Name: robust dna isolation and high-throughput sequencing library construction for herbarium specimens
Uploaded: 2018-03-08T14:30:00.000+00:00
Duration: 13 min 3 s
Description: Watch this Scientific Journal Video about Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens at JoVE.com

我们感谢泰勒 AuBuchon-长老, 约旦 Teisher 和克里斯汀娜 Zudock 协助取样标本, 和密苏里植物园, 以获得植物标本标本的破坏性取样。这项工作得到了国家科学基金会 (DEB-1457748) 的资助。

1. 开始前
<ol><li>通过添加20克 CTAB, 使新的烷基铵溴化 (CTAB) 缓冲区17 , 10 克 polyvinylpyrrolidone (PVP) 40, 100.0 毫升1米三 ph 8.0, 40 毫升四乙酸乙二胺酸 (EDTA) ph 0.5, 8.0 毫升的280.0 米氯化钠, 和5毫升的试剂水一起, 并把总容积1升使用试剂级水。将 pH 值调整为8.0。 注: 附加试剂可添加到 CTAB, 取决于个别分类群中的次生化合物。见艾伦等。18用于添加试剂的详细列表。</li>
	<li>
添加10µL 的β巯基乙醇每5毫升的 CTAB 缓冲区。 注: 这可以分批50毫升, 并储存在室温下3–4周。<ol>
<li>在65摄氏度的水浴中加热 CTAB 溶液。</li>
	</ol>
</li>
	<li>冷却迫击炮和杵在-20 °c 至少20分钟。</li>
	<li>标签4套 2 x n 2 毫升管 (其中 n = 样本数)。把1套标记的管子放在冰上。</li>
	<li>在冰上或在-20 摄氏度冰箱中冷却异丙醇。</li>
	<li>从冰箱中移除固相可逆固定化 (SPRI) 珠 (参见材料表), 并允许它们平衡室温 (至少30分钟)。</li>
	<li>准备80% 乙醇。</li>
	<li>选择标本标本提取和检索〜1厘米2, 或10毫克, 每标本组织, 最好叶材料。</li>
</ol>2. DNA 提取
<ol><li>用 prechilled 迫击炮和杵研磨 ~ 1 厘米2预选的标本室组织。加入液氮和30–50镁消毒砂。研磨直到组织变成细粉。 注:10 毫克或更多的组织是可取的, 但较少也在某些情况下工作。一旦液氮蒸发, 添加更多的需要, 直到组织完全地面。另一个常见的破坏细胞和组织的方法是使用珠搅拌器。然而, 这种方法被发现对这些实验所用的标本没有很好的效果。</li>
	<li>将冷冻粉末转移到两个2毫升管 (增加不超过一半的管的体积)。将600µL 的暖 CTAB 溶液加入每管, 并通过反转和涡流彻底混合管。 注: 由于标本室材料的数量和质量往往很低, 两次重复执行 DNA 分离有助于获得较高的产量。</li>
	<li>在65摄氏度水浴中孵化样品, 1–1.5 h, 涡流每15分钟。</li>
	<li>离心机样品在 1万 x g 5 分钟. 将上清液转移到一组新的标记管 (~ 500 µL)。使用标准的非氯化处理程序丢弃颗粒。</li>
	<li>添加4µL 的 RNase (10 毫克/毫升) 每管和混合通过反转或吹打。在热块或水浴中孵育37摄氏度的样品15分钟。</li>
	<li>当管子达到室温后, 添加一个相等体积 (500 µL) 的 25:24:1 phenol:chloroform:isoamyl 酒精混合物。通过吹打和/或反演彻底混合。离心管在 1.2万 x g 15 分钟. 将水层 (上层) 转移到一组新的标记管 (~ 400 µL)。将有机层丢弃到氯化废料容器中。 注: 如果植物材料中预计有大量的次生化合物, 步骤2.6 可能会重复。</li>
	<li>添加等量 (400 µL) 的24:1 氯仿: 异戊醇混合物。通过吹打和/或反演彻底混合。离心管在 1.2万 x g 15 分钟. 将水层 (上层) 转移到一组新的 prechilled, 标记管 (~ 300 µL)。将有机层丢弃到氯化废料容器中。</li>
	<li>将 prechilled 异丙醇的等量容积 (~ 300 µL) 和2.5 米醋酸钠的12µL 添加到每个管中。孵育样品在-20 °c 为 30–60 min。 注: 潜伏期可以延长 (直至夜间孵化), 但 DNA 质量会降低样品孵化的时间。</li>
	<li>取出冰箱里的样品, 将管子离心在 1.2万 x g 的15分钟内, 轻轻地取出并丢弃上清液, 不干扰颗粒。用新鲜的70% 乙醇 (大约300–500µL), 用悬浮液清洗颗粒。对于每一个加倍的样品, 把两个单独的小球合并成一个与伴随的乙醇。 注: 样品应先用乙醇合并成一管, 然后再进行。不需要单独用乙醇清洗每个样品。</li>
	<li>离心管在 1.2万 x g 10 分钟去除和丢弃上清, 不干扰颗粒。空气干燥颗粒。 注: 使用干热块 (不超过65摄氏度) 可快速干燥样品。确保样品不会过度干燥, 因为这样可以降低 DNA 的最终产量。</li>
	<li>将分离的 DNA 悬浮在50µL 的 1x TE 中。贮存在-20 °c 冷藏柜中, 用于长期贮存或4°c, 供下星期使用。</li>
</ol>3. 质量控制 (QC)
<ol><li>
用琼脂糖凝胶进行质量检查。
	<ol>
<li>通过添加54克三基、27.5 克硼酸和3.75 克 EDTA 二钠盐, 制备1x 三/硼酸/edta (no) 缓冲液, 使总容积达到5升, 使用试剂级水。</li>
		<li>通过添加1克琼脂糖到100毫升 1x, 制备1% 琼脂糖凝胶。微波溶液直到没有琼脂糖可见。添加0.01% 核酸凝胶染色 (请参阅材料表)。让瓶子冷却, 直到它是温暖的触摸。搅拌均匀。将琼脂糖倒入凝胶托盘中, 让它凝固。</li>
		<li>混合3µL 的样品, 2 µL 的试剂级水, 1 µL 6x 的负载染料。将样品装入凝胶基质中, 注意它们的顺序。</li>
		<li>在 60–70 v 的60–70中运行样品, 在紫外光下对凝胶进行正确的曝光和聚焦。 注意: 存在一个清晰的高分子量带是高质量 dna 的标志, 而涂片通常表明 dna 降解。大多数标本标本都被降解。</li>
	</ol>
</li>
	<li>
运行 dsDNA 量化分析 (见材料表), 以确定双链 DNA 的数量。
	<ol>
<li>使用2µL 的样品进行分析。 注: 对标本室材料的定量分析不需要稀释, 因为它们的数量往往很少。成功的图书馆已经从这一步的 dsDNA 总的1.26。</li>
	</ol>
</li>
</ol>4. DNA 剪切
注意: 这是商业双绞 fragmentase 协议的优化版本 (请参见材料表)。
<ol><li>将 dsDNA 碎片酶放在涡流后的冰上3秒。</li>
	<li>在无菌0.2 毫升聚合酶链反应 (PCR) 管, 混合1–16µL 分离的 DNA 与2µL 伴随的碎片反应缓冲。通过添加核酸酶自由水, 将总体积提高到18µL。添加2µL dsDNA 破碎酶和涡旋的混合物为 3 s。 注: 所需 dna 数量因 dna 浓度而异 (瞄准管中的 200)。</li>
	<li>将样品孵化37摄氏度, 8.5 分钟。然后添加5µL 0.5 米 EDTA 的管。 注意: 此步骤需要在潜伏期结束后立即执行, 以终止反应并防止 DNA 样品过度剪切。</li>
</ol>5. 珠清理
<ol><li>融汇 SPRI 珠涡流。</li>
	<li>通过添加25µL 核酸酶游离水, 将被剪切 DNA 的总体积提高到50µL。添加45µL 室温 SPRI 珠 (90% 卷) 到50µL 的剪切 DNA, 并彻底混合吹打上下。 注: 在总试样体积的90% 处添加珠子是为了去除最小的 DNA 片段, 通常低于200基对。</li>
	<li>让样品孵化5分钟. 把管子放在磁板上, 让它们坐5分钟. 小心地取出并丢弃上清。 注意: 小心不要打扰珠子, 因为它们含有所需的 DNA 目标。</li>
	<li>在磁性支架上加入200µL 新鲜80% 乙醇的管子。在室温下孵化三十年代, 然后仔细去除并丢弃上清。重复此步骤一次。空气干燥的珠子5分钟, 而管是在磁性立场与它的盖子打开。 注意: 避免过度干燥的珠子。这可能导致 DNA 的回收率降低。</li>
	<li>把管子从磁铁上取下。洗脱的 DNA 从珠子变成55µL 的 0.1x TE, 并彻底混合吹打上下。在室温下孵育5分钟, 将管子放在磁性支架上, 等待溶液转晴 (~ 2 分钟)。</li>
	<li>把上清的52µL。对样品进行 DNA 定量分析, 以检查回收和进入图书馆准备的初始浓度。 注: 图书馆的总 dsDNA 估计不到1.25 吴, 但在每种情况下 reamplification 是必需的。</li>
</ol>6. 图书馆准备工作
注意: 这是一个商业上可用的库套件的修改版本 (请参见材料目录协议)。
<ol><li>
结束准备
	<ol>
<li>添加3µL 的限制性和磷酸盐尾矿酶, 和7µL 的伴随反应缓冲50µL 清洗, 剪切 DNA。通过上下吹打彻底混合。旋转管子以除去气泡。 注: 总容积应为60µL。</li>
		<li>将样品放在 thermocycler 中, 以下列程序:30 分钟在20°c, 30 分钟在65°c, 然后举行在4°c。 注: 加热盖设置为≥75°c</li>
	</ol>
</li>
	<li>
适配器结扎
	<ol>
<li>稀释适配器25–50折叠 (工作适配器集中的0.6–0.3 µM)。添加30µL 结扎主混合, 1 µL 结扎增强器, 2.5 µL 适配器的高通量短读测序的管。 注: 总容积应为93.5 µL。</li>
		<li>通过上下吹打彻底混合。旋转管子以除去气泡。将试管孵化20摄氏度, 15 分钟。</li>
		<li>添加3µL 的商业混合物的尿 dna glycosylase (UDG) 和 DNA glycosylase 裂解酶限制性 VIII (见材料表) 的管。确保总容积为96.5 µL. 混合彻底和孵化37摄氏度15分钟使用 thermocycler。 注: 盖子应设置为≥47°c。原始版本的商业协议有大小选择后, 适配器结扎步骤, 其次是珠清理作为最后一步。该协议实现了更高的收益率, 切换这些步骤的顺序, 并实现大小选择作为最后一步。</li>
	</ol>
</li>
	<li>
清除酶和小碎片
	<ol>
<li>融汇磁珠由涡流。</li>
		<li>添加78µL SPRI 磁性珠 (80% 卷), 并彻底混合吹打上下。 注: 在总试样体积的80% 处添加珠子, 以去除最小的 DNA 片段, 这些碎片通常少于250基对。更严格地去除小的 DNA 片断是 (i) 去除多余的适配器和 (ii) 在以下步骤强调放大更大的片断。</li>
		<li>让样品孵化5分钟. 把管子放在磁板上5分钟. 小心地移除并丢弃含有所需尺寸范围外的 DNA 的上清。 注意: 注意不要干扰含有所需 DNA 靶的珠子。</li>
		<li>在磁性支架上加入200µL 新鲜80% 乙醇的管子。在室温下孵化三十年代, 然后仔细去除并丢弃上清。重复此步骤一次。空气干燥的珠子5分钟, 而管是在磁性立场与盖子打开。 注意: 避免过度干燥的珠子。这可能导致 DNA 的回收率降低。</li>
		<li>把管子从磁铁上取下。洗脱通过添加17µL 0.1x TE, 并通过上下吹打彻底混合, 将 DNA 目标从珠子中提取出来。</li>
		<li>在室温下孵育5分钟, 将管子放在磁性支架上, 等待溶液转晴 (~ 2 分钟)。</li>
		<li>把上清的15µL。</li>
	</ol>
</li>
	<li>
PCR 扩增
	<ol>
<li>添加25µL 高保真 PCR 主混合, 5 µL 高吞吐量短读测序库准备 5 ' 底漆, 5 µL 的高通量短读测序库准备 3 ' 底漆, 到15µL 清洗适配器-结扎 DNA。 注: 总容积应为50µL。</li>
		<li>和涡流混合。使用在表 1中找到的设置将示例放入 thermocycler 中: thermocycler 放大设置。 注: 由于输入 DNA 数量少, 需要大量的循环。</li>
	</ol>
</li>
</ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>循环步骤</td>
			<td>临时.</td>
			<td>时间</td>
			<td>周期</td>
		</tr>
<tr>
<td>初始变性</td>
			<td>98°c</td>
			<td>三十年代</td>
			<td>1</td>
		</tr>
<tr>
<td>变性</td>
			<td>98°c</td>
			<td>十年代</td>
			<td>12</td>
		</tr>
<tr>
<td>退火/延伸</td>
			<td>65°c</td>
			<td>七十五年代</td>
			<td>12</td>
		</tr>
<tr>
<td>最终扩展名</td>
			<td>65°c</td>
			<td>5分钟</td>
			<td>1</td>
		</tr>
<tr>
<td>举行</td>
			<td>4°c</td>
			<td></td>
			<td></td>
		</tr>
</tbody></table>表 1: PCR 协议变性、退火和延长时间和温度.对本协议中的试剂进行了温度和时间的优化。如果试剂被改变, 温度和时间应该再优化。
<ol start="5"><li>
所需库大小的大小选择 注: 此珠子步骤将删除目标范围上方和下方的碎片。<ol>
<li>融汇 SPRI 珠涡流。</li>
		<li>添加25µL (50% 体积) 室温磁性珠, 并彻底混合吹打上下。让样品孵化5分钟. 把管子放在磁板上, 让它们坐5分钟. 小心地移除和转移上清到一组新的标记管。 注意: 此卷可以根据所需的库大小进行调整。上清含有所需大小的 DNA 片段。在第一个珠孵化, 珠子是绑定较大的库片段。它们被移除, 以集中于400–600基对范围中的那些。上清中含有较小的碎片。</li>
		<li>添加6µL 室温 SPRI 珠到上清, 并彻底混合吹打上下。让样品孵化5分钟. 把管子放在磁板上, 让它们坐5分钟。 注意: 该卷可以根据所需的库大小进行调整, 按照步骤6.5.2。</li>
		<li>小心取出并丢弃上清。 注意: 注意不要干扰含有所需 DNA 的珠子。在第二个珠孵化, 珠子是绑定到留下的碎片后, 最初删除的最大的 DNA 片段。这组片段通常在所需的大小范围内。</li>
		<li>在磁性支架上加入200µL 新鲜80% 乙醇的管子。在室温下孵化三十年代, 然后仔细去除并丢弃上清。重复.空气干燥的珠子5分钟, 而管是在磁性立场与盖子打开。 注意: 避免过度干燥的珠子。这可能导致 DNA 的回收率降低。</li>
		<li>把管子从磁铁上取下。洗脱的 DNA 目标从珠子成33µL 0.1x TE, 并彻底混合吹打上下。</li>
		<li>在室温下孵育5分钟, 将管子放在磁性支架上, 等待溶液转晴 (~ 2 分钟)。拉出30µL 的上清, 转移到2毫升管 (参见材料表) 进行存储。 注意: 图书馆可以保存在-20 ˚C, 用于长期存储。</li>
	</ol>
</li>
	<li>
质量控制
	<ol>
<li>在 DNA 库上运行质量控制测试。请参阅步骤3.1 和3.2。 注: 对于 DNA 库, 在 96 v 上运行凝胶45分钟。</li>
	</ol>
</li>
	<li>
库 reamplification: 如果库数量不足, 则可选。 注意: 使用以下步骤可以 reamplified 10 nM 以下的样本库浓度。低浓度库的 Reamplification 可以实现排序的可行结果, 但 Reamplification 可能导致基本组成多样性的适度转移, 尽管收集的数据 (表 3) 表明, 对于某些度量标准来说, 这是微不足道的。.<ol>
<li>用 0.1x reamplification 稀释普通的引物10倍。</li>
		<li>添加25µL 高保真 PCR 主混合, 5 µL 稀释通用 reamplification 底漆 1 (AATGATACGGCGACCACCGA), 5 µL 稀释通用 reamplification 底漆 2 (CAAGCAGAAGACGGCATACGA) 到15µL 低浓度的图书馆。注: 总容积应为50µL。</li>
		<li>和涡流混合。使用在表 1中找到的设置将示例放入 thermocycler 中: thermocycler 放大设置 注: 由于输入 DNA 数量少, 需要大量的循环。</li>
		<li>珠清理</li>
		<li>融汇 SPRI 珠涡流。添加45µL 室温 SPRI 珠 (90% 卷), 并彻底混合吹打上下。</li>
		<li>让样品孵化5分钟. 把管子放在磁板上5分钟。</li>
		<li>小心地除去并丢弃含有不需要的 DNA 的上清。 注意: 注意不要干扰含有所需 DNA 靶的珠子。</li>
		<li>在磁性支架上加入200µL 新鲜80% 乙醇的管子。在室温下孵化三十年代, 然后仔细去除并丢弃上清。重复此步骤一次。</li>
		<li>空气干燥的珠子5分钟, 而管是在磁性立场与它的盖子打开。 注意: 避免过度干燥的珠子。这可能导致 DNA 目标的恢复降低。</li>
		<li>把管子从磁铁上取下。洗脱的 DNA 目标从珠子成33µL 0.1x TE, 并彻底混合吹打上下。</li>
		<li>在室温下孵育5分钟, 将管子放在磁性支架上, 等待溶液转晴 (~ 2 分钟)。</li>
		<li>把上清的30µL。这些库可以存储在-20 摄氏度, 用于长期存储。</li>
	</ol>
</li>
	<li>
质量控制
	<ol>
<li>在 DNA 库上运行质量控制测试。请参阅步骤3.1 和3.2。对于 DNA 库, 在 96 v 上运行凝胶45分钟。 注: 如果在凝胶中看到双带, 这可能是从 reamplification 步骤的底漆耗尽的后果。可以通过重复6.9 移除带区, 但在表 1中描述的 PCR 程序中只使用一个周期。</li>
	</ol>
</li>
</ol>

<ol>
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作者声明他们没有竞争的兴趣。

本协议是一种综合可靠的方法, 用于 DNA 分离和测序库的干燥标本的制备。该方法的一致性和最小的需要改变它的基础上标本质量, 使它可伸缩的大型植物标本的排序项目。为低收益库包含一个可选的 reamplification 步骤, 允许包含低质量、低数量、稀有或历史上重要的样本, 否则将不适合排序。
初始 DNA 产量的重要性 标本室衍生的 dna 通常被降级为最初的标本保存<...

植物标本集是一个潜在的有价值的物种和基因组多样性的来源的研究, 包括系统学 1, 2, 3, 人口遗传学 4, 5, 保护生物6, 入侵物种生物7和特征演变8。获得丰富多样的物种、种群、地理位置和时间点的能力突出了 "宝箱"9 , 即标本馆。从历史上看, 植物标本所衍生的 DNA 退化的性质阻碍了 PCR 的项目, 往往贬低研究人员只使用高拷贝中发现的标记, 如叶绿体基因组的区域或核糖体的内部转录间隔 (其)rna.标本和 DNA 的质量因保存9、10的方法而有很大的差异, 在干燥过程中使用的热的双绞碎和破碎是最常见的损害形式, 造成所谓的 90% DNA 锁定, 已使基于 PCR 的研究11。除了碎片, 植物标本组学的第二个最普遍的问题是污染, 例如从内生真菌中提取的13或真菌在收集后死后被采集, 但在标本室中安装12之前, 虽然这个问题可以解决 bioinformatically 给出正确的真菌数据库 (见下文)。第三个和更不常见的问题是通过胞嘧啶脱氨作用 (C/G→T/A)14 进行序列修改, 虽然估计在标本标本11 中的低 (~ 0.03%)。随着高通量测序 (高温超导) 的出现, 碎片问题可以通过短读取和排序深度12,15来克服, 从而允许从许多质量较低的样本中获取基因组级数据。DNA, 甚至有时允许整个基因组排序15。
标本室标本越来越多地被使用, 是系统发育项目的一个更大的组成部分16。目前的挑战是使用植物标本标本为高温超导始终获得足够的双链 DNA, 一个必要的先决条件, 排序协议, 从众多物种及时, 不需要优化的方法, 个别标本.本文介绍了一种利用现有方法进行 DNA 提取和库标本制备的协议, 并对其进行修改, 以实现快速、可复制的结果。该方法允许从标本到24个样本库的完整处理, 在13小时, 具有8小时的动手时间, 或 16 h, 与 9 h 的实际操作时间, 当需要可选的 reamplification 步骤。同时处理更多的样品是可以实现的, 虽然限制因素是离心能力和技术技能。该协议的目的是只需要典型的实验室设备 (thermocycler, 离心机和磁性支架), 而不是专门的设备, 如喷雾器或 sonicator, 为剪切 DNA。
在高通量测序实验中, DNA 质量、片段大小和数量是限制标本标本使用的因素。其他隔离标本室 dna 和创建高通量测序库的方法表明, 使用10的 DNA16的效用不大;然而, 它们需要实验性地确定图书馆准备所需的最佳 PCR 周期数。当处理极少量可行的双链 DNA (dsDNA) 时, 这种方法变得不切实际, 因为有些标本标本只为单个库的制备提供了足够的 dna。这里提出的方法使用单一数量的周期, 不管样本质量如何, 所以在库优化步骤中没有丢失 DNA。相反, 当库不满足排序所需的最小金额时, 将调用 reamplification 步骤。许多植物标本是罕见的, 并拥有很少的材料, 使得难以证明破坏性抽样在许多情况下。为了对付这一问题, 所提出的协议允许 dsDNA 的输入大小小于1.25 到库的准备过程, 扩大了可行样本的范围, 以高通量测序, 并尽量减少对样本的破坏性抽样的需要。
下面的协议已经为牧草进行了优化, 并在标本室标本上对数以百计的不同物种进行了测试, 尽管我们预计该协议可以应用于许多其他植物群。它包括一个可选的恢复步骤, 可用于保存低质量和/或稀有标本。根据200余种标本标本, 本协议适用于组织投入和质量低的标本, 可通过最小的破坏性抽样保存稀有标本。这里表明, 该协议可以提供高质量的库, 可以为基于 phylogenomics 的项目排序。

<table><tbody><tr><td>Veriti Thermal Cycler</td><td>Applied Biosystems</td><td>4452300</td><td>96 well&amp;nbsp;</td></tr><tr><td>Gel Imaging System</td><td>Azure Biosystems</td><td>c300</td><td></td></tr><tr><td>Microfuge 20 Series</td><td>Beckman Coulter</td><td>B30137</td><td></td></tr><tr><td>Digital Dry Bath</td><td>Benchmark Scientific</td><td>BSH1001</td><td></td></tr><tr><td>Electrophoresis System</td><td>EasyCast</td><td>B2</td><td></td></tr><tr><td>PURELAB flex 2 (Ultra pure water)</td><td>ELGA&amp;nbsp;</td><td>89204-092</td><td></td></tr><tr><td>DNA LoBind Tube&amp;nbsp;</td><td>Eppendorf</td><td>30108078</td><td>2 ml</td></tr><tr><td>Mini centrifuge</td><td>Fisher Scientific</td><td>12-006-901</td><td></td></tr><tr><td>Vortex-Genie 2</td><td>Fisher Scientific</td><td>12-812</td><td></td></tr><tr><td>Mortar</td><td>Fisher Scientific</td><td>S02591</td><td>porcelain</td></tr><tr><td>Pestle</td><td>fisher Scientific</td><td>S02595</td><td>porcelain</td></tr><tr><td>Centrifuge tubes</td><td>fisher Scientific</td><td>21-403-161</td><td></td></tr><tr><td>Microwave</td><td>Kenmore</td><td>405.7309231</td><td></td></tr><tr><td>Qubit Assay Tubes</td><td>Invitrogen</td><td>Q32856</td><td></td></tr><tr><td>0.2 ml Strip tube and Cap for PCR</td><td>VWR</td><td>20170-004</td><td></td></tr><tr><td>Qubit 2.0 Fluorometer</td><td>Invitrogen</td><td>Q32866</td><td></td></tr><tr><td>Balance</td><td>Mettler Toledo</td><td>PM2000</td><td></td></tr><tr><td>Liquid Nitrogen Short-term Storage</td><td>Nalgene</td><td>F9401</td><td></td></tr><tr><td>Magnetic-Ring Stand&amp;nbsp;</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>AM10050</td><td>96 well&amp;nbsp;</td></tr><tr><td>Water Bath</td><td>VWR</td><td>89032-210</td><td></td></tr><tr><td>Hot Plate Stirrers</td><td>VWR</td><td>97042-754</td><td></td></tr><tr><td>Liquid Nitrogen</td><td>Airgas</td><td>UN1977</td><td></td></tr><tr><td>1 X TE Buffer</td><td>Ambion</td><td>AM9849</td><td>pH 8.0</td></tr><tr><td>CTAB</td><td>AMRESCO</td><td>0833-500G</td><td></td></tr><tr><td>2-MERCAPTOETHANOL</td><td>AMRESCO</td><td>0482-200ML</td><td></td></tr><tr><td>Ribonuclease A</td><td>AMRESCO</td><td>E866-5ML</td><td>10 mg/ml solution</td></tr><tr><td>Agencourt AMPure XP</td><td>Beckman Coulter</td><td>A63882</td><td></td></tr><tr><td>Sodium Chloride</td><td>bio WORLD</td><td>705744</td><td></td></tr><tr><td>Isopropyl Alcohol</td><td>bio WORLD</td><td>40970004-1</td><td></td></tr><tr><td>Nuclease Free water</td><td>bio WORLD</td><td>42300012-2</td><td></td></tr><tr><td>Isoamyl Alcohol</td><td>Fisher Scientific</td><td>A393-500</td><td></td></tr><tr><td>Sodium Acetate Trihydrate</td><td>Fisher Scientific</td><td>s608-500</td><td></td></tr><tr><td>LE Agarose</td><td>GeneMate</td><td>E-3120-500</td><td></td></tr><tr><td>100bp PLUS DNA Ladder</td><td>Gold Biotechnology</td><td>D003-500</td><td></td></tr><tr><td>EDTA, Disodium Salt</td><td>IBI Scientific</td><td>IB70182</td><td></td></tr><tr><td>Qubit dsDNA HS Assay Kit</td><td>Life Technologies</td><td>Q32854</td><td></td></tr><tr><td>TRIS</td><td>MP Biomedicals</td><td>103133</td><td>ultra pure</td></tr><tr><td>Gel Loading Dye Purple (6 X)</td><td>New England BioLabs</td><td>B7024S</td><td></td></tr><tr><td>NEBNext dsDNA Fragmentase</td><td>New England BioLabs</td><td>M0348L</td><td></td></tr><tr><td>NEBNext Ultra II DNA Library Prep Kit for Illumina&amp;nbsp;</td><td>New England BioLabs</td><td>E7645L</td><td></td></tr><tr><td>NEBNext Multiplex Oligos for Illumina</td><td>New England BioLabs</td><td>E7600S</td><td>Dual Index Primers Set 1</td></tr><tr><td>NEBNext Q5 Hot Start HiFi PCR Master Mix</td><td>New England BioLabs</td><td>M0543L</td><td></td></tr><tr><td>Mag-Bind RXNPure Plus</td><td>Omega bio-tek</td><td>M1386-02</td><td></td></tr><tr><td>GelRed 10000 X</td><td>Pheonix Research</td><td>41003-1</td><td></td></tr><tr><td>Phenol solution</td><td>SIGMA Life Science</td><td>P4557-400ml</td><td></td></tr><tr><td>PVP40</td><td>SIGMA-Aldrich</td><td>PVP40-50G</td><td></td></tr><tr><td>Chloroform</td><td>VWR</td><td>EM8.22265.2500</td><td></td></tr><tr><td>Ethanol</td><td>Koptec</td><td>V1016</td><td>200 Proof</td></tr><tr><td>Silica sand</td><td>VWR</td><td>14808-60-7</td><td></td></tr><tr><td>Reamplification primers</td><td>Integrated DNA Technologies</td><td>see text</td><td></td></tr><tr><td>Sequencher v.5.0.1</td><td>GeneCodes</td><td></td><td></td></tr></tbody></table>

robust dna isolation and high-throughput sequencing library construction for herbarium specimens

标本室是一种宝贵的植物材料来源, 可用于各种生物研究。植物标本标本的使用与一些挑战, 包括样本保存质量, 退化的 DNA, 和破坏性抽样稀有标本。为了更有效地利用大型测序工程中的植物标本库材料, 需要一种可靠、可伸缩的 DNA 分离和制备方法。本文展示了一种健壮的, 从不需要对单个样本进行修改的标本标本中进行的 DNA 隔离和高通量库构建的鲁棒的起始到端协议。本协议是为低品质干燥的植物材料量身定做的, 利用现有的方法, 优化组织磨削, 修改库尺寸选择, 并为低产量库引入可选的 reamplification 步骤。低产量 DNA 库的 Reamplification 可以拯救来自不可替代和潜在价值的植物标本标本的样本, 否定额外的破坏性取样的需要, 不引入可识别的顺序偏差, 共同系统进化应用。该议定书已在数以百计的草种上进行了测试, 但预计在验证后能适应其他植物血统的使用。这个协议可以受到极退化的 DNA 的限制, 在那里碎片不存在于所需的大小范围内, 而在某些植物材料中存在的次生代谢物会抑制干净的 dna 分离。总的来说, 该协议引入了一种快速而全面的方法, 允许 DNA 隔离和图书馆准备24样本在少于13小时, 只有8小时的主动动手时间与最小的修改。

DNA 分离与最终图书馆产量 在这项研究中, 使用五十种不同的样本, 从1920年和2012年最年轻的人 (表 2) 中, 证明了隔离标本室 DNA 和恢复高质量测序库的协议的有效性。对于每个样品, 大约10毫克的叶组织被用于 DNA 分离。绿色的叶子组织是有利的, 如果有, 并且没有被选择的有明显的真菌污染的组织。成功的隔离可以使用黄色或褐色的组织, 虽然产量...

Watch this Scientific Journal Video about Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens at JoVE.com

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

植物标本标本的鲁棒 DNA 分离与高通量测序库建设

本文阐述了从植物标本室材料中提取 dna 隔离和高通量测序库的详细协议, 包括抢救异常质量较差的 dna。

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 ...

robust-dna-isolation-high-throughput-sequencing-library-construction

Universidad San Sebastian

Research

JoVE Journal

Genetics

10.5K 次观看.  Donald Danforth Plant Science Center. 本文阐述了从植物标本室材料中提取 dna 隔离和高通量测序库的详细协议, 包括抢救异常质量较差的 dna。

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

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

Purification of High Molecular Weight Genomic DNA from Powdery Mildew for Long-Read Sequencing

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics of these pathogens, in part due to a lack of well-developed genetic and genomic resources. These organisms have large, repetitive genomes, which have made genome sequencing and assembly prohibitively difficult. Here, we describe methods for the collection, extraction, purification and quality control assessment of high molecular weight genomic DNA from one powdery mildew species, Golovinomyces cichoracearum. The protocol described includes mechanical disruption of spores followed by an optimized phenol/chloroform genomic DNA extraction. A typical yield was 7 &#181;g DNA per 150 mg conidia. The genomic DNA that is isolated using this procedure is suitable for long-read sequencing (i.e., &#62; 48.5 kbp). Quality control measures to ensure the size, yield, and purity of the genomic DNA are also described in this method. Sequencing of the genomic DNA of the quality described here will allow for the assembly and comparison of multiple powdery mildew genomes, which in turn will lead to a better understanding and improved control of this agricultural pathogen.

Described here is a method for the extraction, purification, and quality control of genomic DNA from the obligate biotrophic fungal pathogen, powdery mildew, for use in long-read genome sequencing.

超高分子量基因组DNA的分离纯化白粉病对长读测序

The powdery mildew fungi are a group of economically important fungal plant pathogens. Relatively little is known about the molecular biology and genetics ...

遗传学

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 ...

Empirical, Metagenomic, and Computational Techniques Illuminate the Mechanisms by which Fungicides Compromise Bee Health

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered &#34;bee-safe,&#34; there is mounting evidence that fungicide residues in pollen are associated with bee declines (for both honey and bumble bee species). While the mechanisms remain relatively unknown, researchers have speculated that bee-microbe symbioses are involved. Microbes play a pivotal role in the preservation and/or processing of pollen, which serves as nutrition for larval bees. By altering the microbial community, it is likely that fungicides disrupt these microbe-mediated services, and thereby compromise bee health. This manuscript describes the protocols used to investigate the indirect mechanism(s) by which fungicides may be causing colony decline. Cage experiments exposing bees to fungicide-treated flowers have already provided the first evidence that fungicides cause profound colony losses in a native bumble bee (Bombus impatiens). Using field-relevant doses of fungicides, a series of experiments have been developed to provide a finer description of microbial community dynamics of fungicide-exposed pollen. Shifts in the structural composition of fungal and bacterial assemblages within the pollen microbiome are investigated by next-generation sequencing and metagenomic analysis. Experiments developed herein have been designed to provide a mechanistic understanding of how fungicides affect the microbiome of pollen-provisions. Ultimately, these findings should shed light on the indirect pathway through which fungicides may be causing colony declines.

Microbial consortia within bumble bee hives enrich and preserve pollen for bee larvae. Using next generation sequencing, along with laboratory and field-based experiments, this manuscript describes protocols used to test the hypothesis that fungicide residues alter the pollen microbiome, and colony demographics, ultimately leading to colony loss.

经验、基因和计算技术阐明了杀菌剂危害蜜蜂健康的机制

Growers often use fungicide sprays during bloom to protect crops against disease, which exposes bees to fungicide residues. Although considered ...

Ultralow Input Genome Sequencing Library Preparation from a Single Tardigrade Specimen

Tardigrades are microscopic animals that enter an ametabolic state called anhydrobiosis when facing desiccation and can return to their original state when water is supplied. The genomic sequencing of microscopic animals such as tardigrades risks bacterial contamination that sometimes leads to erroneous interpretations, for example, regarding the extent of horizontal gene transfer in these animals. Here, we provide an ultralow input method to sequence the genome of the tardigrade, Hypsibius dujardini, from a single specimen. By employing rigorous washing and contaminant exclusion along with an efficient extraction of the 50 ~ 200 pg genomic DNA from a single individual, we constructed a library sequenced with a DNA sequencing instrument. These libraries were highly reproducible and unbiased, and an informatics analysis of the sequenced reads with other H. dujardini genomes showed a minimal amount of contamination. This method can be applied to unculturable tardigrades that could not be sequenced using previous methods.

Contamination during the genomic sequencing of microscopic organisms remains a large problem. Here, we show a method to sequence the genome of a tardigrade from a single specimen with as little as 50 pg of genomic DNA without whole genome amplification to minimize the risk of contamination.

单缓步动物标本的超低输入基因组测序库制备

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

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 ...

<meta charset="utf8"></head><body>药用植物的 DNA 条形码：鉴定和系统发育分析

Application of DNA Barcoding to Identify Medicinal Plants

Medicinal plants are valuable resources globally and are used worldwide to maintain health and treat disease; however, the presence of adulteration obstructs their development. DNA barcoding, a technique for species identification by standard DNA regions, facilitates prompt and accurate identification of traditional medicinal plants. The process of DNA barcoding entails six basic steps: 1) processing the medicinal plants, 2) extracting high-quality total DNA from the medicinal plants using centrifugal column method, 3) amplifying target DNA region internal transcribed spacer 2 (ITS2) with universal primers of plants and performing Sanger sequencing, 4) splicing and aligning sequence to obtain the target sequence, 5) matching the barcode sequence against the barcode library for identification, 6) aligning sequence, comparing intraspecific and interspecific variation, constructing phylogenetic neighbor-joining tree. As shown in the results, the universal primer can amplify the target region. Basic Local Alignment Search Tool (BLAST) demonstrates the percentage identified was 100%, and the neighbor-joining tree demonstrates that the splicing sequences were clustered with the A. sinensis OR879715.1 clade, and the clade support value is 100. This protocol provides a reference for applying DNA barcoding technology as an effective method to identify medicinal plants and adulterants.

<meta charset="utf8"></head><body>作者聚焦：利用 DNA 条形码技术提高药用植物鉴定的效率

The present protocol describes the use of DNA barcoding technology to authenticate plant-based medicinal materials, and the medicinal plant Angelica sinensis (Oliv.) Diels was identified as an example.

DNA 条形码在药用植物鉴定中的应用

Medicinal plants are valuable resources globally and are used worldwide to maintain health and treat disease; however, the presence of adulteration ...

Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.

Gel-seq enables researchers to simultaneously prepare libraries for both DNA- and RNA-seq at negligible added cost starting from 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries.

凝胶序列: 一种同时测序库的方法用水凝胶基质制备 DNA 和 RNA

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the ...

植物标本标本的鲁棒 DNA 分离与高通量测序库建设

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

超高分子量基因组DNA的分离纯化白粉病对长读测序

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

经验、基因和计算技术阐明了杀菌剂危害蜜蜂健康的机制

单缓步动物标本的超低输入基因组测序库制备

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

DNA 条形码在药用植物鉴定中的应用

DNA 条形码在药用植物鉴定中的应用

DNA 条形码在药用植物鉴定中的应用

凝胶序列: 一种同时测序库的方法用水凝胶基质制备 DNA 和 RNA

植物标本标本的鲁棒 DNA 分离与高通量测序库建设

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

超高分子量基因组DNA的分离纯化白粉病对长读测序

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

经验、基因和计算技术阐明了杀菌剂危害蜜蜂健康的机制

单缓步动物标本的超低输入基因组测序库制备

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

DNA 条形码在药用植物鉴定中的应用

DNA 条形码在药用植物鉴定中的应用

DNA 条形码在药用植物鉴定中的应用

凝胶序列: 一种同时测序库的方法用水凝胶基质制备 DNA 和 RNA

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