res. 387 (2), 97-121 (1997).</li><li>Liu, Q., Swiderski, P., Sommer, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Truncated+amplification:+a+method+for+high-fidelity+template-driven+nucleic+acid+amplification.">Truncated amplification: a method for high-fidelity template-driven nucleic acid amplification.</a> BioTechniques. 33 (1), 129-139 (2002).</li><li>Kaur, M., Zhang, Y., Liu, W. H., Tetradis, S., Price, B. D., Makrigiorgos, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligation+of+a+primer+at+a+mutation:+a+method+to+detect+low+level+mutations+in+DNA.">Ligation of a primer at a mutation: a method to detect low level mutations in DNA.</a> Mutagenesis. 17 (5), 365-374 (2002).</li><li>Albitar, 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=High+Sensitivity+Testing+Shows+Multiclonal+Mutations+in+Patients+with+CLL+Treated+with+BTK+Inhibitor+and+Lack+of+Mutations+in+Ibrutinib-Naive+Patients.">High Sensitivity Testing Shows Multiclonal Mutations in Patients with CLL Treated with BTK Inhibitor and Lack of Mutations in Ibrutinib-Naive Patients.</a> Blood. 126 (23), 716 (2015).</li></ol>

The authors have nothing to disclose.

这里所描述的WTB-PCR测定法使用与设计的延伸部（ 图1）期间阻断WT DNA的扩增阻断寡核苷酸的通用引物组。然后将WTB-PCR产物测序突变分析。 WTB-PCR /桑格的效用在于它的简单性，高灵敏度，以及高通量。使用此处描述的准则，大多数现有的基于桑格测定可简单地通过加入阻断寡核苷酸，大大提高了灵敏度的修改。在这里介绍的例子测定中添加单一阻断寡核苷酸进行PCR的增加的检测的从约16％突变等位基因的极限在WT的背景的以往的测定以&gt; 0.5％，在WTB-PCR测定3。其效果是在临床试验3所示的假阴性64％的减少。确认突变的MYD88存在有显著的诊断和预后IMPLications;虚报的情况下为负可能对整体治疗和病人的管理造成严重后果。与WTB-PCR检测因此非常重要的，尤其是在患者具有相对较低的异常细胞结构。 WTB-PCR技术有很大的不同，有时与LNA，BNA，或PNA介导的PCR夹紧或PCR钳探针测定法2，4，23日 ，24指的是可互换。利用WTB-PCR一些变型涉及qPCR测定这需要对于每个特定突变设计的附加的荧光探针。与此技术相关联甲显著挑战包括需要开发竞争性结合探针WT和突变等位基因之间正确地判断。此外，因为需要一个突变特异性探针，qPCR的方法缺乏检测未知mutatio的能力纳秒。在等位基因特异性方式WTB-PCR块WT扩增的另一种变化。然而代替使用突变特异性引物，阻挡探针特异于WT等位基因抑制完整的引物结合。虽然这种方法不需要特定突变探针定量PCR一样，它没有突变和聚合酶引起的误差可能导致误判增加区分。通过PCR的聚合酶引发的错误而指数扩增可能掩盖罕见突变事件的检测。使用PCR扩增，以富集突变任何方法都有其精度由PCR错误25，26，27的频率的限制。 WTB-PCR /桑格超过许多其它高灵敏度方法的一个基本优点是它可以防止两个WT模板和突变型模板发生由阻挡oligonucleoti靶向的基因区域的外侧，其突变的扩增德。因此，通过聚合酶引入PCR错误与WT DNA一起被有效地滤掉。相反，AS-PCR或定量PCR然而，富集被保留用于通过阻断寡核苷酸覆盖的区域内发生的未知突变。在MYD88的情况下，WTB-PCR允许既L265P和R264 *突变3的检测。其他人已经通过使用WTB-PCR 4，28的多个低频突变的检测同样报告。这使得WTB-PCR理想的研究和临床用途。 随着高灵敏度和消除误报固有的内部控制，WTB-PCR对与拦截器设计很少有额外的步骤适配现有的测序分析的灵活性作出既定的测定许多实验室是有吸引力的选择。同一组中的常规测定法中使用的PCR引物通常可以在WTB-PCR变化来使用。 OT她被强烈建议WTB-PCR-包括UDG处理FFPE组织和适当的PCR后纯化的协议改变的方法，同样转让。阻断剂的设计，因此在实施WTB-PCR测定的关键要素。虽然在这个协议中提出的准则表示在该设计中最相关的因素，各种阻断寡核苷酸应当进行测试，以找到一个块WT扩增高效无二次效果PCR。这包括添加或移除阻断剂的碱来调整的Tm，低聚相对阻挡的位置移动到上述WT模板，并改变寡核苷酸的总长度。阻断剂滴定实验还应当用于建立可接受的测序假象和检测极限的出现之间的平衡。 选择适当的方法，用于检测存在于细胞分钟馏分突变大大在th取决于权证申请和疾病/突变类型。如果突变体的定量是需要的，定量PCR或数字PCR可以提供比WTB-PCR /桑格更可行的解决方案。虽然WTB-PCR主要是定性测定，也可以通过并行地与常规的和WTB-PCR测试的样品以确定所述突变型等位基因频率的粗略估计。因为检测的常规测定的极限是约10 - 20％WT的背景突变等位基因，是合适的结论是由WTB-PCR法检测的突变而不是常规的是存在的浓度小于限制的检测的常规测定。少测定提供的多功能性，简单性和WTB-PCR的鲁棒性。低成本和周转时间短，非常适合评估治疗后残余的疾病或治疗过程中监测出现耐药性突变。此外，检测到以前未描述突变的能力，使WTB-PCR理想的用于研究目的。

Sanger测序传统上一直是测试已知和未知的体细胞突变的金标准。一个Sanger测序的局限性是其检测极限（〜10 -在WT的背景20％突变等位基因）1。灵敏度的这个水平是不合适的检测低的水平的体细胞突变可存在于样品从癌前组织或患者几个循环肿瘤细胞，或当骨髓（BM）是片状。这也使得评估治疗后残留病或由单独2常规测序疗法难以期间检测新兴的抗性突变。通过与锁核酸（LNA）代替传统的PCR介导的野生型在Sanger测序阻断PCR（WTB-PCR），在WT的背景高达0.1％的突变体等位基因的灵敏度，可以实现2，3，4。在（ - 14 NT〜10）阻塞（LNA）的寡核苷酸优先结合WT DNA的DNA WT从而防止扩增WTB-PCR，富集突变体等位基因是通过添加短来实现的。然后将突变体富集WTB-PCR产物进行测序。通过阻断WT DNA，而不是选择用于特定突变WTB-PCR允许存在于分钟的细胞级分的已知和未知突变富集。 多种方法目前用于在小小区的级分检测突变。这包括等位基因特异性PCR，扩增的受阻突变系统（ARMS），变性高效液相色谱（DHPLC），小珠，乳液，放大，和磁性（整经），电场诱导释放和测量（EFIRM），高分辨率熔点等。然而，这些方法大多是由假阳性，并仅检测试验的目的是为4个突变的能力有限</SUP&gt;。 WTB-PCR，但是，允许用户可视化测序痕迹使多个突变类型的检测，并且可以在排除由于伪像或脱氨基事件误报帮助。下一代测序（NGS）可以提供一个合适的替代常规测序，但是，显着更大的成本，复杂性和较长测定时间使其对于许多疾病类型的几个不同的分子标记物或用于监测患者对治疗的新兴的不必要的选项耐药性突变。具有小于5％的突变体等位基因频率。此外，当检测到高的假阳性率变体可造成问题对于基于扩增子-NGS 5,6。 在这里，我们证明在由WTB-PCR在通过Albitar 等人描述筛选在髓样分化因子88基因的突变来实现灵敏度的增加。 3 MYD88 mutatioNS是在华氏巨球蛋白血症（WM）未知的意义（IGM-MGUS）的，IgM单克隆丙种球蛋白病，脾边缘区淋巴瘤（SMZL）重要的诊断和预后因素，和弥漫性大B细胞淋巴瘤（DLBCL）。 MYD88突变在几乎所有情况下的WM和患者的免疫球蛋白M（IgM抗体）分泌型MGUS约50％的发现。与此相反，MYD88突变仅在0-6％的患者SMZL发现是多发性骨髓瘤7，8缺席。因为重叠形态学，免疫表型，细胞遗传学，和WM和SMZL或IgM多发性骨髓瘤通常可以鉴别诊断复杂之间临床特点，一个MYD88突变的存在可作为一种有用的鉴定因子9。 MYD88突变也与较大的疾病负担在DLBCL患者和总生存期差治疗后7相关， </SUP&gt; 10。此外，因为MYD88突变在活化的B细胞样（ABC）DLBCL比更频繁地发现生发中心B细胞样（GCB）DLBCL或原发性纵隔B细胞淋巴瘤（PMBL），MYD88突变状态可以用作替代标记为ABC亚型11,12。 这里提供的详细协议用作从该新测定法可以开发或大多数现有的测序测定法可以容易地适合于准确地检测各种样品类型低频突变的模板。该方法也可用于监测和检测可能在肿瘤中或甚至同时患者正在与靶向治疗或抗生素治疗可能发展细菌发展抗性突变。此外，它解决和补救许多与突变富集特别是在福尔马林固定石蜡包埋（FFPE）组织相关的问题。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 or 2 ml Safe-Lock microcentrifuge tubes</td>
			<td>Eppendorf</td>
			<td>05-402-25</td>
			<td></td>
		</tr>
		<tr>
			<td>100% alcohol</td>
			<td>VWR</td>
			<td>89370-084</td>
			<td>Histology grade; 91.5% Ethanol, 5% Isopropyl alcohol, 4.5% Methyl alcohol</td>
		</tr>
		<tr>
			<td>3730XL sequencer</td>
			<td>ABI</td>
			<td></td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Agencourt AMPure XP</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td>For magnetic bead PCR purification</td>
		</tr>
		<tr>
			<td>Aluminum sealing foils</td>
			<td>GeneMate</td>
			<td>T-2451-1</td>
			<td>For PCR and cold storage</td>
		</tr>
		<tr>
			<td>BigDye Terminator v3.1 Cycle sequencing kit</td>
			<td>Life Technologies</td>
			<td>4337455</td>
			<td>For bi-directional sequencing. With 5X Sequencing Buffer</td>
		</tr>
		<tr>
			<td>Centrifuge 5804 Series</td>
			<td>Eppendorf</td>
			<td></td>
			<td>A-2-DWP rotor (for PCR plate)</td>
		</tr>
		<tr>
			<td>Cold plate for 96 well plates</td>
			<td>Eppendorf</td>
			<td>Z606634</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse, RNAse-free, ultra-pure water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs (100mM)</td>
			<td>Invitrogen</td>
			<td>10297-117</td>
			<td></td>
		</tr>
		<tr>
			<td>DynaMag-96 Side-Skirted Magnet</td>
			<td>Thermo Fisher Scientific</td>
			<td>12027</td>
			<td>For use in PCR Purification.</td>
		</tr>
		<tr>
			<td>Ethanol Absolute&#160;</td>
			<td>Sigma</td>
			<td>E7023</td>
			<td>200 proof, for molecular biology</td>
		</tr>
		<tr>
			<td>Exiqon website Oligo Tools</td>
			<td></td>
			<td></td>
			<td><a href="http://www.exiqon.com/oligo-tools">www.exiqon.com/oligo-tools</a></td>
		</tr>
		<tr>
			<td>FastStart Taq DNA polymerase (5 U/ul)</td>
			<td>Roche</td>
			<td>12032937001</td>
			<td>With10X concentrated PCR reaction buffer, with 20 mM MgCl2&#160;</td>
		</tr>
		<tr>
			<td>Gel electrophoresis apparatus</td>
			<td></td>
			<td></td>
			<td>2% agarose gel</td>
		</tr>
		<tr>
			<td>GeneRead DNA FFPE extraction Kit&#160;</td>
			<td>Qiagen</td>
			<td>180134</td>
			<td>Contains uracil DNA glycosylase necessary for reducing sequencing artifacts</td>
		</tr>
		<tr>
			<td>Hi-Di Formamide</td>
			<td>ABI</td>
			<td>4311320</td>
			<td>For sequencing.</td>
		</tr>
		<tr>
			<td>LNA oligonucleotide</td>
			<td>Exiqon</td>
			<td>500100</td>
			<td>5&#39;-TCAGA+AG+C+G+A+C+T+G+A+T+CC/invdT/ (+N = LNA bases)</td>
		</tr>
		<tr>
			<td>M13-F Sequencing Primer</td>
			<td>ABI</td>
			<td></td>
			<td>5&#39;-tgt aaa acg acg gcc agt</td>
		</tr>
		<tr>
			<td>M13-R Sequencing Primer</td>
			<td>ABI</td>
			<td></td>
			<td>5&#39;-cag gaa aca gct atg acc</td>
		</tr>
		<tr>
			<td>Mastercycler Pro S Thermocycler</td>
			<td>Eppendorf</td>
			<td>E950030020</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge Model 5430</td>
			<td>Eppendorf</td>
			<td></td>
			<td>FA-45-30-11 rotor (for 1.5/2 ml microcentrifuge tubes)</td>
		</tr>
		<tr>
			<td>NanoDrop 2000 Spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PCR forward primer</td>
			<td>IDT</td>
			<td></td>
			<td>5&#39;-tgt aaa acg acg gcc agt TGC CAG GGG TAC TTA GAT GG</td>
		</tr>
		<tr>
			<td>PCR reverse primer</td>
			<td>IDT</td>
			<td></td>
			<td>5&#39;-cag gaa aca gct atg acc GGT TGG TGT AGT CGC AGA CA</td>
		</tr>
		<tr>
			<td>PCR plates</td>
			<td>GeneMate</td>
			<td>T-3107-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettors</td>
			<td></td>
			<td></td>
			<td>20, 200, 1000 &#181;l</td>
		</tr>
		<tr>
			<td>Plate septa, 96 well</td>
			<td>ABI</td>
			<td>4315933</td>
			<td></td>
		</tr>
		<tr>
			<td>QIAamp DNA Mini Kit</td>
			<td>Qiagen</td>
			<td>51304</td>
			<td>For BM aspirate and peripheral blood</td>
		</tr>
		<tr>
			<td>SeqScape Sortware v3.0</td>
			<td>ABI</td>
			<td>4474978</td>
			<td>For sequencing analysis</td>
		</tr>
		<tr>
			<td>Slide basket</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Acetate (3M, pH 5.2)&#160;</td>
			<td>Sigma</td>
			<td>S7899</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile filtered pipette tips&#160;</td>
			<td></td>
			<td></td>
			<td>20, 200, 1000 &#181;l</td>
		</tr>
		<tr>
			<td>Thermomixer C&#160;</td>
			<td>Eppendorf</td>
			<td>5382000023</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex genie</td>
			<td>Scientific Industries</td>
			<td>SI-0236</td>
			<td></td>
		</tr>
		<tr>
			<td>Wash reservoir</td>
			<td></td>
			<td></td>
			<td>~1000 ml</td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>VWR</td>
			<td>89370-088</td>
			<td>Histology grade</td>
		</tr>
	</tbody>
</table>

wild-type blocking pcr combined with direct sequencing as a highly sensitive method for detection of low-frequency somatic mutations

治疗后评估残留病时，筛选治疗期间出现的抗性突变精确检测和低频突变的鉴定可能是有问题时，或者当患者有几个循环肿瘤细胞。野生型阻断PCR，接着测序分析提供了高灵敏度，灵活性和简单性作为用于检测这些低频突变的方法。通过加入设计锁核酸寡核苷酸到一个新的或先前建立的常规的基于PCR的测序测定法自定义，在1,000 WT等位基因背景大约1突变等位基因的灵敏度，可以实现（1:1,000）。与脱氨事件相关联的伪影的测序在福尔马林固定的石蜡包埋的组织中发现可以部分地通过使用尿嘧啶DNA糖基化酶的过程中提取步骤补救。这里的优化的协议是特定用于检测MYD88突变，但可以作为一个模板来设计任何WTB-PCR测定。的WTB-PCR测定相对于其他常用的测定法，用于检测低频突变，包括等位基因特异性PCR和实时定量PCR包括假阳性的发生较少，更大的灵活性和易于实施的优点，并同时检测已知的能力和未知的突变。

延伸期间WTB-PCR的概念的概述示于图1。因为在阻滞剂-DNA杂交的单个核苷酸错配大幅降低其熔化温度（ΔTM = 20 - 30℃）中，WT等位基因的扩增而突变体的模板DNA是免费完成延伸部17被阻断。以这种方式，而WT DNA线性扩增的突变的DNA指数扩增。 由WTB-PCR获得的突变体富集的示范显示在图2。从患者和无突变基因组DNA通过测试常规和WTB-PCR并测序以证明由WTB-PCR可实现的典型的富集和缺乏在WT DNA误报。在WTB-PCR MMX使用阻断剂的工作浓度应通过滴定实验和s来确定HOULD实现突变体富集所需水平的同时不导致误报或完全阻断WT DNA的扩增。 WTB-PCR产物的测序分析显示为突变等位基因富集和检测的超过0.5％的突变体等位基因的野生型的背景在常规PCR 3 16％的限制。 这种增加在临床检测灵敏度的效果可能取决于肿瘤细胞的测试样品中的相对量。在一个方法的比较研究中，这里所描述的WTB-PCR分析表明，MYD88突变的64％将被患者WM或MGUS 3常规测试被错过。 如果使用阻断剂浓度太高，或者如果后PCR purifica甲特性脱落在信号强度（ 图3）经常看到重刑无法删除之前的双向测序拦截。当磁珠纯化代替酶纯化后者是证明。虽然酶纯化是具有更大的样本数中工作时的吸引力的选择，是不合适的应用与WTB-PCR，因为它未能从溶液测序之前除去阻断剂。 序列伪像的一个例子在FFPE衍生DNA经常发现作为胞嘧啶脱氨的结果（C:G&gt; T:A）在图4中3，18，19，20呈现。虽然脱氨基胞嘧啶的实际原因是了解甚少，即丰富了突变等位基因任何基于PCR的测定法将检测这些低频伪像21，22。误报由于DEAmination最好由高品质的模板材料开始避免;在许多情况下，这是不可能的，在提取过程中与尿嘧啶DNA糖基化酶（UDG）处理可以在限制脱氨伪像的频率和强度帮助。萃取（作为DNA FFPE试剂盒的一部分）期间UDG处理FFPE组织的切除脱氨基化胞嘧啶残基，从而防止人工诱导的C:G&gt; T:一个突变。然而，经常发生在CpG二核苷酸的5-甲基胞嘧啶残基被脱氨基胸腺嘧啶，不能由UDG切除。将得到的测序假象是相当可识别并经常出现作为串联突变如图4所示。如果样品已被萃取，UDG处理可以以相对低的损耗DNA的二次萃取来实现。 <img alt="图1" src="/files/ftp_upload/55130/55130fig1.jpg" /> 图1:概念奥雅纳WTB-PCR的rview。在拦截器-DNA杂交的单个核苷酸错配降低多达30℃的Tm。通过设计阻断寡核苷酸具有10为T 米 - 15℃以上的扩展过程中的温度，WT DNA的扩增，同时允许突变体DNA的扩增被阻断。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55130/55130fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55130/55130fig2.jpg" /> 图2:从患者和无突变基因组DNA通过测试常规和WTB-PCR并测序以证明由WTB-PCR可实现的典型的富集。用于实现WTB-PCR阻断剂的最终浓度被选择以实现最大的突变体富集同时不引起假阳性的WT DNA或阻断amplificaWT DNA完全的灰。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55130/55130fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55130/55130fig3.jpg" /> 图3: 当酶PCR纯化来代替磁珠特征脱落在信号强度看出。这可能是因为酶纯化未能删除之前双向测序阻断剂。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55130/55130fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55130/55130fig4.jpg" /> 图4:C:G&gt; T:在FFPE组织出现测序假象时胞嘧啶或甲基化胞嘧啶脱氨基被vIA福尔马林固定，以分别尿嘧啶或胸腺嘧啶。尿嘧啶DNA糖基化酶（UDG）可在此之前WTB-PCR切除尿嘧啶帮助减少测序假象。然而，从脱氨基5-甲基胞嘧啶，这经常发生在CpG岛所得胸腺嘧啶，不能由UDG切除。降低在WTB-PCR中使用阻断剂的浓度可以有助于减少不是由UDG处理补救测序假象的发生。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55130/55130fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations at JoVE.com

Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations

Wild-type blocking PCR followed by direct sequencing offers a highly sensitive method of detection for low frequency somatic mutations in a variety of sample types.

野生类型阻止PCR结合直接测序作为低频体细胞突变的检测的高灵敏度的方法

Accurate detection and identification of low frequency mutations can be problematic when assessing residual disease after therapy, screening for emerging ...

wild-type-blocking-pcr-combined-with-direct-sequencing-as-highly

Nanyang Technological University

Research

JoVE Journal

Genetics

11.6K 次观看.  NeoGenomics Laboratories. Wild-type blocking PCR followed by direct sequencing offers a highly sensitive method of detection for low frequency somatic mutations in a variety of sample types.

视频: Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

单细胞测序拷贝数改变的检测

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

科研

遗传学

Detection of microRNA Expression in Peritoneal Membrane of Rats Using Quantitative Real-time PCR

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been investigated in various organs and tissues in rat. However, standard methods for the purification of miRNAs and detection of their expression in rat peritoneal membrane have not been well established. We have developed an effective and reliable method to purify and quantify miRNAs using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) in rat peritoneal membrane. This protocol consists of four steps: 1) purification of peritoneal membrane sample; 2) purification of total RNA including miRNA from peritoneal membrane sample; 3) reverse transcription of miRNA to produce cDNA; and 4) qRT-PCR to detect miRNA expression. Using this protocol, we successfully determined that the expression of six miRNAs (miRNA-142-3p, miRNA-21-5p, miRNA-221-3p, miRNA-223-3p, miRNA-327, and miRNA-34a-5p) increased significantly in the peritoneal membrane of a rat peritoneal fibrosis model compared with those in control groups. This protocol can be used to study the profile of miRNA expression in the peritoneal membrane of rats in many pathological conditions.

Here we present a protocol for the detection of microRNA expression in rat peritoneal membrane using quantitative real-time reverse-transcription polymerase chain reaction. This method is suitable for studying the microRNA expression profile in rat peritoneal membrane in several pathological conditions.

使用定量实时PCR检测大鼠腹膜中的微小RNA表达

MicroRNAs (miRNAs) are small noncoding RNAs that regulate messenger RNA expression post-transcriptionally. The miRNA expression profile has been ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

植物标本标本的鲁棒 DNA 分离与高通量测序库建设

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

基于纠错 DNA 和 RNA 测序的稀有事件检测方法

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

Lentiviral Mediated Production of Transgenic Mice: A Simple and Highly Efficient Method for Direct Study of Founders

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a routine procedure is widely utilized throughout the world and its main limitation resides in the poor efficacy of transgene integration, resulting in a low yield of founder animals. Only few percent of animals born after implantation of injected fertilized oocytes have integrated the transgene. In contrast, lentiviral vectors are powerful tools for integrative gene transfer and their use to transduce fertilized oocytes allows highly efficient production of founder transgenic mice with an average yield above 70%. Furthermore, any mouse strain can be used to produce transgenic animal and the penetrance of transgene expression is extremely high, above 80% with lentiviral mediated transgenesis compared to DNA microinjection. The size of the DNA fragment that can be cargo by the lentiviral vector is restricted to 10 kb and represents the major limitation of this method. Using a simple and easy to perform injection procedure beneath the zona pellucida of fertilized oocytes, more than 50 founder animals can be produced in a single session of microinjection. Such a method is highly adapted to perform, directly in founder animals, rapid gain and loss of function studies or to screen genomic DNA regions for their ability to control and regulate gene expression in vivo.

Here, we present a protocol to promote transgene integration and production of founder transgenic mice with high efficacy by a simple injection of a lentiviral vector in the perivitelline space of a fertilized oocyte.

慢病毒载体介导的转基因小鼠生产: 一种简单高效的创始人直接研究方法

For almost 40 years, pronuclear DNA injection represents the standard method to generate transgenic mice with random integration of transgenes. Such a ...

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are&#160;therefore potentially replication-competent. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5&#39; and 3&#39; LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required.

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

用于下一代测序的近全长 HIV-1 Proviruses 的扩增

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

单液滴数字聚合酶链反应对热点地区突变的综合和同步检测

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.

基于数字PCR的沙门氏菌健康高通量分析竞争指数

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...

Validating Whole Genome Nanopore Sequencing, using Usutu Virus as an Example

Whole genome sequencing can be used to characterize and to trace viral outbreaks. Nanopore-based whole genome sequencing protocols have been described for several different viruses. These approaches utilize an overlapping amplicon-based approach which can be used to target a specific virus or group of genetically related viruses. In addition to confirmation of the virus presence, sequencing can be used for genomic epidemiology studies, to track viruses and unravel origins, reservoirs and modes of transmission. For such applications, it is crucial to understand possible effects of the error rate associated with the platform used. Routine application in clinical and public health settings require that this is documented with every important change in the protocol. Previously, a protocol for whole genome Usutu virus sequencing on the nanopore sequencing platform was validated (R9.4 flowcell) by direct comparison to Illumina sequencing. Here, we describe the method used to determine the required read coverage, using the comparison between the R10 flow cell and Illumina sequencing as an example.

We previously validated a protocol for amplicon-based whole genome Usutu virus (USUV) sequencing on a nanopore sequencing platform. Here, we describe the methods used in more detail and determine the error rate of the nanopore R10 flow cell.

以乌苏图病毒为例，验证全基因组纳米孔测序

Whole genome sequencing can be used to characterize and to trace viral outbreaks. Nanopore-based whole genome sequencing protocols have been described for ...

Optimization for Sequencing and Analysis of Degraded FFPE-RNA Samples

Gene expression analysis by RNA sequencing (RNA-seq) enables unique insights into clinical samples that can potentially lead to mechanistic understanding of the basis of various diseases as well as resistance and/or susceptibility mechanisms. However, FFPE tissues, which represent the most common method for preserving tissue morphology in clinical specimens, are not the best sources for gene expression profiling analysis. The RNA obtained from such samples is often degraded, fragmented, and chemically modified, which leads to suboptimal sequencing libraries. In turn, these generate poor quality sequence data that may not be reliable for gene expression analysis and mutation discovery. In order to make the most of FFPE samples and obtain the best possible data from low quality samples, it is important to take certain precautions while planning experimental design, preparing sequencing libraries, and during data analysis. This includes the use of appropriate metrics for precise sample quality control (QC), identifying the best methods for various steps during the sequencing library generation, and careful library QC. In addition, applying correct software tools and parameters for sequence data analysis is critical in order to identify artifacts in RNA-seq data, filter out contamination and low quality reads, assess uniformity of gene coverage, and measure the reproducibility of gene expression profiles among biological replicates. These steps can ensure high accuracy and reproducibility for profiling of very heterogeneous RNA samples. Here we describe the various steps for sample QC, library preparation and QC, sequencing, and data analysis that can help to increase the amount of useful data obtained from low quality RNA, such as that obtained from FFPE-RNA tissues.

This method describes the steps to improve the quality and quantity of sequence data that can be obtained from formalin-fixed paraffin-embedded (FFPE) RNA samples. We describe the methodology to more accurately assess the quality of FFPE-RNA samples, prepare sequencing libraries, and analyze the data from FFPE-RNA samples.

降解FFPE-RNA样品测序与分析优化

Gene expression analysis by RNA sequencing (RNA-seq) enables unique insights into clinical samples that can potentially lead to mechanistic understanding ...