这项工作得到了台湾国家科学技术委员会的支持。[NSTC 111-2314-B-006-003]

1. Poly（A） RNA纯化
注：在进行poly（A） RNA纯化之前，使用用DNase I处理的总RNA，并通过毛细管或常规凝胶电泳评估检查总RNA的质量和完整性。研究人员应该能够识别高分子量场中的 28S 和 18S rRNA 核糖体条带以及低分子量场中的 5.8S rRNA 带，而电泳图中没有任何明显的拖尾带。纯化步骤基本上遵循制造商的说明，并在特定步骤中进行了细微的修改。有关本协议中使用的所有材料和仪器的详细信息，请参阅 材料表 。
<ol><li>制备<ol><li>在进行以下程序之前，在室温下预热试剂、洗涤缓冲液、oligo（dT）珠子和裂解缓冲液30分钟。</li>
		<li>为了分离富集poly（A）的RNA，通过将足量的RNA转移到无核酸酶的1.5mL微量离心管中，制备10-20μg高质量总RNA的等分试样作为起始原料。在 70°C 的加热块中孵育5分钟，然后在冰上保持2 分钟。 注：作为起始材料的总RNA的量应根据所需的mRNA量和所用细胞的性质进行适当调整。根据经验，富集poly（A）的RNA可能占总RNA丰度的1-5%。10 μg 高质量总 RNA 的等分试样应提供 100-500 ng 范围内的高质量富集 poly（A） RNA，用于下游亚硫酸氢盐转化。</li>
		<li>同时，完全重悬oligo（dT）磁珠。将所需量的珠子悬浮液转移到新的 1.5 mL 微量离心管中，然后放在磁铁上 3 分钟，直到磁珠形成沉淀。 注：使用 50 μL oligo（dT） 磁珠处理 10 μg 总 RNA 起始量。根据所使用的细胞，可以进一步优化此步骤中的磁珠体积。</li>
		<li>除去上清液，用等量（步骤1.1.3中使用的珠子悬浮液的体积）重悬寡核苷酸（dT）珠子，并在除去上清液之前置于磁铁上3分钟。 注意： 不要过度干燥珠子;用户应快速继续执行步骤 1.2.1。</li>
	</ol></li>
	<li>第一轮净化<ol><li>将裂解缓冲液加入 RNA 样品管（体积比 4：1）并充分混合，然后转移到 oligo（dT） 磁珠中。 注：如果 RNA 样品体积为 20 μL，则加入 80 μL 裂解缓冲液。</li>
		<li>通过移液至少 10 次，将裂解缓冲液混合物和 oligo（dT） 微球完全重悬。</li>
		<li>让样品在室温下连续旋转孵育15分钟。</li>
		<li>将样品放在磁铁上5分钟或直到上清液澄清;弃去上清液。</li>
		<li>向珠子中加入 600 μL 洗涤缓冲液 1，小心混合以重悬珠子。</li>
		<li>将试管放在磁铁上5分钟或直到上清液澄清，弃去上清液，重复洗涤步骤1.2.5一次。</li>
		<li>加入 300 μL 洗涤缓冲液 2 洗涤珠子，并轻轻重悬珠子。</li>
		<li>将试管放在磁铁上5分钟或直到上清液澄清，弃去上清液，重复洗涤步骤1.2.7一次。</li>
		<li>向样品管中加入30μL冷的10mM Tris-HCl（洗脱缓冲液），并在 70°C 下孵育 5分钟。</li>
		<li>快速将试管转移到磁铁上，当悬浮液在20秒或更长时间后澄清时，将含有洗脱的poly（A）富集RNA的上清液转移到新的1.5mL微量离心管中。</li>
	</ol></li>
	<li>第二轮净化<ol><li>向洗脱的RNA样品中加入120μL（洗脱样品体积的4倍）裂解缓冲液并充分混合。</li>
		<li>用等量的裂解缓冲液洗涤第一轮纯化中使用的珠子，如步骤1.1.4，移液10次以重悬珠子，然后将它们放在磁铁上5分钟，然后弃去上清液。</li>
		<li>将RNA：裂解缓冲液混合物转移到洗涤的珠子中，并通过移液至少10x彻底混合。</li>
		<li>将样品在室温下连续旋转孵育15分钟。</li>
		<li>重复步骤1.2.4至1.2.8以除去盐和其他RNA亚型。 注：洗涤缓冲液的体积可减少至 300 μL 洗涤缓冲液 1 和 150 μL 洗涤缓冲液 2。</li>
		<li>向样品管中加入25μL（所需体积）冷的10mM Tris-HCl（洗脱缓冲液），并在 70°C 下孵育 5分钟。</li>
		<li>快速将试管转移到磁铁上，当悬浮液在20秒或更长时间后澄清时，将含有洗脱的poly（A）富集RNA的上清液转移到新的1.5mL微量离心管中。</li>
		<li>将 2.2 μL 转移到 8 联管中，使用 RNA 高灵敏度测定法对 RNA 数量和质量进行质量评估。</li>
		<li>将样品在-20°C下短期储存，在-70°C下储存较长时间。 注意：这可以是协议中的暂停点。</li>
	</ol></li>
</ol>2.亚硫酸氢盐转化
注：离心步骤均在室温下进行。这些程序基本上是按照制造商的说明执行的，但有一个额外的步骤 2.2，用于在亚硫酸氢盐反应步骤 2.3 之前添加加标对照 mRNA 序列。
<ol><li>将 19 μL 纯化的富集 poly（A） RNA 样品转移到 0.2 mL 8 联管中。</li>
	<li>加入1.0μL加标对照，其以预定的1：10,000数量比（即，0.1ng荧光素酶mRNA：1μg纯化的poly（A）富集RNA）无任何修饰）到步骤2.1的RNA样品中。 注：加标对照序列可以是萤火虫荧光素酶RNA、肾荧光素酶或不含甲基化胞嘧啶的 体外 转录RNA序列。</li>
	<li>向试管中加入 130 μL 转化摄政剂，轻轻倒置数次以充分混合。</li>
	<li>将试管放入PCR机中，在70°C下加热10分钟，然后在64°C下孵育45分钟，然后最后一步冷却至4°C。</li>
	<li>将色谱柱放在收集管上，将 250 μL RNA 结合缓冲液转移到色谱柱上。</li>
	<li>将步骤2.4中的样品转移到步骤2.5中的色谱柱中，并彻底移液。</li>
	<li>向色谱柱中加入 400 μL 95-100% 乙醇，迅速盖上盖子，倒置数次。</li>
	<li>在25°C下以10,000× g 离心30秒，弃去流出物。</li>
	<li>向色谱柱中加入 200 μL RNA 洗涤缓冲液。</li>
	<li>在25°C下以10,000× g 离心30秒，弃去流出物。</li>
	<li>加入 200 μL 脱磺缓冲液，在室温下孵育 30 分钟。</li>
	<li>在25°C下以10,000× g 离心30秒，弃去流出物。</li>
	<li>加入400μLRNA洗涤缓冲液，在25°C下以10,000× g 离心30秒，弃去流出物。重复此步骤一次。</li>
	<li>在25°C下再次以10,000× g 离心2分钟，以完全除去残留的洗涤缓冲液。 注：洗脱前的另一个离心步骤2.14 2分钟是完全除去洗涤缓冲液。洗涤步骤执行两次，以洗掉色谱柱上亚硫酸氢盐试剂和脱磺缓冲液中的高盐组分。</li>
	<li>将色谱柱转移到新的 1.5 mL 微量离心管中。</li>
	<li>向色谱柱中加入 20-30 μL 无核酸酶的 H2O，并在室温下静置 1 分钟。</li>
	<li>在25°C下以10,000× g 离心30秒。</li>
	<li>将 2.2 μL 转移到 8 条管中，以评估 RNA 的数量和质量。</li>
	<li>将亚硫酸氢盐处理的RNA样品在-20°C下短期储存，在-70°C下储存较长时间。 注意：这可以是协议中的暂停点。</li>
</ol>3. 亚硫酸氢盐处理的mRNA文库制备
注：按照文库制备说明方案第 4 部分用于纯化的 mRNA 或 rRNA 耗尽的 RNA。第一个启动步骤应遵循 FFPE RNA 方案，因为亚硫酸氢盐处理会使 RNA 片段化。在层流罩中执行每一步，并将反应混合物添加到冰冷冷却架上。
<ol><li>引发起始RNA<ol><li>转移所需量的亚硫酸氢盐处理的 mRNA 样品，总量最大为 5 μL，或加入无核酸酶的 H2O 使总量为 5 μL。 注：建议将至少 10 ng 亚硫酸氢盐处理的 mRNA 作为输入，用于此类 RNA 文库制备并生成 4nM 文库产物的最终摩尔浓度。</li>
		<li>向样品中加入 1 μL 随机引物（丁香），并通过移液混合。</li>
		<li>将样品管置于预设在65°C的PCR机中，并在105°C下盖上盖子5分钟，并在4°C下保持。</li>
	</ol></li>
	<li>第一链cDNA合成<ol><li>向样品中加入 4 μL 合成反应缓冲液（淡紫色）、8 μL 链特异性试剂（棕色）和 2 μL 合成酶混合物（棕色），总体积为 20 μL。 通过移液至少 10 倍并快速旋转彻底混合。</li>
		<li>将样品管置于预设的PCR机中，在25°C下10分钟，42°C下50分钟，70°C下15分钟，并在4°C下保持。 注：对于超过200个碱基的插入物，建议42°C孵育期为50分钟;对于低于200个碱基的插入片段，建议在42°C孵育15分钟。</li>
	</ol></li>
	<li>第二链cDNA合成<ol><li>加入 8 μL 含有 dUTP 混合物（橙色）、4 μL 合成酶混合物（橙色）和 48 μL 无核酸酶 H2O 的合成反应缓冲液，通过移液至少 10 次彻底混合并快速旋转。</li>
		<li>将样品管置于预设在16°C的PCR机中1小时，盖子关闭或≤40°C。</li>
	</ol></li>
	<li>用DNA纯化微球纯化双链cDNA<ol><li>在进行纯化前预热DNA纯化珠30分钟，并涡旋以重悬珠子。</li>
		<li>从步骤3.3.2中向cDNA样品中加入144μL重悬的DNA纯化珠，并通过移液至少10x充分混合。</li>
		<li>在室温下孵育5分钟。</li>
		<li>将样品管放在磁性架上5分钟，直到溶液澄清，然后除去上清液并保留含有DNA的珠子。</li>
		<li>将 200 μL 新鲜制备的 80% 乙醇加入带有珠子的试管中，同时将样品保持在磁架上并在室温下孵育 30 秒。</li>
		<li>除去上清液并重复步骤3.4.5一次，但这次小心地除去所有残留的上清液。</li>
		<li>将珠子风干 1-5 分钟，同时将样品保持在磁铁上。 注意： 不要过度干燥珠子;颜色应为深棕色。</li>
		<li>从磁架中取出样品，并通过向磁珠中加入 53 μL 0.1x TE（10 mM Tris-HCl 和 1 mM EDTA，pH 8.0）缓冲液来洗脱 DNA。通过移液至少10次彻底混合，并在室温下孵育2分钟。</li>
		<li>将试管放在磁架上5分钟或直到溶液澄清。</li>
		<li>将 50 μL 含有双链 cDNA 的上清液转移到新的 8 条管中。 注意：此时可以停止方案，样品可以储存在-30°C。</li>
	</ol></li>
	<li>cDNA文库的末端制备<ol><li>向洗脱的双链 cDNA 样品中加入 7 μL 末端精纯反应缓冲液（绿色）和 3 μL 末端精纯酶混合物（绿色）。</li>
		<li>以 50 μL 体积设置移液 10 次，在不产生气泡的情况下混合混合物，然后快速旋转样品。</li>
		<li>将样品置于PCR机器中，在20°C孵育30分钟，在65°C孵育30分钟，并在4°C下保持。</li>
	</ol></li>
	<li>接头连接<ol><li>根据制造商的说明稀释适配器（红色）。</li>
		<li>向样品中加入 2.5 μL 稀释的接头、1 μL 连接增强剂（红色）和 30 μL 连接预混液（红色）。</li>
		<li>以 80 μL 体积设置移液 10 次混合混合物，而不会产生气泡，然后快速旋转样品。</li>
		<li>将样品置于PCR机中，并在20°C下孵育15分钟。</li>
		<li>向样品中加入 3 μL 尿嘧啶 DNA 糖基化酶和 DNA 糖基化酶裂解酶核酸内切酶 VIII（蓝色）的混合物并彻底移液。</li>
		<li>将样品放回PCR机中，并在37°C下孵育15分钟，盖子设置为75°C。</li>
	</ol></li>
	<li>纯化连接反应处理的cDNA 注意：使用 SPRIselect 磁珠或可选的 AMpure XP 磁珠的尺寸选择都可用于此目的18.<ol><li>在进行纯化之前预热珠子30分钟，并涡旋以重悬它们。</li>
		<li>将 25 μL 重悬珠转移到步骤 3.6.6 中的 cDNA 样品中，并通过移液至少 10 次彻底混合。</li>
		<li>在室温下孵育5分钟。</li>
		<li>将样品管放在磁性架上5分钟，直到溶液澄清，然后将上清液转移到新的8条管中并丢弃珠子。</li>
		<li>向上清液中加入 10 μL 重悬珠，并通过移液至少 10 次彻底混合。</li>
		<li>将样品管放在磁性架上5分钟，直到溶液澄清，然后弃去上清液，不要干扰珠子。</li>
		<li>将 200 μL 新鲜制备的 80% 乙醇加入带有珠子的试管中，同时将样品保持在磁架上并在室温下孵育 30 秒。</li>
		<li>除去上清液并重复步骤3.7.7一次，但这次小心地除去所有残留的上清液。</li>
		<li>将珠子风干 1-5 分钟，同时将样品保持在磁铁上。 注意： 不要过度干燥珠子;颜色应为深棕色。</li>
		<li>从磁架中取出样品，并通过向磁珠中加入 17 μL 0.1x TE 缓冲液来洗脱 DNA。通过移液至少10次彻底混合，并在室温下孵育2分钟。</li>
		<li>将试管放在磁架上5分钟，直到溶液澄清。</li>
		<li>将含有靶标末端制备的 dsDNA 的 15 μL 上清液转移到新的 8 条管中。</li>
	</ol></li>
	<li>接头连接DNA的PCR富集<ol><li>向接头连接的 DNA 样品中加入 25 μL 预混液（蓝色）、5 μL 5' 接头引物和 5 μL 3' 接头引物。通过移液至少 10 次彻底混合。</li>
		<li>将样品放入PCR仪中，按照以下步骤进行PCR扩增：（1）98°C30秒，（2）98°C10秒，（3）65°C75秒，（4）65°C5分钟，（5）保持在4°C;其中步骤 （2） 和 （3） 应重复 N+2 循环数，其中 N 等于说明书中的 RNA 输入量。这意味着由于在步骤 3.7 中对样品进行双面尺寸选择，因此需要 2 个额外的循环。 注意：例如，建议在 9-10 个循环的 PCR 中使用 8 ng 纯化的 mRNA 起始量;但在步骤 3.7 中使用双侧、大小选择、连接的 cDNA，推荐的 PCR 循环为 11-12 个循环。</li>
	</ol></li>
	<li>PCR扩增文库的纯化<ol><li>在进行纯化之前，将DNA纯化珠预热30分钟，并涡旋以重悬珠。</li>
		<li>将 45 μL （0.9x） 重悬的珠子转移到步骤 3.8.2 中的 PCR 产物中，并通过移液至少 10x 彻底混合。</li>
		<li>在室温下孵育5分钟。</li>
		<li>将样品管放在磁架上5分钟，直到溶液澄清，然后除去上清液并保留含有DNA的珠子。</li>
		<li>将 200 μL 新鲜制备的 80% 乙醇加入到带有珠子的试管中，将样品留在磁架上，并在室温下静置 30 秒。</li>
		<li>取出上清液并重复步骤3.9.5一次，但这次小心地除去所有残留的上清液。</li>
		<li>将珠子风干 1-5 分钟，同时将样品保持在磁铁上。 注意： 不要过度干燥珠子;颜色应为深棕色。</li>
		<li>从磁架上取下样品，向珠子中加入 22 μL 0.1x TE 缓冲液以洗脱 DNA。通过移液至少10次彻底混合，并在室温下孵育2分钟。</li>
		<li>将试管放在磁性架上，静置5分钟或直到溶液在室温下澄清。</li>
		<li>将 20 μL 上清液转移到新的 8 条管中。</li>
		<li>将 2.2 μL 转移到 8 联管中，以评估文库数量和质量。 注：文库的数量也可以通过qPCR检测（参见 材料表）。</li>
		<li>将bsRNA-seq文库样品储存在-20°C。</li>
	</ol></li>
</ol>

创建用于转录组范围作图的稳健亚硫酸氢盐-mRNA 文库

<ol>
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NSUN2 as the methyltransferase and ALYREF as an m5C reader.</a> Cell Research. 27, 606 (2017).</li><li>Chen, X., 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=5-Methylcytosine+promotes+pathogenesis+of+bladder+cancer+through+stabilizing+mRNAs.">5-Methylcytosine promotes pathogenesis of bladder cancer through stabilizing mRNAs.</a> Nature Cell Biology. 21 (8), 978-990 (2019).</li><li>Guo, 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=Disease+activity-associated+alteration+of+mRNA+m(5)+C+methylation+in+CD4(+)+T+cells+of+systemic+lupus+erythematosus.">Disease activity-associated alteration of mRNA m(5) C methylation in CD4(+) T cells of systemic lupus erythematosus.</a> Frontiers in Cell and Developmental Biology. 8 (5), 430 (2020).</li><li>Song, H., 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=Biological+roles+of+RNA+m5C+modification+and+its+implications+in+cancer+immunotherapy.">Biological roles of RNA m5C modification and its implications in cancer immunotherapy.</a> Biomarker Research. 10 (1), 15 (2022).</li><li>Xue, C., Zhao, Y., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+RNA+cytosine-5+methylation:+detection,+regulatory+mechanisms,+biological+functions+and+links+to+cancer.">Advances in RNA cytosine-5 methylation: detection, regulatory mechanisms, biological functions and links to cancer.</a> Biomarker Research. 8 (1), 43 (2020).</li><li>Helm, M., Motorin, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+RNA+modifications+in+the+epitranscriptome:+predict+and+validate.">Detecting RNA modifications in the epitranscriptome: predict and validate.</a> Nature Reviews Genetics. 18 (5), 275-291 (2017).</li><li>Guo, 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=Advances+in+mRNA+5-methylcytosine+modifications:+Detection,+effectors,+biological+functions,+and+clinical+relevance.">Advances in mRNA 5-methylcytosine modifications: Detection, effectors, biological functions, and clinical relevance.</a> Molecular Therapy - 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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=Widespread+occurrence+of+5-methylcytosine+in+human+coding+and+non-coding+RNA.">Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA.</a> Nucleic Acids Research. 40 (11), 5023-5033 (2012).</li><li>Han, X., 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=Dynamic+DNA+5-hydroxylmethylcytosine+and+RNA+5-methycytosine+reprogramming+during+early+human+development.">Dynamic DNA 5-hydroxylmethylcytosine and RNA 5-methycytosine reprogramming during early human development.</a> Genomics, Proteomics &amp; Bioinformatics. , (2022).</li><li>Amort, 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=Long+non-coding+RNAs+as+targets+for+cytosine+methylation.">Long non-coding RNAs as targets for cytosine methylation.</a> RNA biology. 10 (6), 1003-1008 (2013).</li><li>Sun, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+NSUN2+deficiency+on+the+mRNA+5-methylcytosine+modification+and+gene+expression+profile+in+HEK293+cells.">Effects of NSUN2 deficiency on the mRNA 5-methylcytosine modification and gene expression profile in HEK293 cells.</a> Epigenomics. 11 (4), 439-453 (2019).</li><li>Huang, T., Chen, W., Liu, J., Gu, N., Zhang, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+identification+of+mRNA+5-methylcytosine+in+mammals.">Genome-wide identification of mRNA 5-methylcytosine in mammals.</a> Nature Structural and Molecular Biology. 26 (5), 380-388 (2019).</li><li>Rieder, D., Amort, T., Kugler, E., Lusser, A., Trajanoski, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=meRanTK:+methylated+RNA+analysis+ToolKit.">meRanTK: methylated RNA analysis ToolKit.</a> Bioinformatics. 32 (5), 782-785 (2015).</li><li>Kraus, A. J., Brink, B. G., Siegel, T. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+and+specific+oligo-based+depletion+of+rRNA.">Efficient and specific oligo-based depletion of rRNA.</a> Scientific Reports. 9 (1), 12281 (2019).</li><li>Edelheit, S., Schwartz, S., Mumbach, M. 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作者没有要披露的利益冲突。

在该协议中，通过使用标准化组件实现了聚（A）富集，亚硫酸氢盐转化和文库制备的详细流程。进一步的测序分析提供了目标样品中mRNA 5-甲基胞嘧啶的鉴定。
关键步骤是起始材料（总RNA）的质量，因为RNA的降解会影响poly（A） RNA纯化的回收率。在进行poly（A） RNA纯化步骤之前，应小心处理样品并避免RNase污染。该过程的另一个关键部分是文库制备中的PCR循环次数。循环数的?...

在 150 多种转录后修饰1 中，已在各种类型的 RNA 中鉴定出 5-甲基胞嘧啶 （m5C） 修饰，包括核糖体 RNA、转移 RNA、信使 RNA、micro RNA、长链非编码 RNA、vault RNA、增强子 RNA 和小 cajal 体特异性 RNA2。RNA m 5 C 与多种生物学和病理机制有关，例如调节植物根系发育3、病毒基因表达4 和癌症进展5。该协议的目的是提供简化的管道，以表征处于不同发育阶段或疾病环境中的生物样品的转录组范围的mRNA m5C修饰谱。开发了全转录组亚硫酸氢盐测序来表征亚硫酸氢盐转化 RNA 中碱基对分辨率为 6、7、8、9 的位置和定量胞嘧啶甲基化水平。这在研究 m5C 与参与细胞生物调控机制的基因表达和 RNA 命运的关联时特别有用。在哺乳动物细胞中，有两种已知的 m 5 C 读取器：ALYREF 可以识别细胞核的 m 5 C 并充当 mRNA 细胞核到胞质溶胶转运蛋白10，而 YBX1 可以识别细胞质中的 m5C 并增加 mRNA 稳定性11。系统性红斑狼疮 CD4+ T 细胞中报告了与免疫通路相关的异常 m5C mRNA12。研究表明，mRNA m5C 修饰与癌症免疫调节和癌症进展之间存在关联13,14。因此，在mRNA上绘制m5C修饰图谱可以为阐明潜在的调控机制提供关键信息。
为了研究RNA m 5 C修饰在某些生物学条件下的功能作用，基于亚硫酸氢盐转化（bsRNA-seq）和基于抗体亲和富集的方法，如m 5 C-RIP-seq、miCLIP-seq和5-Aza-seq，可以与高通量测序平台相结合，在转录组范围内使用m5C修饰对靶区和序列进行高效检测15，16.该协议的优势在于，由于基于抗体亲和富集的方法依赖于高质量抗体的可用性，因此该协议的优势在于单碱基分辨率下提供了全面的RNA m 5 C景观，并且可以实现m5C甲基化景观的单片段分辨率17。
所有 RNA 样品都将使用 oligo（dT） 微球进行两轮 mRNA 富集、三个亚硫酸氢盐反应循环和测序文库制备。为了监测 RNA 质量，将在 mRNA 纯化和亚硫酸氢盐反应程序前后通过毛细管凝胶电泳检查每个 RNA 样品，以评估片段分布。纯化的文库将通过其 PCR 扩增子质量进行检查，通过毛细管凝胶电泳检查 DNA 大小分布片段，并在测序前通过基于荧光染料的定量测定检查其总量。该系统还可用于分析广谱生物样品，如农产品、分离的病毒粒子、细胞系、模式生物和病理标本。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agilent 2100 Electrophoresis Bioanalyzer System</td>
			<td>Agilent, Santa Clara, CA</td>
			<td></td>
			<td>RNA quality detection</td>
		</tr>
		<tr>
			<td>AMpure XP beads</td>
			<td>Beckman Coulter</td>
			<td>A63881</td>
			<td>purify DNA</td>
		</tr>
		<tr>
			<td>Bioanalyzer DNA high sensitivity kit</td>
			<td>Agilent, Santa Clara, CA</td>
			<td>5067-4626</td>
			<td>DNA quality dection</td>
		</tr>
		<tr>
			<td>Bioanalyzer RNA 6000 Pico kit</td>
			<td>Agilent, Santa Clara, CA</td>
			<td>5067-1513</td>
			<td>RNA quality dection</td>
		</tr>
		<tr>
			<td>DiaMag02 - magnetic rack</td>
			<td>Diagenode, Denville, NJ</td>
			<td>B04000001</td>
			<td>assist library preparation</td>
		</tr>
		<tr>
			<td>DiaMag1.5 - magnetic rack</td>
			<td>Diagenode, Denville, NJ</td>
			<td>B04000003</td>
			<td>assist poly(A) RNA purificaion</td>
		</tr>
		<tr>
			<td>Dynabeads mRNA DIRECT purification kit</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>61011</td>
			<td>poly(A) RNA purificaion; Wash Buffer 1 and Wash Buffer 2</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>J.T.Baker</td>
			<td>64-17-5</td>
			<td></td>
		</tr>
		<tr>
			<td>EZ RNA methylation kit</td>
			<td>Zymo, Irvine, CA</td>
			<td>R5002</td>
			<td>bisulfite treatment</td>
		</tr>
		<tr>
			<td>Firefly luciferase mRNA</td>
			<td>Promega, Madison, WI, USA</td>
			<td>L4561</td>
			<td>spike in control seqeunce&#160;</td>
		</tr>
		<tr>
			<td>KAPA Library Quantification Kits</td>
			<td>Roche, Switzerland</td>
			<td>KK4824</td>
			<td>library quantification</td>
		</tr>
		<tr>
			<td>Nanodrop spectrophotometer</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td></td>
			<td>Total RNA quantity detection</td>
		</tr>
		<tr>
			<td>NEBNext multiplex Oligos for illumina (index Primer set1)</td>
			<td>New England Biolabs, Ipswich, MA</td>
			<td>E7335S</td>
			<td>library preparation</td>
		</tr>
		<tr>
			<td>NEBNext Ultra <img alt="Equation 1" src="/files/ftp_upload/65352/65352eq01.jpg" style="margin: auto;" /> Directional RNA Library Prep Kit for Illumina</td>
			<td>New England Biolabs, Ipswich, MA</td>
			<td>E7760S</td>
			<td>library preparation</td>
		</tr>
		<tr>
			<td>Nuclease-free Water</td>
			<td>Thermo Fisher Scientific</td>
			<td>AM9932</td>
			<td></td>
		</tr>
		<tr>
			<td>P2 pipetman</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>4641010</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 2.0 fluorometer&#160;</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td></td>
			<td>RNA quantity detection</td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS Assay Kit</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>Q32854</td>
			<td>DNA quantity detection</td>
		</tr>
		<tr>
			<td>Qubit RNA HS Assay Kit</td>
			<td>Thermo Fisher Scientific, Waltham, MA</td>
			<td>Q32852</td>
			<td>RNA quantity detection</td>
		</tr>
	</tbody>
</table>

enrichment of mrna and bisulfite-mrna library preparation for next-generation sequencing

各种类型的 RNA 转录本中的 RNA 转录后修饰与真核细胞中的多种 RNA 调控有关。异常的 RNA 5-甲基胞嘧啶修饰和 RNA 甲基转移酶的失调表达已被证明与包括癌症在内的多种疾病有关。开发了全转录组亚硫酸氢盐测序，以表征亚硫酸氢盐转化的 RNA 在碱基对分辨率下的位置和定量胞嘧啶甲基化水平。在此，该协议详细介绍了两轮poly（A）RNA纯化，三个亚硫酸氢盐反应循环和文库制备的程序，以允许mRNA 5-甲基胞嘧啶修饰位点的转录组范围定位。主反应后RNA数量和质量的评估对于监测RNA完整性至关重要，也是确保高质量测序文库的关键步骤。理想情况下，这些程序可以在三天内完成。通过该方案，使用高质量的总RNA作为输入，实际上可以建立强大的亚硫酸氢盐-mRNA文库，用于从目标样品进行二代测序。

按照本报告中的程序从细胞系19 中生成一系列bsRNA-seq文库 （图1）。在对细胞系样品进行总RNA纯化并进行DNase处理并通过凝胶电泳和紫外-可见分光光度法（A260/A280）检查质量后，RNA样品可以进行poly（A）RNA富集。为了确定双重纯化是否可以去除大部分核糖体RNA，通过毛细管电泳总RNA测定法评估poly（A） RNA的纯化效率，该测定法可以自动计算rRNA污...

Watch this Scientific Journal Video about Decoding RNA Methylation's Role in Pancreatic Cancer - A Single-Base Resolution Study at JoVE.com

作者聚焦：解码 RNA 甲基化在胰腺癌中的作用 - 一项单碱基分辨率研究 

该协议提供了一种易于遵循的工作流程，可使用标准化设备对感兴趣的生物样品进行poly（A）RNA纯化、亚硫酸氢盐转化和文库制备。

mRNA和亚硫酸氢盐-mRNA文库的富集制备用于二代测序

RNA post-transcriptional modifications in various types of RNA transcripts are associated with diverse RNA regulation in eukaryotic cells. Aberrant RNA ...

本研究旨在研究 RNA 甲基化，特别是 5-甲基胞嘧啶修饰在胰腺癌中的作用。我们想要回答的问题是某些RNA甲基转移酶的上调或下调如何影响癌细胞表型，以及RNA底物通过哪些表型。本研究使用标准化设备以单碱基分辨率提供全面的 RNA m5C 图景。从这项使用亚硫酸氢盐RNA seq的研究中，结果将RNA m5C修饰与癌细胞分化不良的关联联系起来。这应该为未来的研究铺平道路，以辨别RNA转录本中特定m5C的乘客与疾病驱动因素的影响。通过这种技术，我们可以识别可能参与肿瘤进展的一组差异甲基化RNA。此外，单分子碱基实验可以提供特异性RNA m5C的更详细的调控作用。

enrichment-mrna-bisulfite-mrna-library-preparation-for-next

Research

JoVE Journal

Biology

Enrichment of mRNA and Bisulfite-mRNA Library Preparation for Next-Generation Sequencing

965 次观看.  National Cheng Kung University. 本研究旨在研究 RNA 甲基化，特别是 5-甲基胞嘧啶修饰在胰腺癌中的作用。我们想要回答的问题是某些RNA甲基转移酶的上调或下调如何影响癌细胞表型，以及RNA底物通过哪些表型。本研究使用标准化设备以单碱基分辨率提供全面的 RNA m5C 图景。从这项使用亚硫酸氢盐RNA seq的研究中，结果将RNA m5C修饰与癌细胞分化不良的关联联系起来。这应该为未来的研究铺平道路，以辨...

视频: 作者聚焦：解码 RNA 甲基化在胰腺癌中的作用 - 一项单碱基分辨率研究 

Fluorescence-microscopy Screening and Next-generation Sequencing: Useful Tools for the Identification of Genes Involved in Organelle Integrity

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the subcellular distribution of a specific tagged fluorescent marker in the secretory pathway. Arabidopsis is a powerful biological model for genetic studies because of its genome size, generation time, and conservation of molecular mechanisms among kingdoms. The array genotyping as an approach to map the mutation in alternative to the traditional method based on molecular markers is advantageous because it is relatively faster and may allow the mapping of several mutants in a really short time frame. This method allows the identification of proteins that can influence the integrity of any organelle in plants. Here, as an example, we propose a screen to map genes important for the integrity of the endoplasmic reticulum (ER). Our approach, however, can be easily extended to other plant cell organelles (for example see1,2), and thus represents an important step toward understanding the molecular basis governing other subcellular structures.

A fundamental quest in cell biology is to define the mechanisms that underlie the identity of the organelles that make eukaryotic cells. Here we propose a method to identify the genes responsible for the morphological and functional integrity of plant organelles using fluorescence microscopy and next-generation sequencing tools.

荧光显微镜筛选和新一代测序：参与细胞器完整性的基因鉴定的有用工具

This protocol describes a fluorescence microscope-based screening of Arabidopsis seedlings and describes how to map recessive mutations that alter the ...

科研

生物学

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

的16S核糖体RNA基因的扩增子的新一代测序

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

有针对性的DNA甲基化分析通过新一代测序

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

逆转录病毒整合位点的扩增，新一代测序和基因组DNA制图

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

3' End Sequencing Library Preparation with A-seq2

Studies in the last decade have revealed a complex and dynamic variety of pre-mRNA cleavage and polyadenylation reactions. mRNAs with long 3&#39; untranslated regions (UTRs) are generated in differentiated cells whereas proliferating cells preferentially express transcripts with short 3&#39;UTRs. We describe the A-seq protocol, now at its second version, which was developed to map polyadenylation sites genome-wide and study the regulation of pre-mRNA 3&#39; end processing. Also this current protocol takes advantage of the polyadenylate (poly(A)) tails that are added during the biogenesis of most mammalian mRNAs to enrich for fully processed mRNAs. A DNA adaptor with deoxyuracil at its fourth position allows the precise processing of mRNA 3&#39; end fragments for sequencing. Not including the cell culture and the overnight ligations, the protocol requires about 8 h hands-on time. Along with it, an easy-to-use software package for the analysis of the derived sequencing data is provided. A-seq2 and the associated analysis software provide an efficient and reliable solution to the mapping of pre-mRNA 3&#39; ends in a wide range of conditions, from 106 or fewer cells.

3&#039; End Sequencing Library Preparation with A-seq2

This protocol describes a method for mapping pre-mRNA 3&#39; end processing sites.

3 A-seq2 的末端测序库的研制

Studies in the last decade have revealed a complex and dynamic variety of pre-mRNA cleavage and polyadenylation reactions. mRNAs with long 3&#39; ...

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to poorly folding proteins or random open reading frames. We have developed a strategy where, by using TEM-1 &#946;-lactamase (the enzyme conferring ampicillin resistance) on a genomic scale, we can select collections of correctly folded protein domains from the coding portion of the DNA of any intronless genome. The protein fragments obtained by this approach, the so called &#34;domainome&#34;, will be well expressed and soluble, making them suitable for structural/functional studies.
By cloning and displaying the &#34;domainome&#34; directly in a phage display system, we have showed that it is possible to select specific protein domains with the desired binding properties (e.g., to other proteins or to antibodies), thus providing essential experimental information for gene annotation or antigen identification.
The identification of the most enriched clones in a selected polyclonal population can be achieved by using novel next-generation sequencing technologies (NGS). For these reasons, we introduce deep sequencing analysis of the library itself and the selection outputs to provide complete information on diversity, abundance and precise mapping of each of the selected fragment. The protocols presented here show the key steps for library construction, characterization, and validation.

The protocols described allow the construction, characterization and selection (against the target of choice) of a &#34;domainome&#34; library made from any DNA source. This is achieved by a research pipeline that combines different technologies: phage display, a folding reporter and next generation sequencing with a web tool for data analysis.

Interactome: Domainome 图书馆建设、噬菌体展示和下一代测序的验证与选择协议

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to ...

Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective and widely available vaccines are lacking for a majority of these diseases, necessitating the development of novel disease mitigation strategies. To this end, a promising avenue of disease control involves targeting the vector microbiome, the community of microbes inhabiting the vector. The vector microbiome plays a pivotal role in pathogen dynamics, and manipulations of the microbiome have led to reduced vector abundance or pathogen transmission for a handful of vector-borne diseases. However, translating these findings into disease control applications requires a thorough understanding of vector microbial ecology, historically limited by insufficient technology in this field. The advent of next-generation sequencing approaches has enabled rapid, highly parallel sequencing of diverse microbial communities. Targeting the highly-conserved 16S rRNA gene has facilitated characterizations of microbes present within vectors under varying ecological and experimental conditions. This technique involves amplification of the 16S rRNA gene, sample barcoding via PCR, loading samples onto a flow cell for sequencing, and bioinformatics approaches to match sequence data with phylogenetic information. Species or genus-level identification for a high number of replicates can typically be achieved through this approach, thus circumventing challenges of low detection, resolution, and output from traditional culturing, microscopy, or histological staining techniques. Therefore, this method is well-suited for characterizing vector microbes under diverse conditions but cannot currently provide information on microbial function, location within the vector, or response to antibiotic treatment. Overall, 16S next-generation sequencing is a powerful technique for better understanding the identity and role of vector microbes in disease dynamics.

Here we present a next-generation sequencing protocol for 16S rRNA sequencing which enables identification and characterization of microbial communities within vectors. This method involves DNA extraction, amplification and barcoding of samples through PCR, sequencing on a flow-cell, and bioinformatics to match sequence data to phylogenetic information.

下一代 16S rRNA 扩增子测序的蜱菌群鉴定

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

使用两步PCR和下一代16S rRNA基因测序进行微生物群分析

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

A Magnetic-Bead-Based Mosquito DNA Extraction Protocol for Next-Generation Sequencing

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high quality and quantity DNA from a well-preserved individual mosquito sufficient for downstream whole genome sequencing. However, reliance on an expensive automated DNA extraction instrument can be prohibitive for many laboratories. Here, the study provides a budget-friendly magnetic-bead-based DNA extraction protocol, which is suitable for low to medium throughput. The protocol described here was successfully tested using individual Aedes aegypti mosquito samples. The reduced costs associated with high quality DNA extraction will increase the application of high throughput sequencing to resource limited labs and studies.

Described here is a DNA extraction protocol using magnetic beads to produce high quality DNA extractions from mosquitoes. These extractions are suitable for a downstream next-generation sequencing approach.

用于下一代测序的基于磁珠的蚊子DNA提取协议

A recently published DNA extraction protocol using magnetic beads and an automated DNA extraction instrument suggested that it is possible to extract high ...

评估非鳞状非小细胞肺癌患者的基因组改变

Ultra-Fast Amplicon-Based Next-Generation Sequencing in Non-Squamous Non-Small Cell Lung Cancer

The number of molecular alterations to be tested for targeted therapy of non-squamous non-small cell lung cancer (NS-NSCLC) patients has significantly increased these last few years. The detection of molecular abnormalities is mandatory for the optimal care of advanced or metastatic NS-NSCLC patients, allowing targeted therapies to be administrated with an improvement in overall survival. Nevertheless, these tumors develop mechanisms of resistance that are potentially targetable using novel therapies. Some molecular alterations can also modulate the treatment response. The molecular characterization of NS-NSCLC has to be performed in a short turnaround time (TAT), in less than 10 working days, as recommended by the international guidelines. In addition, the origin of the tissue biopsies for genomic analysis is diverse, and their size is continuously decreasing with the development of less invasive methods and protocols. Consequently, pathologists are being challenged to perform effective molecular technics while maintaining an efficient and rapid diagnosis strategy. Here, we describe the ultra-fast amplicon-based next-generation sequencing (NGS) workflow used in daily routine practice at diagnosis for NS-NSCLC patients. We showed that this system is able to identify the current molecular targets used in precision medicine in thoracic oncology in an appropriate TAT.

作者聚焦：非鳞状非小细胞肺癌分子生物标志物检测的进展 

The increase of molecular biomarkers to be tested for non-squamous non-small cell lung cancer (NS-NSCLC) care management has prompted the development of fast and reliable molecular detection methods. We describe a workflow for genomic alteration assessment for NS-NSCLC patients using an ultra-fast-next generation sequencing (NGS) approach.

基于扩增子的超快速二代测序在非鳞状非小细胞肺癌中的应用

The number of molecular alterations to be tested for targeted therapy of non-squamous non-small cell lung cancer (NS-NSCLC) patients has significantly ...