作者感谢 Nozomi、Takai 和 Nahoko 在基因组测序中的技术支持。这项工作得到了日本促进科学学会 (jsp) 研究员的资助, KAKENHI 为年轻科学家提供援助 (22681029 号), 以及 KAKENHI 的科研资助 (B), 来自 jsp 的17H03620 号, 由一个来自住友基金会 (140340 号) 基础科学研究项目的赠款, 部分是由山形县政府和日本鹤的研究基金提供的。以小球藻为原料, 提供水熊蟲的小球藻。

1. 准备
<ol><li>用蒸馏水 (DW) 制备2% 琼脂糖凝胶, 作为90毫米塑料培养皿中的溶剂, 10 毫升1% 青霉素/链霉素鸡尾酒与 DW。凝胶可以储存 2-3 周的孵化器设置在18摄氏度。 注: 避免任何贮存的凝胶低于10摄氏度, 因为低温会收缩琼脂糖凝胶, 造成一个微小的缝隙之间的凝胶和培养皿壁, 其中水熊蟲可以被困。</li>
</ol>2. 样品制备和污染物排除
<ol><li>收集一个单一的缓步动物, 把它放在准备好的琼脂板上, 并用 DW 清洗 2x-3x, 以除去剩余的粒子。</li>
	<li>孵化的缓步动物在 18-22 °c 24 小时, 以消除任何多余的食物从肠道。</li>
	<li>将饥饿的缓步动物在青霉素/链霉素抗生素中放置 2-6 小时, 去除任何细菌污染, 并使用 P10 吸管将被净化的动物放在干净的滑动玻璃上。</li>
	<li>在显微镜下观察缓步动物500X 放大倍数, 并确认没有残留细菌。 注: 显微镜的高放大倍数是最佳的, 但光学显微镜可以在不应用盖玻片的情况下交替使用。</li>
	<li>收集的人使用的 P10 吸管最多5µL 的液体, 将其放入低结合聚合酶链反应 (PCR) 管, 并尽可能多地去除多余的液体。 注意: 管内, PCR 管和吸管提示应该是低约束力, 以减少 DNA 的损失。 注意: 尽快进行同质化, 因为动物的快速脱水或死亡会严重损害基因组 DNA。</li>
</ol>3. 均匀化和 DNA 提取
<ol><li>融汇动物以下列方法之一获得其基因组 DNA (gDNA)。 注意: 在下面的 DNA 提取步骤中, 使用材料表中所示的指定套件是非常关键的, 因为其他套件 (无论是柱形还是珠基) 在这个极低的输入中是不有效的。在使用前, 基因组裂解缓冲液已经提供了0.5% β巯基乙醇。<ol>
<li>融汇的动物与冻融循环18。<ol>
<li>紧接着步骤 2.5, 添加100µL 的裂解缓冲液的 PCR 管含有缓步动物。</li>
			<li>将 PCR 管放置在液氮中10分钟, 10 分钟, 将其移动到加热到37摄氏度的热块中。重复此步骤2x。 注: 本步骤可在均匀化不足或手动粉碎后进行重复。</li>
		</ol>
</li>
		<li>手动粉碎动物。<ol>
<li>紧接着步骤 2.5, 在显微镜下, 用吸管的 P10 尖端挤压该动物, 并将其压在 PCR 管壁上, 然后立即添加100µL 的溶解缓冲液。 注意: 在显微镜下观察这个过程是非常重要的, 因为缓步动物很容易溜掉, 而且低效的粉碎会导致无法提取 gDNA。确保缓步动物角质层断裂, 使裂解缓冲液渗入机体。</li>
		</ol>
</li>
	</ol>
</li>
	<li>在室温下孵化管30分钟以进行裂解。 注: 有效裂解至少需要30分钟, 但孵化时间可能更长。</li>
	<li>将裂解混合物的完整容积 (100 µL) 转移到干净的1.5 毫升低结合微细。</li>
	<li>将100µL 溶解缓冲剂添加到用于均匀化的低结合 PCR 管中, 现在是空的, 在吹打混合物后, 将其转移到步骤3.3 中使用的1.5 毫升低约束微细。重复此步骤2x。</li>
	<li>与步骤3.4 中一样, 在低结合 PCR 管中加入300µL 裂解缓冲液, 并在吹打后将混合物移动到1.5 毫升低束缚微细。1.5 毫升低结合微细应包含600µL 的混合物后, 这一步。 注意: 这些步骤是为了尽量减少 gDNA 的损失, 通过多次洗涤样品来降低链到 PCR 管壁的损耗。</li>
	<li>将600µL 的裂解混合物添加到放置在收集管中的旋柱上, 并将其离心在 1万 x g 处, 1 分钟。</li>
	<li>将流量重新应用到柱上, 并将其离心在 1万 x g 处1分钟。 注意: 此步骤对于确保大多数 gDNA 绑定到该列至关重要。</li>
	<li>将500µL 的洗涤缓冲器添加到旋转柱上, 并将其离心在 1万 x g 上1分钟. 将旋转柱转移到干净的 1.5 mL 微细。</li>
	<li>应用20µL 10 毫米三盐酸, pH 8.5, 到自旋柱, 在室温下等待5分钟, 并离心它在 1万 x g 为1分钟。 注: 洗脱缓冲液不能含有 EDTA, 因为它干扰了库的制备酶。</li>
	<li>重新应用流通过自旋柱, 并在室温孵化5分钟后, 离心机1分钟 1万 x g。 注意: 此步骤对于确保绑定到列的 gDNA 的最大洗脱至关重要。</li>
</ol>4. 图书馆建设顺序
<ol><li>
DNA 碎片
	<ol>
<li>将 gDNA 淋洗剂组成的15µL 转移到15µL 微细, 用于 DNA 碎片化, 并使用桌面离心将管离心为1分钟。 注意: 在材料表中显示的指定微细对低输入是最佳的。低体积超声波的碎片是至关重要的, 由于 DNA 浓度极低, 这不能代替酶分裂。</li>
		<li>碎片 gDNA 到 550 bp。 注: 我们使用的 550 bp 设置如下: 峰值事件功率 = 30 W, 责任因数 = 20%, 周期每突发 = 50, 治疗时间 = 二十三年代。</li>
		<li>经过彻底的吹打, 转移10µL 的碎片 DNA 混合物到一个干净的低结合 PCR 管。 注意: 实验可以在这里停止。保存在4摄氏度或-20 摄氏度的 DNA。</li>
	</ol>
</li>
	<li>
图书馆建设顺序 注: 由于输入 DNA 量低, 在下列过程中使用所述材料表中的指定套件是绝对关键的。<ol>
<li>添加2µL 的模板准备缓冲区和1µL 的模板制备酶, 并将其与吸管彻底混合。</li>
		<li>在热循环仪上执行模板准备反应, 条件如下:22 °c 为25分钟, 55 °c 20 分钟, 4 °c 举行, 和一个加热盖子在 101-105 °c。一旦反应完成后, 继续下一步。</li>
		<li>添加1µL 的图书馆合成缓冲器和1µL 的图书馆合成酶的模板制备反应产品和孵化混合物在22°c 40 分钟 (与4°c 举行)。一旦反应完成后, 继续下一步。</li>
		<li>增加30µL 的图书馆放大主混合 (25 µL 的图书馆放大缓冲器, 1 µL 的图书馆扩增酶, 4 µL 核酸酶水) 和5µL 的索引试剂。</li>
		<li>用吸管将所有东西彻底混合, 然后用台式离心机将混合物简单地离心。</li>
		<li>执行 PCR 与表 1所列条件。</li>
	</ol>
</li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>温度</td>
			<td>时间</td>
			<td>周期</td>
		</tr>
<tr>
<td>72˚C</td>
			<td>3分钟</td>
			<td></td>
		</tr>
<tr>
<td>85˚C</td>
			<td>2分钟</td>
			<td></td>
		</tr>
<tr>
<td>98˚C</td>
			<td>2分钟</td>
			<td></td>
		</tr>
<tr>
<td>98˚C</td>
			<td>20秒</td>
			<td rowspan="3">4个周期</td>
		</tr>
<tr>
<td>67˚C</td>
			<td>20秒</td>
		</tr>
<tr>
<td>72˚C</td>
			<td>40秒</td>
		</tr>
<tr>
<td>98˚C</td>
			<td>20秒</td>
			<td rowspan="2">16个周期</td>
		</tr>
<tr>
<td>72˚C</td>
			<td>50秒</td>
		</tr>
<tr>
<td>4˚C</td>
			<td>举行</td>
			<td></td>
		</tr>
</tbody></table>表 1: PCR 条件。
<ol start="3"><li>
PCR 产品的纯化 注: 此步骤可替代其他纯化方法。如果使用另一种方法, 请确保使用分光光度计检查生成的 DNA 纯度。吸光度比为260/280 和260/230 必须在1.8 以上。<ol>
<li>添加50µL 磁珠, 吸管的解决方案 10x, 并离心机它与桌面离心。</li>
		<li>在室温下孵化溶液2分钟。</li>
		<li>在磁性支架上孵育5分钟, 或直到溶液完全清除, 然后取出上清。</li>
		<li>添加200µL 新鲜制备的80% 乙醇的低结合 PCR 管在磁铁立场, 等待三十年代, 并删除上清。重复此步骤2x。不要打扰珠子。</li>
		<li>简单地将低结合 PCR 管与台式离心机分离, 并去除磁性支架上多余的乙醇。空气干燥的珠子, 但避免 overdrying。</li>
		<li>并用重悬15µL 10 毫米三盐酸, pH 8.5, 彻底吸管溶液, 使磁性珠均匀分布, 在室温下孵化管2分钟, 并在短暂离心后, 孵化在一个磁性立场2分钟, 直到解决方案是明确的。 注: 洗脱缓冲器不应含有 EDTA, 因为它干扰测序化学。</li>
		<li>转移上清, 不干扰颗粒, 到一个新的低结合 PCR 管。 注意: 实验可以在这里停止。保存的 DNA 在4摄氏度或在-20 摄氏度长时间储存。</li>
	</ol>
</li>
</ol>5. DNA 的质量检查、定量和序列
注: 由于 DNA 含量低, 本步骤前未进行质量检查。
<ol><li>
DNA 库大小分布的验证 注: 其他高灵敏度的电泳系统可以使用数字报告的大小分布。<ol>
<li>返回电泳缓冲试剂和凝胶盒 (25-1000 bp 在范围内) 到室温。</li>
		<li>添加3µL 电泳缓冲试剂与1µL 的测序库, 并将其彻底混合1分钟与涡, 并简要离心他们与台式离心机。</li>
		<li>与相关软件进行电泳和验证库大小分布。主要的碎片峰值应该从大约300到 1000 bp 不等。</li>
	</ol>
</li>
	<li>
DNA 量化 注: 其他基于荧光的方法或定量 PCR 也可以使用, 但应避免分光光度法, 因为它是不够准确, 以量化测序库。<ol>
<li>添加796µL 溶液缓冲器和4µL 荧光试剂, 并将其彻底混合。将190µL 的工作溶液分配给两个化验管和197µL 到一个化验管。</li>
		<li>将已知的 DNA 浓度的10µL 标准添加到每个检测管中, 其中包含190µL 的工作溶液, 3 µL 准备好的库到含有197µL 工作溶液的检测管。</li>
		<li>将管子简单地涡旋, 然后将其离心在台式离心机上。使用具有3µL 设置的荧光计量化 DNA。</li>
	</ol>
</li>
	<li>
DNA 库序列 注意: 测序平台必须与指定的库结构套件兼容。<ol>
<li>根据制造商的协议准备排序库。</li>
		<li>将试剂盒和流动单元设置为测序仪, 并按照制造商的协议输入顺序运行信息。</li>
		<li>运行排序。 注: 我们已经进行了两个排序运行: 一个样本/运行, 以及四样本复用一次运行。</li>
	</ol>
</li>
</ol>6. 计算分析
<ol><li>基调用, 如果需要, 解读取。</li>
	<li>使用 FastQC19验证序列数据的质量。 注意: 为了更深入地验证获得的数据, 请参阅我们以前的报告16。</li>
</ol>

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C., 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=Evidence+for+extensive+horizontal+gene+transfer+from+the+draft+genome+of+a+tardigrade.">Evidence for extensive horizontal gene transfer from the draft genome of a tardigrade.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (52), 15976-15981 (2015).</li><li>Arakawa, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+evidence+for+extensive+horizontal+gene+transfer+from+the+draft+genome+of+a+tardigrade.">No evidence for extensive horizontal gene transfer from the draft genome of a tardigrade.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (22), E3057 (2016).</li><li>Arakawa, K., Yoshida, Y., Tomita, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+sequencing+of+a+single+tardigrade+Hypsibius+dujardini+individual.">Genome sequencing of a single tardigrade Hypsibius dujardini individual.</a> Scientific Data. 3, 160063 (2016).</li><li>Yoshida, Y., 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=Comparative+genomics+of+the+tardigrades+Hypsibius+dujardini+and+Ramazzottius+varieornatus.">Comparative genomics of the tardigrades Hypsibius dujardini and Ramazzottius varieornatus.</a> PLoS Biology. 15 (7), e2002266 (2017).</li><li>He, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Total+RNA+Extraction+from+C.+elegans.">Total RNA Extraction from C. elegans.</a> Bio-protocol. Bio101, e47 (2011).</li><li>Andrews, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> FastQC a quality-control tool for high-throughput sequence data. , (2015).</li><li>Hashimoto, 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=Extremotolerant+tardigrade+genome+and+improved+radiotolerance+of+human+cultured+cells+by+tardigrade-unique+protein.">Extremotolerant tardigrade genome and improved radiotolerance of human cultured cells by tardigrade-unique protein.</a> Nature Communications. 7, 12808 (2016).</li><li>Zimin, A. V., 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=The+MaSuRCA+genome+assembler.">The MaSuRCA genome assembler.</a> Bioinformatics. 29 (21), 2669-2677 (2013).</li><li>Li, H., Durbin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+accurate+short+read+alignment+with+Burrows-Wheeler+transform.">Fast and accurate short read alignment with Burrows-Wheeler transform.</a> Bioinformatics. 25 (14), 1754-1760 (2009).</li><li>Okonechnikov, K., Conesa, A., Garcia-Alcalde, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Qualimap+2:+advanced+multi-sample+quality+control+for+high-throughput+sequencing+data.">Qualimap 2: advanced multi-sample quality control for high-throughput sequencing data.</a> Bioinformatics. 32 (2), 292-294 (2016).</li><li>Horikawa, D. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+rearing+system+of+the+extremotolerant+tardigrade+Ramazzottius+varieornatus:+a+new+model+animal+for+astrobiology.">Establishment of a rearing system of the extremotolerant tardigrade Ramazzottius varieornatus: a new model animal for astrobiology.</a> Astrobiology. 8 (3), 549-556 (2008).</li></ol>

作者没有什么可透露的。

细菌污染对微生物的基因组测序构成威胁。虽然以前关于缓步动物基因组测序的研究已经通过广泛的信息学方法12、20来筛选出污染, 但我们对单个个体的基因组进行了排序, 以尽量减少污染物的风险。由于单个缓步动物包含大约 50-200 pg 基因组 DNA16和包裹在厚层甲壳素外骨骼, 排除污染物和高质量的 DNA 提取是本议定书的关键点。现有的...

水熊蟲是微小的动物, 可以进入一个 ametabolic 的状态称为 anhydrobiosis 时, 面对干燥。他们由水的吸收1,2恢复。在 ametabolic 状态下, 水熊蟲能够耐受各种极端环境, 其中包括极端温度3和压力4,5, 高剂量的紫外线光6, X 射线和伽玛射线7,8, 宇宙空间9。基因组数据是研究 anhydrobiosis 分子机制的不可缺少的基础。
以前试图序列的水熊蟲基因组已经显示细菌污染的迹象10,11,12,13,14。基因组测序从这些小有机体需要大量的动物和容易细菌污染;因此, 我们以前已经建立了一个排序协议使用一个超低的输入方法从一个单一的缓步动物样本, 以最小化15的风险。利用这些数据, 我们进一步进行了高品质的重测序和重组dujardini16,17的基因组。在这里, 我们详细描述了这个方法的基因组测序从一个单一的缓步动物个体(图 1)。这种测序方法的验证超出了本文的重点, 在我们上一份报告16中已经作了深入的讨论。
该方法由两部分组成: 分离出具有最低污染的单一缓步动物, 以及高质量的象形文字 DNA 水平提取。缓步动物是饥饿和冲洗彻底与水, 以及抗生素, 并观察下显微镜下500X 倍放大, 以确保清除任何细菌污染。以前的估计和测量表明, 单个的缓步动物包含大约 50-200 pg 的基因组 DNA16, 这是通过裂解甲壳素外骨骼的冻融循环或手工均匀化提取。这种基因组 dna 被提交到图书馆建设和排序的 dna 测序仪。另外一项信息学分析显示, 与以前的缓步动物测序项目相比, 高质量的测序以及较低的污染水平。

<table><tbody><tr><td>SZ61 microscope</td><td>OLYMPUS</td><td></td><td></td></tr><tr><td>BactoAgar</td><td>Difco Laboratories</td><td>214010</td><td></td></tr><tr><td>Penicillin Streptomycin (10,000 U/mL)</td><td>Gibco by life technologies</td><td>15140-148</td><td></td></tr><tr><td>VHX-5000 System</td><td>Keyence</td><td></td><td></td></tr><tr><td>0.2mL Silicone coating tube</td><td>Bio Medical Science</td><td>BC-bmb20200</td><td></td></tr><tr><td>Quick-DNA Microprep Kit</td><td>ZYMO Research</td><td>D3021</td><td>Use of this kit is absolutey critical; see step 3.1</td></tr><tr><td>1.5 mL microtube</td><td>greiner bio-one</td><td>616-201</td><td>See 4.1.1</td></tr><tr><td>HIgh speed refrigerated micro centrifuge</td><td>TOMY</td><td>MX-307</td><td></td></tr><tr><td>Covaris M220</td><td>Covaris Inc.</td><td>4482277</td><td></td></tr><tr><td>ThruPLEX DNA-Seq kit</td><td>Rubicon Genomics</td><td>CAT. NO. R400406</td><td>Use of this kit is absolutey critical; see step 4.2</td></tr><tr><td>Thermal Cycler</td><td>Bioer Technology</td><td>TC-96GHbC</td><td></td></tr><tr><td>AMPure XP reagent</td><td>BECKMAN COULTER Life Science</td><td>A63881</td><td></td></tr><tr><td>Ethanol</td><td>Wako</td><td>054-027335</td><td></td></tr><tr><td>EB buffer</td><td>QIAGEN</td><td>19086</td><td></td></tr><tr><td>2200 TapeStation</td><td>Agilent</td><td>G2965AA&amp;nbsp;</td><td></td></tr><tr><td>D1000 Reagents</td><td>Agilent</td><td>5067-5583</td><td></td></tr><tr><td>D1000 ScreenTape</td><td>Agilent</td><td>5067-5582</td><td></td></tr><tr><td>Qubit dsDNA BR Buffer/Reagent</td><td>ThermoFisher Scientific</td><td>Q32850</td><td></td></tr><tr><td>Cubee Mini-Centrifuge</td><td>RecenttecGenereach</td><td>R5-AQBD01aqbd</td><td></td></tr><tr><td>MiSeq 600 cycle v3</td><td>Illumina Inc.</td><td>MS-102-3003</td><td></td></tr><tr><td>MiSeq Sequencer</td><td>Illumina Inc.</td><td>SY-410-1003</td><td></td></tr></tbody></table>

ultralow input genome sequencing library preparation from a single tardigrade specimen

水熊蟲是微小的动物, 进入一个 ametabolic 的状态称为 anhydrobiosis 时, 面对干燥, 可以返回到他们的原始状态, 当水供应。像水熊蟲这样的微小动物的基因组测序有可能导致细菌污染, 例如, 关于这些动物水平基因转移的程度。在这里, 我们提供了一个超低的输入方法序列的基因组的缓步动物, Hypsibius dujardini, 从一个单一的标本。通过使用严格的洗涤和污染物排除以及高效提取单个个体的 50 ~ 200 pg 基因组 DNA, 我们构建了一个用 DNA 测序仪测序的库。这些图书馆是高度重现性和无偏见的, 并对序列读取与其他H. dujardini基因组的信息学分析显示了少量的污染。此方法可应用于无法用以前的方法排序的 unculturable 水熊蟲。

污染物排除:
该协议包括彻底清洗缓步动物和抗生素治疗灭菌, 以减少污染。它还包括一个视觉检查过程, 以确保这些过程的完整性。在验证过程 (协议步骤 2.4) 中所做的显微镜图像如图 2所示。当观察到500X 放大, 细菌细胞可以被视为小颗粒移动周围的缓步动物个人。
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Watch this Scientific Journal Video about Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen at JoVE.com

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

单缓步动物标本的超低输入基因组测序库制备

微生物基因组测序过程中的污染仍然是一个大问题。在这里, 我们展示了一种方法来序列的缓步动物的基因组从一个单一的样本, 只有 50 pg 的基因组 DNA 没有整个基因组的放大, 以减少污染的风险。

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state ...

ultralow-input-genome-sequencing-library-preparation-from-single

Universidad San Sebastian

Research

JoVE Journal

Genetics

8.7K 次观看.  Keio University. 微生物基因组测序过程中的污染仍然是一个大问题。在这里, 我们展示了一种方法来序列的缓步动物的基因组从一个单一的样本, 只有 50 pg 的基因组 DNA 没有整个基因组的放大, 以减少污染的风险。

视频: Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

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 ...

科研

环境科学

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 ...

遗传学

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a protocol for performing the chromatin conformation capture technique in situ Hi-C on staged Drosophila melanogaster embryo populations. The result is a sequencing library that allows the mapping of all chromatin interactions that occur in the nucleus in a single experiment. Embryo sorting is done manually using a fluorescent stereo microscope and a transgenic fly line containing a nuclear marker. Using this technique, embryo populations from each nuclear division cycle, and with defined cell cycle status, can be obtained with very high purity. The protocol may also be adapted to sort older embryos beyond gastrulation. Sorted embryos are used as inputs for in situ Hi-C. All experiments, including sequencing library preparation, can be completed in five days. The protocol has low input requirements and works reliably using 20 blastoderm stage embryos as input material. The end result is a sequencing library for next generation sequencing. After sequencing, the data can be processed into genome-wide chromatin interaction maps that can be analyzed using a wide range of available tools to gain information about topologically associating domain (TAD) structure, chromatin loops, and chromatin compartments during Drosophila development.

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

This work describes a protocol for the generation of high resolution in situ Hi-C libraries from tightly staged pre-gastrulation Drosophila melanogaster embryos.

大范围早期果蝇胚胎的全基因组染色质构象捕获库的生成

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a ...

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription reflect changes in protein synthesis, but many exceptions have been observed. Recently, a technique called ribosome profiling (or Ribo-Seq) has emerged as a powerful method that allows identification, with high accuracy, which regions of mRNA are translated into proteins and quantification of translation at the genome-wide level. Here, we present a generalized protocol for genome-wide quantification of translation using Ribo-Seq in budding yeast. In addition, combining Ribo-Seq data with mRNA abundance measurements allows us to simultaneously quantify translation efficiency of thousands of mRNA transcripts in the same sample and compare changes in these parameters in response to experimental manipulations or in different physiological states. We describe a detailed protocol for generation of ribosome footprints using nuclease digestion, isolation of intact ribosome-footprint complexes via sucrose gradient fractionation, and preparation of DNA libraries for deep sequencing along with appropriate quality controls necessary to ensure accurate analysis of in vivo translation.

Translational regulation plays an important role in the control of protein abundance. Here, we describe a high-throughput method for quantitative analysis of translation in the budding yeast Saccharomyces cerevisiae.

核糖体剖面对芽殖酵母中翻译的全基因组定量

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

生物化学

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

一种在裂变酵母中的深度固序辅助自发抑制屏

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

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 ...

研究稀缺细胞中启动子相互作用组的综合方法

An Integrated Workflow to Study the Promoter-Centric Spatio-Temporal Genome Architecture in Scarce Cell Populations

Spatiotemporal gene transcription is tightly regulated by distal regulatory elements, such as enhancers and silencers, which rely on physical proximity with their target gene promoters to control transcription. Although these regulatory elements are easy to identify, their target genes are difficult to predict, since most of them are cell-type specific and may be separated by hundreds of kilobases in the linear genome sequence, skipping over other non-target genes. For several years, Promoter Capture Hi-C (PCHi-C) has been the gold standard for the association of distal regulatory elements to their target genes. However, PCHi-C relies on the availability of millions of cells, prohibiting the study of rare cell populations such as those commonly obtained from primary tissues. To overcome this limitation, low input Capture Hi-C (liCHi-C), a cost-effective and customizable method to identify the repertoire of distal regulatory elements controlling each gene of the genome, has been developed. liCHi-C relies on a similar experimental and computational framework as PCHi-C, but by employing minimal tube changes, modifying&#160;the reagent concentration and volumes, and swapping or eliminating steps, it accounts&#160;for minimal material loss during library construction. Collectively, liCHi-C enables the study of gene regulation and spatiotemporal genome organization in the context of developmental biology and cellular function.

作者聚焦:研究稀缺细胞群中以启动子为中心的时空基因组结构的集成工作流程  

Gene expression is regulated by interactions of gene promoters with distal regulatory elements. Here, we descirbe how low input Capture Hi-C (liCHi-C) allows the identification of these interactions in rare cell types, which were previously unmeasurable.

研究稀缺细胞群中以启动子为中心的时空基因组结构的集成工作流程

Spatiotemporal gene transcription is tightly regulated by distal regulatory elements, such as enhancers and silencers, which rely on physical proximity ...

Ultra-long Read Sequencing for Whole Genomic DNA Analysis

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes and analysis of complex structural variants. Nanopore platforms perform single-molecule sequencing by directly measuring the current changes mediated by DNA passage through the pores and can generate hundreds of kilobase (kb) reads with minimal capital cost. This platform has been adopted by many researchers for a variety of applications. Achieving longer sequencing read lengths is the most critical factor to leverage the value of nanopore sequencing platforms. To generate ultra-long reads, special consideration is required to avoid DNA breakages and gain efficiency to generate productive sequencing templates. Here, we provide the detailed protocol of ultra-long DNA sequencing including high molecular weight (HMW) DNA extraction from fresh or frozen cells, library construction by mechanical shearing or transposase fragmentation, and sequencing on a nanopore device. From 20-25 &#181;g of HMW DNA, the method can achieve N50 read length of 50-70 kb with mechanical shearing and N50 of 90-100 kb read length with transposase mediated fragmentation. The protocol can be applied to DNA extracted from mammalian cells to perform whole genome sequencing for the detection of structural variants and genome assembly. Additional improvements on the DNA extraction and enzymatic reactions will further increase the read length and expand its utility.

Long-read sequences greatly facilitate the assembly of complex genomes and characterization of structural variation. We describe a method to generate ultra-long sequences by nanopore-based sequencing platforms. The approach adopts an optimized DNA extraction followed by modified library preparations to generate hundreds of kilobase reads with moderate coverage from human cells.

用于全基因组 DNA 分析的超长读数测序

Third generation single-molecule DNA sequencing technologies offer significantly longer read length that can facilitate the assembly of complex genomes ...

单缓步动物标本的超低输入基因组测序库制备

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一种在裂变酵母中的深度固序辅助自发抑制屏

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研究稀缺细胞群中以启动子为中心的时空基因组结构的集成工作流程

研究稀缺细胞群中以启动子为中心的时空基因组结构的集成工作流程

用于全基因组 DNA 分析的超长读数测序

单缓步动物标本的超低输入基因组测序库制备

摘要

探索更多视频

此视频中的章节

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

植物标本标本的鲁棒 DNA 分离与高通量测序库建设

大范围早期果蝇胚胎的全基因组染色质构象捕获库的生成

核糖体剖面对芽殖酵母中翻译的全基因组定量

一种在裂变酵母中的深度固序辅助自发抑制屏

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

研究稀缺细胞群中以启动子为中心的时空基因组结构的集成工作流程

研究稀缺细胞群中以启动子为中心的时空基因组结构的集成工作流程

研究稀缺细胞群中以启动子为中心的时空基因组结构的集成工作流程

用于全基因组 DNA 分析的超长读数测序

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