This work was funded by a National Institutes of Health R01 (DC009453 support to CWB).

注意:在此之前提供唾液样本所有受试者知情同意书符合指引处理在全国儿童医院人类受试者。 1，唾液的采集和存储<ol><li>此前唾液的收集，确保拍摄对象的嘴是免费的食物或其他异物所具有的主题冲洗他们的嘴的水，避免进食或收集样本前，喝了30分钟。 </li><li>用2.5毫升的DNA稳定缓冲液2，确保避免触及上限或管内打开一个15毫升的离心管中，并有课题吐唾液2.5毫升放入缓冲液。注:收集多个2.5毫升唾液可导致样品降解从样品的DNA的稳定缓冲液的不足比率。收集太少唾液会减少从协议预期产量。为了评估珍藏册，使用编号为梯度对T的侧面他管的。 </li><li>更换盖和通过反转混合，直到混合物均化。剧烈摇动是没有必要的。储存在室温下将样品用于短期存储或4℃下长期储存（&gt; 3个月）。 </li></ol> 2，前期准备和细胞裂解<ol><li>在此之前开始开采，加热水浴至37℃，并准备一个冰桶。三的15毫升锥形离心管中，将需要为每个提取的样品。三管将被用于保持细胞和蛋白质沉淀，最终提取基因组DNA，并在异丙醇和乙醇上清液。 </li><li>从存储器中检索的样品，并翻转试样几次然后涡流在中等速度持续15秒。 </li><li>免除2.5毫升样品放入一个干净的15ml离心管中，加入5毫升细胞裂解液。通过倒置混合样品50次，并在室温下孵育30分钟。 </li></ol> 3，RNA的去除<ol><li>加入40μl的RNase溶胶ution在100毫克/毫升，并在37℃下进行15分钟。 </li><li>从37℃水浴中取出样品，并在冰上冷却3分钟。 <ol><li>的RNA酶A孵育后，提高水浴温度至65℃的协议的DNA再水合步骤。 </li></ol></li></ol> 4，蛋白质和脂肪去除<ol><li>加入50微升蛋白酶K溶液20毫克/毫升，颠倒混匀数次，并在室温下孵育至少30分钟。注:这是一个可能的暂停点的协议。在加入蛋白酶K溶液中后，可将样品储存于4℃直至萃取就可以完成。储存在4℃下长达24小时未显示出对萃取率或DNA质量显著效果。长期存放在这个阶段并没有进行评估。 </li><li>添加1.7毫升的蛋白沉淀溶液，涡旋混合20秒，在高速，并置于冰上10分钟。 </lI&gt; <li>一旦样品已在冰上冷却10分钟，离心以3,000 XG 10分钟，4℃。沉淀的蛋白质必须形成一个紧密的小球继续。如果沉淀不紧或溶液仍是浑浊的样品可以在冰上冷却5分钟以上，并离心分离重复进行。样品必须保持在冰上，以确保紧密的颗粒。 </li></ol> 5，分离和基因组DNA纯化<ol><li>到一个干净的15ml离心管，移液管5毫升异丙醇和8微升纯糖原溶液20毫克/毫升。 </li><li>倾从步骤4.3到含有异丙醇和糖原溶液的管含有基因组DNA的上清液，剩下的沉淀的蛋白质沉淀物的后面。一旦上清液已经加入，轻轻在3000 XG和4℃下混匀50次颠倒和离心30分钟。 </li><li>慢慢倒入上清到一个干净的15ml试管中。除去上清液后，加入1 ml 70％的乙醇，以通过缓慢摇动，然后轻轻移动的乙醇在沉淀沉淀数次洗涤沉淀。保留在管中的乙醇。 </li><li>初始洗涤后，离心样品在2000 XG 1分钟和20℃。此离心步骤可以在任4°C或20°C下进行。温度的无显著效果已被证实对于此步骤。 </li><li>以下将沉淀的初始洗涤并离心，慢慢倾从管的乙醇洗涤并弃去，然后通过重复步骤5.3和5.4进行第二次洗涤。 </li><li>除去来自第二洗涤上清液后，使沉淀物在空气中干燥15分钟。 <ol><li>如果样品没有完全干燥，风干另外15分钟。 </li></ol></li></ol> 6补液的基因组DNA <ol><li>一旦样品已干燥，加入300微升的Tris-EDTA至再水化干燥的gDNA粒料。 </li><li>涡样品5秒，中速和发生在65℃的热水浴中搅拌1小时。 </li><li>从水浴中取出样品，并培育在o / n RT。 注意:所有的产品和试剂使用中列出的材料表，以及表4中 。 </li></ol>

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The authors have nothing to disclose.

本程序是一个优化的DNA提取的协议，已大大改善了高分子量DNA的产量相比，标准的方法，而不损害DNA的质量。与对产量的大部分影响最大的关键步骤是5.2步，其中包括在乙醇沉淀比任何出版协议审查这里，除了一个没有广泛分布11较长的离心步骤。检测DNA的质量没有改变与该较长的离心关联，表明大部分从唾液采集的可用的DNA是不是退化和高的分子量。 唾液收集在样品质量的限制，例如为外国污染物的潜力，需要在采集阶段被最小化，并且过多的蛋白质的样品中的存在，可以是一个潜在的感染的迹象。大量的蛋白质或外国的污染物可以保留在最终提取的DNA，从而使定量准确。如果有剩余的蛋白或污染物后补液（步骤6）被怀疑，样品清理可以通过启动在蛋白质沉淀步骤以缩放，以反映样品输入量的试剂量来进行。协议的另一个限制是时间的长短来执行的步骤需要。多个样品可以并行地运行;然而，建议不超过24平行萃取同时运行。这是用于蛋白质沉淀步骤，其中所述样品必须保持冷，以确保紧密的颗粒和运行超过24个样品可以允许粒料时重新溶解尤为重要。 这里提出的方案是最经济有效的方法考虑（ 见表3）。而该协议不使用试剂从PUREGENE提取试剂盒，更小的体积比建议的PUREGENE协议用于不影响萃取效率，这是这种减少的试剂，其驱动节约成本相对于该协议。需要注意的是计算的费用提取使用的标价为每一个项目，并不能反映任何折扣。与优化的协议每提取成本可通过公司的销售代表进一步与大宗订单或折扣减少。 证据也被提供，该协议是适用于下一代测序使用。所获得的数据提供唾液样本进行人类遗传学研究许多疾病的效用，其中血液是不是经常用进一步的证据。而血液和细胞系的DNA仍是用于测试遗传物质的优选的来源，唾液的收集是一个可行的选择时，这样的源不可用，当患者人数会受他们的集合或放血不可用或不实际的。

获得高质量的DNA对人类遗传研究是在疾病基因的发现进程至关重要。血，虽然需要侵入性手术，也比唾液采集更昂贵，是有利的，用于创建永生化细胞系的DNA的无限源，或iPS细胞的功能性研究，有时血液的DNA时使用的细胞系是不可用的。然而，获得的血液需要经过培训的采血和血液有一个较短的半衰期比口水1。从唾液DNA是较便宜，容易得到，因为它可以被收集，并通过邮件发送，而不需要用于抽血，由此增加潜在受试者池远远超出医院和实验室2的集水面积。当受试者具有给人一种唾液样品代替血液3,4的选择研究登记可以得到改进。左右的数量，从唾液DNA的质量问题可能已限制了其广泛我们ë尽管许多研究最近的研究显示全唾液的适用性，平均每毫升4.3×10 5个细胞，用于对较早的口腔拭子的方法未获得显著量唾液2，3，4，5的DNA检测， 6，虽然适度的文学存在呈现出唾液的DNA是否适合进行基因分型应用，包括基于微阵列的方法，8，9，10，没有研究探讨了新一代测序（NGS）。为优化这种唾液DNA提取协议的目的是最大限度地提高数量和质量的遗传学应用，很容易与普通试剂和耗材实验室实现了一个具有成本效益的方式。 从唾液DNA提取需要几个步骤:1）收集并存储，2）细胞裂解，3）核糖核酸酶处理，4）沉淀蛋白，5）用乙醇沉淀，6）DNA的补液。该DNA的稳定缓冲液，所述的此前2，没有充分改变功能。没有尝试优化核糖核酸酶处理和DNA补液措施作出。对于每一个剩下的步骤，可能影响产量的几个变量进行鉴定。每个变量都被单独操纵和提高产量和质量统计评估。对于被证实可以改善产率和/或DNA质量的变量的最优值被包括在最终的协议。

<table><tbody><tr><td>15 ml Centrifuge Tubes</td><td>Fisher</td><td>12-565-268</td><td></td></tr><tr><td>Cell Lysis Solution</td><td>Qiagen</td><td>158908</td><td></td></tr><tr><td>Proteinase K</td><td>Sigma</td><td>P6556</td><td></td></tr><tr><td>Protein Precipitation Solution</td><td>Qiagen</td><td>158912</td><td></td></tr><tr><td>Isopropanol</td><td>Fisher</td><td>A416-4</td><td></td></tr><tr><td>Glycogen</td><td>EZ-BioResearch</td><td>S1003</td><td></td></tr><tr><td>70% Ethanol</td><td>Fisher</td><td>04-355-305</td><td></td></tr><tr><td>Tris-EDTA (TE)</td><td>Fisher</td><td>BP2473-1</td><td></td></tr><tr><td>NaCl</td><td>Fisher</td><td>AC194090010</td><td></td></tr><tr><td>Tris HCl</td><td>Fisher</td><td>BP1757-100</td><td></td></tr><tr><td>EDTA (0.5 M) Solution</td><td>Fisher</td><td>03-500-506</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate</td><td>Fisher</td><td>BP166-100&amp;nbsp;</td><td></td></tr><tr><td>Analog Vortex Mixer</td><td>Fisher</td><td>02-215-365</td><td></td></tr><tr><td>Centrifuge 5810R</td><td>Eppendorf</td><td>5811 000.010</td><td></td></tr></tbody></table>

collection and extraction of saliva dna for next generation sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

为了确定最佳参数提取DNA进行了一系列配对的DNA提取的。单唾液样品被分裂并与两个可能的值对于给定的变量1的各部分进行测试。至少8个重复的每个配对测试被执行（ 例如 ，一个单唾液样品等分，以测试提取具有和不具有初始50℃温育）。优化是基于四个标准指标:总DNA的收量，二百八十零分之二百六十〇值时，二百三十分之二百六十值，和电泳的DNA以评估碎片的目视检查。变量不是所有可能的组合进行了评估统计学的相互作用（N = 169的组合），而是选择单独评估每个变量的边际效应。影响采用多路重复测量方差分析测试，估计影响来源于相当于回归方程。所有显著效果总结在表1中 ，示出为AVER产量年龄变化或DNA的质量（260/280和260/230）（每毫升唾液中输入纳克/微升）。 细胞裂解（步骤2）中的优化通过评估:1）的存在/不存在50℃下温育（1小时）的细胞溶解，以确保蛋白酶K降解和细胞裂解由存储缓冲介导的去完成，2前）存在/不存在同质通过涡旋步骤（中速，15秒），以及3）裂解溶液的温育时间（5和30分钟）。在30分钟的细胞裂解孵化的平均3.5％（P &lt;0.01），但没有其他细胞裂解变量对产量显著效果提高产量。涡旋由统计显著（P &lt;0.001），但实际上小的0.03下降260/280比例。 蛋白质沉淀（步骤4）前面的蛋白酶K消化破坏氨基酸链，提高蛋白质沉淀效率和释放捕获的DNA。蛋白酶K的量是变化的10倍。离心温度从20℃降低至4℃。增加蛋白酶K的量引起的产率（8.7％），也稍微提高了260/280和260/230比率均具有统计学显著下降。 乙醇沉淀（步骤5）的检测协议的最后阶段。糖原载体（0,8微升）的量是变化的，因为是总离心时间（5与30分钟11）。只有离心时间显著影响产量，以290％的平均增幅。较长的自旋也减少了260/280比略（0.05）。在实验中观察到的糖原对产​​量无显著效果;虽然在这些提取的DNA的总量是足够大，使糖原通常不会被使用。尽管缺乏这些样品中的效果，但仍然建议使用糖原，以减少产量减少的危险，每当唾液输入体积比此处或如存在给定的低e是任何其它理由相信产量将是低的。 代表DNA样品的目视检查（ 图1）表明，该提取的DNA不大大分段为任何唾液DNA提取过程，而是表现出适当的高分子量带不表示降解的DNA的拖尾现象。经过RNA酶消化，该协议产生的1.74的平均260/280。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51697/51697fig1highres.jpg" width="500" /> 图唾液的DNA 1。质量。四种提取程序被应用到相同的唾液采集。 （一 ）样品进行电泳在0.8％琼脂糖凝胶（250 ng的DNA）。的唾液DNA提取协议的所有变化导致的高分子量（&gt; 20 kb的）的DNA，没有证据退化。里:1的DNA laddeR，2＆3 Oragene小坑L2P协议样本，4＆5 Gentra PUREGENE体液协议，6＆7不用RNA去除步骤的优化的协议，8及9与RNA移除步骤的优化的协议。泳道2-7是直接在同一唾液样品类似的协议。泳道8和9表明，RNA去除步骤不引入DNA降解。 （B）的样品进行电泳在2％的琼脂糖凝胶（150毫微克DNA）。轻微的RNA峰是观察到的在泳道2中的凝胶，通过图7（约定如上述）的底部附近。泳道8和9示出的RNA去除步骤的有效性。 RNA的拆卸步骤（用RNA酶A步骤3）是DNA的准确定量的关键。在测试过程中，持续的高RNA含量观察到，如由双链DNA的RNA由量子比特2.0荧光测量的比率来确定。从样本平均，核酸含量无RNA酶A处理由46.6％（±​​0.4）RNA.样品将经历读为"&lt;20毫微克/毫升的"，这是可能的最低读数的量子比特的RNA检测的RNA的去除步骤。 通过这种优化的协议获得的DNA是具有足够质量的高通量测序当附加的RNaseA步骤施加。为了实现定向重测序数据，自定义安捷伦SureSelect靶向序列富集试剂盒适用于24个样本，针对2.6 MB的顺序。高通量测序对每个通道的12条形码（索引）的样品进行的。顺序读取被BWA对齐到hg19参考基因组12，用的是最好的做法很难过滤参数，然后进行应用GATK 13个碱基的质量分数校准，插入缺失会师，重复拆卸，SNP发现和在所有24个样本同时进行基因分型值14。所有24个样本产生高品质的农工商数据（ 表2）。的读取通过ILLUMina的标准过滤器和过Q&gt; 20，91.4％相一致的序列富集靶区，提供&gt; 30倍的覆盖范围平均在目标深度覆盖在Q&gt; 100，以及在必要的限度罕见SNP发现每个样品中。普通股余额为49.9％。与Illumina公司的微阵列比较基因型变异的呼叫产生了98.9％的一致。 <table border="0" cellpadding="0" cellspacing="0" width="352"><tbody><tr><td> 候选变量 </td><td> 纳克/微升 </td><td> 260/280 </td><td> 260/230 </td></tr><tr><td>漩涡</td><td> NS </td><td> -0.03 *** </td><td> NS </td></tr><tr><td> X30分钟细胞裂解孵化</td><td> 3.5％** </td><td> NS </td><td> NS </td></tr><tr><td>蛋白酶K X10 </td><td> -8.7％* </td><td> -0.05 *** </td><td> -0.03 *** </td></tr><tr><td> 30分钟旋</td><td> 290.2％*** </td><td> -0.05 *** </td><td> NS </td></tr><tr><td>糖原</td><td> NS </td><td> NS </td><td> -0.37 *** </td></tr></tbody></table> 表1对数量/质量指标优化变量的大小，所有的影响大小都在列标题中列出的单位。 p值的方差分析:* P &lt;0.05; ** P &lt;0.01; *** P &lt;.001; NS不显著<table border="0" cellpadding="0" cellspacing="0" width="176"><tbody><tr><td> 质量度量 </td><td> 价值 </td></tr><tr><td>目标长度</td><td> 1,708 KB </td></tr><tr><td>目标覆盖&gt; 30倍</td><td> 85.22％ </td></tr><tr><td>在dbSNP的SNP </td><td> 92.55％ </td></tr><tr><td>阵列协议</td><td> 98.99％ </td></tr></tbody></table> 高通量的序列，从靶向序列捕获的表2，质量。 <table border ="0"的cellpadding ="0"CELLSPACING ="0"宽度="668"&gt; <tbody><tr><td rowspan="14"> 全新优化的协议 </td><td> 项目 </td><td> 分配器 </td><td> 产品编号 </td><td> 采购单位 </td><td> 成本/单位 </td><td> 成本/收藏 </td></tr><tr><td> 15ml离心管</td><td>费舍尔</td><td> 12-565-268 </td><td> 500管</td><td> 233.50美元</td><td> 3.2690美元</td></tr><tr><td>细胞裂解液</td><td> QIAGEN（凯杰） </td><td> 158908 </td><td> 1升</td><td> 401.00美元</td><td> 3.2080美元</td></tr><tr><td>蛋白酶K </td><td>西格玛</td><td> P6556 </td><td> 1克</td><td> 713.00美元</td><td> 1.5686美元</td></tr><tr><td>蛋白质沉淀的解决方案</td><td> QIAGEN（凯杰） </td><td> 158912 </td><td> 350毫升</td><td> 350.00美元</td><td> 2.7200美元</td></tr><tr><td>异丙醇</td><td>费舍尔</td><td> A416-4 </td><td>的4×4升的情况下</td><td> 486.71美元</td><td> 0.2434美元</td></tr><tr><td>糖原溶液（20毫克/毫升） </td><td> EZ-BioResearch公司</td><td> S1003 </td><td> 1毫升</td><td> 51.00美元</td><td> 0.8160美元</td></tr><tr><td> 70％乙醇</td><td>费舍尔</td><td> 04-355-305 </td><td>案例4×1加仑。 </td><td> 123.03美元</td><td> 0.0325美元</td></tr><tr><td>的Tris-EDTA（TE） </td><td>费舍尔</td><td> BP2473-1 </td><td> 1升</td><td> 68.67美元</td><td> 0.0412美元</td></tr><tr><td>氯化钠</td><td>费舍尔</td><td> AC194090010 </td><td> 1公斤</td><td> 34.65美元</td><td> 0.000004美元</td></tr><tr><td>三盐酸</td><td>费舍尔</td><td> BP1757-100 </td><td>百毫升</td><td> 57.84美元</td><td> 0.0116美元</td></tr><tr><td> EDTA（0.5M）解决方案</td><td>费舍尔</td><td> 03-500-506 </td><td>百毫升</td><td> 33.60美元</td><td> 0.0134美元</td></tr><tr><td>多德钠ecyl硫酸</td><td>费舍尔</td><td> BP166-100 </td><td> 100克</td><td> 59.95美元</td><td> 0.0060美元</td></tr><tr><td></td><td></td><td></td><td></td><td> 总成本 </td><td> 11.93美元 </td></tr><tr><td rowspan="8"> Oragene </td><td> 项目 </td><td> 分配器 </td><td> 产品编号 </td><td> 采购单位 </td><td> 成本/单位 </td><td> 成本/收藏 </td></tr><tr><td> 15ml离心管</td><td>费舍尔</td><td> 12-565-268 </td><td> 500管</td><td> 233.50美元</td><td> 0.9340美元</td></tr><tr><td> 100％乙醇</td><td>费舍尔</td><td> BP2818-100 </td><td>百毫升</td><td> 34.29美元</td><td> 1.6459美元</td></tr><tr><td> 70％乙醇</td><td>费舍尔</td><td> 04-355-305 </td><td>案例4×1加仑。 </td><td> 123.03美元</td><td> 0.0081美元</td></tr><tr><td>的Tris-EDTA（TE） </td><td>费舍尔</td><td> BP2473-1 </td><td> 1升</td><td> 68.67美元</td><td> 0.0687美元</td></tr><tr><td> 1.5毫升管</td><td>杰纳西</td><td> 22-281A </td><td> 500管</td><td> 22.85美元</td><td> 0.0457美元</td></tr><tr><td> Oragene采集盒</td><td> Oragene </td><td> OG-500 </td><td> 1 </td><td> 25.00美元</td><td> 25.00美元</td></tr><tr><td></td><td></td><td></td><td></td><td> 总成本 </td><td> 27.70美元 </td></tr><tr><td rowspan="14"> PUREGENE </td><td> 项目 </td><td> 分配器 </td><td> 产品编号 </td><td> 采购单位 </td><td> 成本/单位 </td><td> 成本/收藏 </td></tr><tr><td> 15ml离心管</td><td>费舍尔</td><td> 12-565-268 </td><td> 500管</td&gt; <td> 233.50美元</td><td> 0.9340美元</td></tr><tr><td>细胞裂解液</td><td> QIAGEN（凯杰） </td><td> 158908 </td><td> 1升</td><td> 401.00美元</td><td> 4.0100美元</td></tr><tr><td>蛋白酶K </td><td> QIAGEN（凯杰） </td><td> 158918 </td><td> 650毫升</td><td> 73.10美元</td><td> 7.8723美元</td></tr><tr><td>蛋白质沉淀的解决方案</td><td> QIAGEN（凯杰） </td><td> 158912 </td><td> 350毫升</td><td> 350.00美元</td><td> 4.0000美元</td></tr><tr><td>异丙醇</td><td>费舍尔</td><td> A4164 </td><td>的4×4升的情况下</td><td> 486.71美元</td><td> 0.3650美元</td></tr><tr><td>糖原解决方案</td><td> QIAGEN（凯杰） </td><td> 158930 </td><td> 500毫升</td><td> 64.30美元</td><td> 2.5720美元</td></tr><tr><td> 70％乙醇</td><td>费舍尔</td><td> 04-355-305 </td><td>案例4×1加仑。 </td><td> 123.03美元</td><td> 0.0975美元</td></tr><tr><td> DNA的水合溶液</td><td> QIAGEN（凯杰） </td><td> 158914 </td><td>百毫升</td><td> 69.80美元</td><td> 0.1396美元</td></tr><tr><td>氯化钠</td><td>费舍尔</td><td> AC19409-0010 </td><td> 1公斤</td><td> 34.65美元</td><td> 0.000004美元</td></tr><tr><td>三盐酸</td><td>费舍尔</td><td> BP1757-100 </td><td>百毫升</td><td> 57.84美元</td><td> 0.0116美元</td></tr><tr><td> EDTA（0.5M）解决方案</td><td>费舍尔</td><td> 03-500-506 </td><td>百毫升</td><td> 33.60美元</td><td> 0.0134美元</td></tr><tr><td>十二烷基硫酸钠</td><td>费舍尔</td><td> BP166-100 </td><td> 100克</td><td> 59.95美元</td><td> 0.0060美元</td></tr><tr><td></td><td></td><td></td><td></td><td> 总成本 </td><td> 19.99美元 </td></tr></tbody></table> 优化的协议表3的成本比较，从2毫升唾液与其他市售的协议中提取的DNA，一个所需的DNA提取LL试剂和耗材使用标准目录价格在互联网9月30日对被评估，2013年需要注意的是此协议是写从1.25毫升全唾液（提取的DNA 即 2.5唾液和缓冲液，参见步骤2.3），为这个值表示一半的唾液收集管中的总体积。对于成本比较，将2ml被选择，因为这是一个常见的​​市售试剂盒（Oragene）相关联的量。 <table border="0" cellpadding="0" cellspacing="0" width="264"><tbody><tr><td colspan="3"> DNA的稳定缓冲液（250毫升）中 </td></tr><tr><td>组件</td><td>体积（ml） </td><td> [最后] </td></tr><tr><td> 1M NaCl的</td><td> 1.461克</td><td> 0.1M的</td></tr><tr><td>三盐酸</td><td> 2.5 </td><td> 0.01M的</td></tr><tr><td> 0.5M EDTA </td><td> 5 </td><td> 0.01M的</td></tr><tr><td> 10％SDS </td><td> 12.5 &lt;/ TD&gt; <td> 0.014 M </td></tr><tr><td>蛋白酶K溶液（20毫克/毫升） </td><td> 2.5 </td><td> 6.92x10 -6 M </td></tr><tr><td> DDH 2 O </td><td> 227.5 </td><td></td></tr><tr><td colspan="3"> 蛋白酶K溶液（20毫克/毫升） </td></tr><tr><td>组件</td><td>体积（ml） </td><td> [最后] </td></tr><tr><td>蛋白酶K粉</td><td> 500毫克</td><td> 6.92x10 -4 M </td></tr><tr><td> DDH 2 O </td><td> 25 </td><td></td></tr><tr><td colspan="3"> 70％乙醇（500ml）中 </td></tr><tr><td>组件</td><td>体积（ml） </td><td> [最后] </td></tr><tr><td>乙醇95％的</td><td> 368.5 </td><td> 12.63 M </td></tr><tr><td> DDH 2 O </td><td> 131.5 </td><td></td></tr></tbody></table> 表4食谱试剂。

Watch this Scientific Journal Video about Collection and Extraction of Saliva DNA for Next Generation Sequencing at JoVE.com

Collection and Extraction of Saliva DNA for Next Generation Sequencing

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

收集和唾液DNA提取用于新一代测序

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

collection-and-extraction-of-saliva-dna-for-next-generation-sequencing

Universidad San Sebastian

Research

JoVE Journal

Medicine

39.1K 次观看.  The Research Institute at Nationwide Children's Hospital. DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

视频: Collection and Extraction of Saliva DNA for Next Generation Sequencing

Metagenomic Analysis of Silage

Metagenomics is defined as the direct analysis of deoxyribonucleic acid (DNA) purified from environmental samples and enables taxonomic identification of the microbial communities present within them. Two main metagenomic approaches exist; sequencing the 16S rRNA gene coding region, which exhibits sufficient variation between taxa for identification, and shotgun sequencing, in which genomes of the organisms that are present in the sample are analyzed and ascribed to "operational taxonomic units"; species, genera or families depending on the extent of sequencing coverage. 
In this study, shotgun sequencing was used to analyze the microbial community present in cattle silage and, coupled with a range of bioinformatics tools to quality check and filter the DNA sequence reads, perform taxonomic classification of the microbial populations present within the sampled silage, and achieve functional annotation of the sequences. These methods were employed to identify potentially harmful bacteria that existed within the silage, an indication of silage spoilage. If spoiled silage is not remediated, then upon ingestion it could be potentially fatal to the livestock.

Metagenomics was used to investigate the microbiome of silage cattle feed. Analysis was performed by shotgun sequencing. This approach was used to characterize the composition of the microbial community within the cattle feed.

青贮的宏基因组学分析

Metagenomics is defined as the direct analysis of deoxyribonucleic acid (DNA) purified from environmental samples and enables taxonomic identification of ...

科研

遗传学

Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA libraries are needed for optimal results from next-generation sequencing. Environmental samples such as water may have low quality and quantities of DNA as well as contaminants that co-precipitate with DNA. The mechanical and enzymatic processes involved in extraction and library preparation may further damage the DNA. Gel size selection enables purification and recovery of DNA fragments of a defined size for sequencing applications. Nevertheless, this task is one of the most time-consuming steps in the DNA library preparation workflow. The protocol described here enables complete automation of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments. 
In this study, we describe a high-throughput approach to prepare high quality DNA libraries from freshwater samples that can be applied also to other environmental samples. We used an indirect approach to concentrate bacterial cells from environmental freshwater samples; DNA was extracted using a commercially available DNA extraction kit, and DNA libraries were prepared using a commercial transposon-based protocol. DNA fragments of 500 to 800 bp were gel size selected using Ranger Technology, an automated electrophoresis workstation. Sequencing of the size-selected DNA libraries demonstrated significant improvements to read length and quality of the sequencing reads.

This manuscript describes an automated gel size selection approach for purifying DNA fragments for next-generation sequencing. The Ranger Technology provides complete automation of the entire process of agarose gel loading, electrophoretic analysis, and recovery of targeted DNA fragments allowing for high-throughput and high quality next-generation sequencing libraries.

自动凝胶尺寸选择，以提高下一代测序文库的质量，从环境水样准备

Next-generation sequencing of environmental samples can be challenging because of the variable DNA quantity and quality in these samples. High quality DNA ...

环境科学

Optimization and Comparative Analysis of Plant Organellar DNA Enrichment Methods Suitable for Next-generation Sequencing

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic fragments. Organellar genomes also exist in admixtures within a given cell or tissue type (heteroplasmy), and an abundance of subtypes may change throughout development or when under stress (sub-stoichiometric shifting). Next-generation sequencing (NGS) technologies are required to obtain deeper understanding of organellar genome structure and function. Traditional sequencing studies use several methods to obtain organellar DNA: (1) If a large amount of starting tissue is used, it is homogenized and subjected to differential centrifugation and/or gradient purification. (2) If a smaller amount of tissue is used (i.e., if seeds, material, or space is limited), the same process is performed as in (1), followed by whole-genome amplification to obtain sufficient DNA. (3) Bioinformatics analysis can be used to sequence the total genomic DNA and to parse out organellar reads. All these methods have inherent challenges and tradeoffs. In (1), it may be difficult to obtain such a large amount of starting tissue; in (2), whole-genome amplification could introduce a sequencing bias; and in (3), homology between nuclear and organellar genomes could interfere with assembly and analysis. In plants with large nuclear genomes, it is advantageous to enrich for organellar DNA to reduce sequencing costs and sequence complexity for bioinformatics analyses. Here, we compare a traditional differential centrifugation method with a fourth method, an adapted CpG-methyl pulldown approach, to separate the total genomic DNA into nuclear and organellar fractions. Both methods yield sufficient DNA for NGS, DNA that is highly enriched for organellar sequences, albeit at different ratios in mitochondria and chloroplasts. We present the optimization of these methods for wheat leaf tissue and discuss major advantages and disadvantages of each approach in the context of sample input, protocol ease, and downstream application.

The comparison and optimization of two plant organellar DNA enrichment methods are presented: traditional differential centrifugation and fractionation of the total gDNA based on methylation status. We assess the resulting DNA quantity and quality, demonstrate performance in short-read next-generation sequencing, and discuss the potential for use in long-read single-molecule sequencing.

适用于下一代测序的植物细菌DNA富集方法的优化与比较分析

Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic ...

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. ...

癌症研究

Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry

Although human saliva proteome and peptidome have been revealed 1-2 they were majorly identified from tryptic digests of saliva proteins. Identification of indigenous peptidome of human saliva without prior digestion with exogenous enzymes becomes imperative, since native peptides in human saliva provide potential values for diagnosing disease, predicting disease progression, and monitoring therapeutic efficacy. Appropriate sampling is a critical step for enhancement of identification of human indigenous saliva peptidome. Traditional methods of sampling human saliva involving centrifugation to remove debris 3-4 may be too time-consuming to be applicable for clinical use. Furthermore, debris removal by centrifugation may be unable to clean most of the infected pathogens and remove the high abundance proteins that often hinder the identification of low abundance peptidome.
Conventional proteomic approaches that primarily utilize two-dimensional gel electrophoresis (2-DE) gels in conjugation with in-gel digestion are capable of identifying many saliva proteins 5-6. However, this approach is generally not sufficiently sensitive to detect low abundance peptides/proteins. Liquid chromatography-Mass spectrometry (LC-MS) based proteomics is an alternative that can identify proteins without prior 2-DE separation. Although this approach provides higher sensitivity, it generally needs prior sample pre-fractionation 7 and pre-digestion with trypsin, which makes it difficult for clinical use. 
To circumvent the hindrance in mass spectrometry due to sample preparation, we have developed a technique called capillary ultrafiltration (CUF) probes 8-11. Data from our laboratory demonstrated that the CUF probes are capable of capturing proteins in vivo from various microenvironments in animals in a dynamic and minimally invasive manner 8-11. No centrifugation is needed since a negative pressure is created by simply syringe withdrawing during sample collection. The CUF probes combined with LC-MS have successfully identified tryptic-digested proteins 8-11. In this study, we upgraded the ultrafiltration sampling technique by creating a lollipop-like ultrafiltration (LLUF) probe that can easily fit in the human oral cavity. The direct analysis by LC-MS without trypsin digestion showed that human saliva indigenously contains many peptide fragments derived from various proteins. Sampling saliva with LLUF probes avoided centrifugation but effectively removed many larger and high abundance proteins. Our mass spectrometric results illustrated that many low abundance peptides became detectable after filtering out larger proteins with LLUF probes. Detection of low abundance saliva peptides was independent of multiple-step sample separation with chromatography. For clinical application, the LLUF probes incorporated with LC-MS could potentially be used in the future to monitor disease progression from saliva.

Considering saliva sampling for future clinical application, a lollipop-like ultrafiltration (LLUF) probe was fabricated to fit in the human oral cavity. Direct analysis of undigested saliva by NanoLC-LTQ mass spectrometry demonstrated the ability of LLUF probes to remove large proteins and high abundance proteins, and make low-abundant peptides more detectable.

采样使用超探头棒棒糖样的土著人的唾液多肽：简化和增强肽的临床质谱检测

Although human saliva proteome and peptidome have been revealed 1-2 they were majorly identified from tryptic digests of saliva proteins. Identification ...

医学

Application of DNA Fingerprinting using the D1S80 Locus in Lab Classes

In biological sciences, DNA fingerprinting has been widely used for paternity testing, forensic applications and phylogenetic studies. Here, we describe a reliable and robust method for genotyping individuals by Variable Number of Tandem Repeat (VNTR) analysis in the context of undergraduate laboratory classes. The human D1S80 VNTR locus is used in this protocol as a highly polymorphic marker based on variation in the number of repetitive sequences.
This simple protocol conveys useful information for teachers and the implementation of DNA fingerprinting in practical laboratory classes. In the presented laboratory exercise, DNA extraction followed by PCR amplification is used to determine genetic variation at the D1S80 VNTR locus. Differences in the fragment size of PCR products are visualized by agarose gel electrophoresis. The fragment sizes and repeat numbers are calculated based on a linear regression of the size and migration distance of a DNA size standard.
Following this guide, students should be able to:
&#8226;&#160; Harvest and extract DNA from buccal mucosa epithelial cells 
&#8226;&#160; Perform a PCR experiment and understand the function of various reaction components 
&#8226;&#160; Analyze the amplicons by agarose gel electrophoresis and interpret the results 
&#8226;&#160; Understand the use of VNTRs in DNA fingerprinting and its application in biological sciences

Application of DNA Fingerprinting using the D1S80 Locus in Lab Classes

Here we describe a simple protocol to generate DNA fingerprinting profiles by amplifying the VNTR locus D1S80 from epithelial cell DNA.

使用 D1S80 轨迹在实验室类中应用 DNA 指纹

In biological sciences, DNA fingerprinting has been widely used for paternity testing, forensic applications and phylogenetic studies. Here, we describe a ...

Hybrid De Novo Genome Assembly for the Generation of Complete Genomes of Urinary Bacteria using Short- and Long-read Sequencing Technologies

Complete genome sequences provide valuable data for the understanding of genetic diversity and unique colonization factors of urinary microbes. These data may include mobile genetic elements, such as plasmids and extrachromosomal phage, that contribute to the dissemination of antimicrobial resistance and further complicate treatment of urinary tract infection (UTI). In addition to providing fine resolution of genome structure, complete, closed genomes allow for the detailed comparative genomics and evolutionary analyses. The generation of complete genomes de novo has long been a challenging task due to limitations of available sequencing technology. Paired-end Next Generation Sequencing (NGS) produces high quality short reads often resulting in accurate but fragmented genome assemblies. On the contrary, Nanopore sequencing provides long reads of lower quality normally leading to error-prone complete assemblies. Such errors may hamper genome-wide association studies or provide misleading variant analysis results. Therefore, hybrid approaches combining both short and long reads have emerged as reliable methods to achieve highly accurate closed bacterial genomes. Reported herein is a comprehensive method for the culture of diverse urinary bacteria, species identification by 16S rRNA gene sequencing, extraction of genomic DNA (gDNA), and generation of short and long reads by NGS and Nanopore platforms, respectively. Additionally, this method describes a bioinformatic pipeline of quality control, assembly, and gene prediction algorithms for the generation of annotated complete genome sequences. Combination of bioinformatic tools enables the selection of high quality read data for hybrid genome assembly and downstream analysis. The streamlined approach for the hybrid de novo genome assembly described in this protocol may be adapted for the use in any culturable bacteria.

Hybrid De Novo Genome Assembly for the Generation of Complete Genomes of Urinary Bacteria using Short- and Long-read Sequencing Technologies

This protocol details a comprehensive approach for the culturing, sequencing, and de novo hybrid genome assembly of urinary bacteria. It provides a reproducible procedure for the generation of complete, circular genome sequences useful in studying both chromosomal and extrachromosomal genetic elements contributing to urinary colonization, pathogenesis, and antimicrobial resistance dissemination.

使用短读和长读测序技术生成尿细菌完整基因组的杂交从 头 基因组组装

Complete genome sequences provide valuable data for the understanding of genetic diversity and unique colonization factors of urinary microbes. These data ...

Large-Scale SARS-CoV-2 Testing Utilizing Saliva and Transposition Sample Pooling

Identification and isolation of contagious individuals along with quarantine of close contacts, is critical for slowing the spread of COVID-19. Large-scale testing in a surveillance or screening capacity for asymptomatic carriers of COVID-19 provides both data on viral spread and the follow-up ability to rapidly test individuals during suspected outbreaks. The COVID-19 early detection program at Michigan State University has been utilizing large-scale testing in a surveillance or screening capacity since fall of 2020. The methods adapted here take advantage of the reliability, large sample volume, and self-collection benefits of saliva, paired with a cost-effective, reagent conserving two-dimensional pooling scheme. The process was designed to be adaptable to supply shortages, with many components of the kits and the assay easily substituted. The processes outlined for collecting and processing SARS-CoV-2 samples can be adapted to test for future viral pathogens reliably expressed in saliva. By providing this blueprint for universities or other organizations, preparedness plans for future viral outbreaks can be developed.

The protocol is intended to serve as a blueprint for universities and other organizations considering large-scale testing for SARS-CoV-2 or developing preparedness plans for future viral outbreaks.

Identification and isolation of contagious individuals along with quarantine of close contacts, is critical for slowing the spread of COVID-19. ...

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using noninvasive sampling techniques to investigate population genetics and demography of wild populations1. This approach has proven to be especially useful when dealing with rare or elusive species2. While a number of these methods have been developed to sample hair, feces and other biological material from carnivores and medium-sized mammals, they have largely remained untested in elusive small mammals. In this video, we present a novel, inexpensive and noninvasive hair snare targeted at an elusive small mammal, the American pika (Ochotona princeps). We describe the general set-up of the hair snare, which consists of strips of packing tape arranged in a web-like fashion and placed along travelling routes in the pikas&#x2019; habitat. We illustrate the efficiency of the snare at collecting a large quantity of hair that can then be collected and brought back to the lab. We then demonstrate the use of the DNA IQ system (Promega) to isolate DNA and showcase the utility of this method to amplify commonly used molecular markers including nuclear microsatellites, amplified fragment length polymorphisms (AFLPs), mitochondrial sequences (800bp) as well as a molecular sexing marker. Overall, we demonstrate the utility of this novel noninvasive hair snare as a sampling technique for wildlife population biologists. We anticipate that this approach will be applicable to a variety of small mammals, opening up areas of investigation within natural populations, while minimizing impact to study organisms.

We present a noninvasive sampling approach to efficiently collect hair samples from elusive small mammals, as shown for the American pika. We demonstrate the utility of this method by extracting DNA from sampled hair and amplifying several types of molecular markers commonly used in studies of wildlife ecology and conservation.

一种无创性的发采样技术，以获得难以捉摸的小型兽类高质量的DNA

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

生物学

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 ...

收集和唾液DNA提取用于新一代测序

摘要

探索更多视频

此视频中的章节

青贮的宏基因组学分析

自动凝胶尺寸选择，以提高下一代测序文库的质量，从环境水样准备

适用于下一代测序的植物细菌DNA富集方法的优化与比较分析

利用下一代测序检测 CtDNA 中的稀有突变

采样使用超探头棒棒糖样的土著人的唾液多肽：简化和增强肽的临床质谱检测

使用 D1S80 轨迹在实验室类中应用 DNA 指纹

使用短读和长读测序技术生成尿细菌完整基因组的杂交从头基因组组装

Large-Scale SARS-CoV-2 Testing Utilizing Saliva and Transposition Sample Pooling

一种无创性的发采样技术，以获得难以捉摸的小型兽类高质量的DNA

有针对性的DNA甲基化分析通过新一代测序

收集和唾液DNA提取用于新一代测序

摘要

探索更多视频

此视频中的章节

青贮的宏基因组学分析

自动凝胶尺寸选择，以提高下一代测序文库的质量，从环境水样准备

适用于下一代测序的植物细菌DNA富集方法的优化与比较分析

利用下一代测序检测 CtDNA 中的稀有突变

采样使用超探头棒棒糖样的土著人的唾液多肽：简化和增强肽的临床质谱检测

使用 D1S80 轨迹在实验室类中应用 DNA 指纹

使用短读和长读测序技术生成尿细菌完整基因组的杂交从 头 基因组组装

Large-Scale SARS-CoV-2 Testing Utilizing Saliva and Transposition Sample Pooling

一种无创性的发采样技术，以获得难以捉摸的小型兽类高质量的DNA

有针对性的DNA甲基化分析通过新一代测序

使用短读和长读测序技术生成尿细菌完整基因组的杂交从头基因组组装