这项工作得到了国家科学基金会通过职业奖 (赠款号 DBI-1253293) 的支持;国立卫生研究院 (NIH) (赠款编号 HG007233-01、R01-EB019453-01、1R21HG007233、DP2-AR068129-01、R01-HG008978);和国防高级研究项目机构生活铸造项目 (合同编号 HR0011-12-C-0065, N66001-12-C-4211, HR0011-12-C-0066)。

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与此工作流程有关的专利可能被授权给特派团生物, 其中 Adam r 减弱为股东。

SiC 序列微流控流产生了数以千计的细菌细胞的单细胞基因组测序数据。将数字条形码拼接到微凝胶体封装的单元格的基因组上, 可以将数据的在硅片反褶积转化为来自同一单元格的条形码读取组。一个已知成分的微生物群落的控制实验是评价条形码组纯度的必要方法。大量低纯度组表明, 细胞封装率过高, 或有明显的水滴交叉污染发生在微流控处理步骤。根据泊松统计数据, 条形码和单元格应封装在1粒子的目标比, 每10滴, 以限制多个封装事件的速率小于5% 的所有非空滴。比这更高的封装速率会以指数的形式增加双峰的速率, 因此在 dropmaking 过程中对封装率的验证至关重要。用户在单个微凝胶体中应特别小心封装多个单元格, 因为共享同一条形码序列的不同单元格的读取不能 bioinformatically 分隔。如果1单元接收2不同的条形码, 条码组的纯度不受影响, 虽然丰度指标是在按条形码顺序计数时倾斜的。
由于合并条件不理想, 液滴交叉污染也可能出现。在成功的操作中, 微流控合并装置 (图 5) 可以 controllably 1 个条码液滴与1微凝胶体和一定量 PCR 试剂。非理想的流速会导致液滴配对不正确的比率: 例如, 1 条码可以与2微配对。协议中列出的所有流量都是估计的, 可能需要根据设备几何和雾滴大小的细微变化来调整。使用高速录音功能 (> 1万帧/秒) 的摄像头的用户应在微流控操作的开始和结束过程中验证正确的水滴合并。用户如果不使用高速相机, 就可以收集少量的合并输出, 并手动测量显微镜下的水滴大小。液滴尺寸应均匀: 过量的并入条形码或微凝胶体下降表明, 应相应减少回注率。
在处理微和 microdroplets 时, 应采取一些一般的预防措施, 以维护其完整性。微, 虽然机械健壮, 必须充分冷却之前, 打破和洗涤步骤, 以确保完整的凝胶。非球形微表明琼脂糖没有足够的时间凝固。清洗微时, 按所需的速度旋转悬浮液以避免产品损失。琼脂糖凝胶具有与水紧密匹配的折射率, 在管22中可能很难看到, 因此用户在吸入前应仔细识别凝胶液的边界。在实验室手套和油管上, 静态力23的累积使水中油滴容易发生聚结。因此, 我们建议在泵浦启动前, 用徒手装入滴注注射器, 并用防静电枪处理所有回灌线。通过缓慢旋转注射器中的乳液, 并手动吸出较大的水滴, 在顶部积聚, 由于其较大的浮力, 可以去除大的合并液滴。
SiC 序列是第一个证明单细胞基因组测序 > 5万细菌细胞的技术。该平台在现有方法的吞吐量方面具有显著优势, 并能够更深入地取样异构微生物群落。迄今为止, 单细胞基因组测序的微流控技术已使用 microchambers9和 microwells24进行细胞隔离和扩增, 但仅有数以上百的细胞的吞吐量。将单个单元格的流排序为 wellplates5,6不需要专门的微流控工具, 但具有类似的低吞吐量。鉴于来自环境的土壤和水样通常在物种级25、26的 > 1000 中具有 alpha 多样性, 因此, 由于它能够取样大量的生物体, 所以 SiC 序列是非常有利的。SiC 序列工作流适用于实验室文化、自然环境或活体宿主的细胞输入。细胞样品只需要在水悬浮和无大颗粒 (> 10 µm) 适合微流控封装。例如, 以前已将该方法应用于海水样本, 使用一系列洗涤和过滤步骤在封装17之前对单元格进行预处理。
SiC 序列协议从每个单元中生成相对稀疏的排序数据量, 可能不适用于所有应用程序。一些生物信息学算法, 如 "从头" 基因组组装或单核苷酸变体 (SNV) 调用需要更高的覆盖率深度才能有效地工作。相反, 可以通过分类 binning 方法27将条码组聚集在硅片中, 以便可以将算法应用于较大的读取集。碳化硅序列工作流的总体条形码效率相对较低, 在输入样本的可用性较低的情况下也可能带来挑战。SiC 序列依赖于泊松分布的条形码封装步骤, 因此大约10% 的细胞接收到一个分子条码, 并在最后的图书馆准备步骤中被放大。虽然这与其他基于 microdroplet 的条形码方案10类似, 但使用珍贵细胞样本的用户可能难以达到适当的库产量, 因此可能需要在最终的时间内增加 PCR 周期的数量。放大步骤。另一种潜在的解决方案为使用微流控技术的用户是在数字 PCR 步骤后排序正条码滴, 从而使整体条形码效率 > 85%28。
SiC 序列技术的一个潜在的未来方向是调整工作流, 用于哺乳动物细胞, 为新的临床单细胞研究铺平道路。例如, 对单癌细胞复制数变异的分析, 可以进一步了解异质性在肿瘤病理学中的作用2。或者, 将 SiC 序列与现有的方法相结合, 以探测和丰富感兴趣的 DNA 顺序29将使目标单细胞序列的亚群或罕见的细胞株进行排序。通过环境样本, 已知的代谢通路中的基因可以被定位, 并与相邻的基因进行分析, 以确定新的基因组岛。从人类宿主环境中, 低效的致病细菌样本可以被分离和序列在单细胞水平, 以更密切地检查他们的基因型的致病力的起源。

基因组作为细胞身份和功能的蓝图, 包含了有机体的编码潜能的整体。在基因组层面上对细胞生物学的理解可以解释异质细胞群中观察到的表型多样性。这种异质性在生物系统中很明显, 对人类健康和疾病有着广泛的影响。例如, 肿瘤细胞中基因复制数的变化与癌症的演变和扩散有关, 如1,2。在细菌感染中, 在一小部分基因组中存在的致病性岛屿可以水平转移, 并导致抗药性细菌的扩散3,4。在单细胞水平上研究基因组的一个主要挑战是可用的 DNA 数量少, 以及需要分析数以千计的细胞来取样基因型的全部多样性。由于这些原因, 实验吞吐量的限制阻碍了单细胞研究的有效性, 使结果偏向于最丰富的细胞。单单元隔离技术, 如流排序5,6, 光学镊子7, 在大容量凝胶8中嵌入, 微流体9能够处理数以百计的细胞进行测序;但是, 这仅代表大多数示例中的一小部分。单细胞基因组测序的方法具有极高的吞吐量, 可以更深入和更完整地分析细胞种群, 从而阐明基因型多样性在这些社区中的作用。
液滴微流体能够在数以百万计的 picoliter 反应器内对细胞和生物试剂进行高通量的操作。到目前为止, microdroplet 技术已经被用来研究不同组织的细胞之间的差异表达模式10,11,12, 深序列长分子13,14 、15, 并对单个单元格16进行染色质免疫沉淀排序 (芯片序列) 分析。事实上, microdroplets 能够进行高通量的、分割的操作, 使它们能够在单细胞基因组学中应用。然而, 这种技术的发展呈现出自己独特的技术挑战。细胞必须裂解, 纯化, 并以最小的偏差放大, 以均匀的样本细胞数量。另外, 与哺乳动物细胞中的 polyadenylated mRNA 记录不同, 基因组中没有可比的分子母题来促进目标核酸的捕获。由于这些原因, 单细胞基因组测序在 microdroplet 平台中难以实现。
在这项工作中, 我们提供了我们以前报告的单细胞微流控方法的详细协议, 能够在单个实验17中对成千上万个细胞的基因组进行排序。使用这种技术, 称为 SiC 序列, 细菌细胞被封装在微米尺度的水凝胶和单独裂解, tagmented, 并合并 microdroplet 含有一个独特的寡核苷酸条码, 这是拼接到细胞的基因组 DNA 通过单重叠扩展聚合酶链反应 (PCR)。水凝胶作为孤立的容器, 其中高分子量的基因组 DNA 被 sterically 包裹, 允许较小的分子, 如洗涤剂和裂解酶, 以获取和纯化 DNA 之前, 条形码18。此协议在几个小时内处理 > 5万个单元格, 导致条形码库准备好进行排序。按照顺序, 读取是根据他们的单单元条码序列 demultiplexed 的, 结果是由数以百万计的读取组成的数据集, 每个都带有一个蜂窝索引。

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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">3&#34; silicon wafers, P type, virgin test grade</td>
			<td style="width:245px;">University Wafers</td>
			<td style="width:163px;">447</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SU-8 3025 photoresist</td>
			<td style="width:245px;">Microchem</td>
			<td style="width:163px;">17030192</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Spin coater</td>
			<td style="width:245px;">Specialty Coating Systems</td>
			<td style="width:163px;">G3P-8</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Photomasks</td>
			<td style="width:245px;">CadArt Servcies</td>
			<td style="width:163px;">(custom)</td>
			<td>See Supplemental Files for mask designs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PGMEA developer</td>
			<td>Sigma-Aldrich</td>
			<td>484431</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isopropanol</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td>109827</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sylgard 184 silicone elastomer kit</td>
			<td style="width:245px;">Krayden</td>
			<td style="width:163px;">4019862</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Degassing chamber</td>
			<td style="width:245px;">Bel-Art</td>
			<td style="width:163px;">42025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.75 mm biopsy punch</td>
			<td>World Precision Instruments</td>
			<td>504529</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass microscope slides (75 mm x 50 mm)</td>
			<td style="width:245px;">Corning</td>
			<td>294775X50</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Aquapel (hydrophobic glass treatment)</td>
			<td style="width:245px;">Pittsburgh Glass Works</td>
			<td>47100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PE-2 polyethylene tubing</td>
			<td style="width:245px;">Scientific Commodities</td>
			<td>B31695-PE/2</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">1 mL syringes</td>
			<td style="width:245px;">BD</td>
			<td style="width:163px;">309628</td>
			<td style="width:297px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">27 gauge needles</td>
			<td style="width:245px;">BD</td>
			<td style="width:163px;">305109</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">Syringe pump</td>
			<td style="width:245px;">New Era Pump Systems</td>
			<td>NE-501</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">Novec HFE-7500 fluorinated oil (HFE)</td>
			<td style="width:245px;">3M</td>
			<td style="width:163px;">98-0212-2928-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">FC-40 fluorinated oil</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td style="width:163px;">F9755</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PEG-PFPE surfactant</td>
			<td style="width:245px;">Ran Biotechnologies</td>
			<td style="width:163px;">008-FluoroSurfactant</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Space heater</td>
			<td style="width:245px;">Lasko&#160;</td>
			<td style="width:163px;">CD09250&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">Agarose, low gelling temperature</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td style="width:163px;">a9414</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">TE (10X)</td>
			<td style="width:245px;">Rockland</td>
			<td style="width:163px;">mb-007</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">PBS 1X, pH 7.4</td>
			<td style="width:245px;">E&#38;K Scientific Products</td>
			<td>EK-65083</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">OptiPrep (density gradient medium)</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td style="width:163px;">d1556</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">1H,1H,2H-Perfluoro-1-Octanol (PFO)</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td style="width:163px;">370533</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">Span 80 (sorbitane monooleate)</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td style="width:163px;">s6760</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">Hexane</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td style="width:163px;">139386</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">Tween 20 (polysorbate 20)</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td style="width:163px;">p2287</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">Lysozyme Type IV</td>
			<td style="width:245px;">MP Biomedicals</td>
			<td style="width:163px;">195303</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">Mutanolysin</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td>M9901</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">Zymolyase (yeast lytic enzyme)</td>
			<td style="width:245px;">Zymo Research</td>
			<td style="width:163px;">e1004</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">Lysostaphin</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td>L7386</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">Sodium chloride</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">EDTA</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">Tris-HCl, pH 7.5, 1M</td>
			<td style="width:245px;">Invitrogen</td>
			<td style="width:163px;">15567-027</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">Dithiothreitol (DTT)</td>
			<td style="width:245px;">Teknova</td>
			<td style="width:163px;">d9750</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">Lithium dodecyl sulfate</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td style="width:163px;">L9781</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Proteinase K</td>
			<td style="width:245px;">New England Biosciences</td>
			<td style="width:163px;">P8107S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ethanol, 200 Proof (100%)</td>
			<td style="width:245px;">Koptec</td>
			<td>V1001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">SYBR Green I (nucleic acid stain)</td>
			<td style="width:245px;">Invitrogen</td>
			<td style="width:163px;">S7563</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">PEG 6k</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td>81260</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">Triton X-100 (octylphenol ethoxylate)</td>
			<td style="width:245px;">Sigma-Aldrich</td>
			<td style="width:163px;">t8787</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Nextera DNA Library Prep Kit</td>
			<td style="width:245px;">Illumina</td>
			<td>FC-121-1030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">Phusion Hot Start Flex Master Mix (High-Fidelity Hot Start Master Mix)</td>
			<td style="width:245px;">New England Biosciences</td>
			<td style="width:163px;">m05365</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">Platinum Multiplex PCR Master Mix (Taq Master Mix)</td>
			<td style="width:245px;">Applied Biosystems</td>
			<td style="width:163px;">4464263</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">Warmstart 2.0 Bst Polymerase (isothermal polymerase)</td>
			<td style="width:245px;">New England Biosciences</td>
			<td style="width:163px;">m0538m</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">NT buffer from Nextera XT kit (neutralization buffer)</td>
			<td style="width:245px;">Illumina</td>
			<td style="width:163px;">FC-131-1024</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">Cold cathode fluorescent inverter</td>
			<td style="width:245px;">(custom)</td>
			<td style="width:163px;">(custom)</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">DC power supply</td>
			<td style="width:245px;">Mastech</td>
			<td>HY1503D</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">Zerostat 3 anti-static gun</td>
			<td style="width:245px;">Milty</td>
			<td>5036694022153</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:513px;">3D-printed centrifuge syringe holder</td>
			<td style="width:245px;">(custom)</td>
			<td>(custom)</td>
			<td>See Supplemental Files for 3D print file</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zymo DNA Clean &#38; Concentrator-5</td>
			<td style="width:245px;">Zymo Research</td>
			<td>D4003</td>
			<td></td>
		</tr>
	</tbody>
</table>

an ultrahigh-throughput microfluidic platform for single-cell genome sequencing

测序技术经历了从大批量到单细胞分辨率的范式转变, 以响应对生物系统中细胞异质性作用的逐渐理解。然而, 由于处理基因组测序的局限性, 大量人群的单细胞测序受到限制。本文描述了单细胞基因组测序 (SiC 序列) 的方法, 它利用液滴微流体分离、放大和条形码的单细胞基因组。微中的细胞封装允许分离纯化和 tagmentation DNA, 而微流控合并有效地将每个基因组与一个独特的单细胞寡核苷酸条码配对, 允许 > 5万单胞细胞被测序每次运行。测序数据由条形码 demultiplexed, 生成来自单个单元格的读取组。作为单细胞测序的高通量和低偏置方法, SiC 序列将使针对不同细胞群的更广泛的基因组研究得以实现。

SiC 序列实验工作流包含3个用软光刻过程制作的微流控芯片 (图 1)。共流 dropmaker (图 3A) 生成25µm 的数字条码水滴, 用于标记基因组 DNA, 并使用唯一的单细胞标识符。条形码寡核苷酸由 15 bp 退化序列, 由 PCR 手柄两侧的放大 (表 2, 棒底漆)。条码被稀释到 femtomolar 浓度, 以实现单分子封装, 所有的水滴接收0或1条码片段 (s)。含有条形码的水滴放大, 产生了许多双绞型条码 amplicons 的拷贝。核酸染色用于验证成功的放大和量化条形码片段的封装速率 (图 4B)。微是由细菌细胞悬浮液和熔融琼脂糖凝胶在同等流速下产生的 (图 2A)。琼脂糖是准备在两次预期的最终浓度, 因为共流 dropmaking 过程有效稀释水溶液的系数为2。当琼脂糖冷却, 它固化成一个25µm 直径微凝胶体占据球形体积的水滴。
一系列的洗涤和裂解步骤净化微中的高分子量基因组 DNA (图 2B)。在破乳剂后, 在大体积内进行水洗, 稀释微量有机溶剂, 抑制下游酶处理。在显微镜下观察洗涤的微, 以验证细胞封装速率 (图 4A)。在微凝胶体悬浮液中加入含有广泛溶解活性的酶的鸡尾酒, 以消化细菌和真核微生物的细胞壁19。第二种蛋白酶 K 和洗涤剂的治疗会降低蛋白质和 solubilizes 细胞的碎片。
纯化 dna 的 Tagmentation 是在水滴中进行的, 以避免在微18之间扩散小 tagmented dna 片段导致潜在的交叉污染。液滴封装设备 (图 3B) 划分每个微凝胶体与一个缓冲和 tagmentation 酶, 它同时碎片双链 DNA, 同时也 "标记" 它与预先加载的寡核苷酸20。微被装入水滴作为接近包装的微粒, 达到封装率接近1微凝胶体为每下落与少量双峰21(图 4C)。
在微流控流的最后一步 (图 2C) 中, 一个设备执行双重合并操作, 组合1个条码降、1个含微凝胶体的降, 以及在受控的两步过程中的放大组合。首先, 含有 PCR 试剂的液滴被配对, 并与黄色 (图 5) 中显示的条形码下落合并。微流控通道中的盐水电极产生高电场梯度, 触发熔滴的合并。以类似的方式, 第一个合并的水滴与微凝胶体液滴配对, 并在以红色显示的区域中第二次合并。在单重叠扩展 (SOE) PCR 中采集液滴和热循环脱离芯片。条形码和 tagmented 基因组 DNA 重叠的互补端允许只有适当的条形码结构的融合和指数放大。
顺序数据首先通过读取质量进行筛选, 然后根据它们的 15 bp 单单元条码序列对读取进行分组分析。要认为条形码组是有效的, 应包含最少的读数数;这种阈值限制了对具有大量排序数据的单元格的分析, 并从数据集中删除了 PCR 变异的条形码 "孤儿"。在这个示例运行中, 最小值被设置为每组 7.5 kbps (每个50读取150个 bp)。条形码计数与组大小的直方图显示, 有效条码组的重要部分刚好高于阈值大小 (图 6A)。
在一个已知微生物群落组成的控制实验中, 用纯度和相对丰度指标来评价 SiC 序列的质量。在这里, 分析了由3革兰氏阴性菌、5革兰氏阳性菌和2株酵母组成的合成10细胞群落。给定条码组的纯度定义为组中最常见的基因组的读数映射数除以组中读取总数的个数。绝大多数条码组的纯度大于 0.95 (图 6B)。单元格类型的相对丰度是通过计算原始读取数和通过计数条形码组来计算的, 其中, 组分配了与其成员读取的一致性相对应的单元格类型 (图 6C)。大量的读取和条码组跟踪大致相同的比例, 表明细胞的人口正在取样, 使某些物种不包含在不成比例的小或大型条码组。从单个物种中绘制所有条码组的总覆盖范围, 表明整个基因组的覆盖率很高, 且很少或没有辍学区域 (图 7)。覆盖范围的均匀性可以通过规范化覆盖率值的频率分布进行验证, 大多数值围绕平均值 (图 7, 嵌入)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57598/57598fig1.jpg"> 图 1: 通过光刻工艺制备微流控器件.(A) 具有单个特征高度的主模具由自旋涂覆一层 SU-8 光刻胶到硅片上制成。然后用光刻掩模和 UV 光进行图案化, 将暴露的 SU-8 交联。最后, uncrosslinked SU-8 溶解在显影液中。所产生的模具用于铸造与玻璃滑块粘合, 以产生完整的微流控装置。(B) 对于双层设备, 制造过程类似于从自旋涂层和曝光步骤开始。然后重复这些步骤以创建两层设备。<a href="/files/ftp_upload/57598/57598fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57598/57598fig2.jpg"> 图 2:SiC 序列工作流概述.(A) 微生物悬浮液与 dropmaker 装置中的熔融琼脂糖共同流动, 以封装微中的单个细胞。(B) 微受到一系列清洗, 以净化细菌基因组 DNA。溶解酶消化革兰阳性菌和酵母细胞壁, 洗涤剂 solubilizes 细胞碎片。微被重新封装成水滴, tagmentation 以减少交叉污染。(C) 微流控合并结合了数字 PCR 条码、tagmented 微凝胶体基因组, 以及一个速率 > 1 赫的放大组合。非芯片的国企-PCR 拼接一个独特的单细胞条码到 tagmented 基因组, 并有选择地放大完全条形码结构。<a href="/files/ftp_upload/57598/57598fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57598/57598fig3.jpg"> 图 3:用于 dropmaking 和微凝胶体重新封装的微流控设备。(A) 此面板显示一个共流 dropmaker (25 µm 的特征高度)。细胞和熔融琼脂糖被引入到设备中, 在相同的流速, 以产生25µm 液滴在25µm x 25 µm 结。对于数字条码 dropmaking, 细胞入口堵塞, 并引入 PCR 组合进入琼脂糖入口。(B) 此面板显示微凝胶体重新封装设备 (25 µm 的特征高度)。微流入漏斗形入口, 以维持其紧密包装的订购, 并在25µm x 30 µm 路口重新封装之前接收 tagmentation 混合体。<a href="/files/ftp_upload/57598/57598fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57598/57598fig4.jpg"> 图 4: 水滴和微的显微照片.(A) 此面板显示在酶裂解之前洗涤了25µm 微。细菌被荧光染色, 以定量的封装速率。泊松负荷统计数据规定, 单元格的封装速率为 1, 在10滴或更少的情况下, 以最小化多封装事件的频率。(B) 此面板显示了一个荧光显微图像, 25 µm 数字条码水滴处理的核酸染色。含有放大条码片段的水滴产生强烈的荧光信号。(C) 此面板显示微重新封装在50µm 滴中。微的封闭包装使封装速率接近1凝胶每滴, 很少双峰。<a href="/files/ftp_upload/57598/57598fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57598/57598fig5.jpg"> 图 5: 单细胞基因组条形码的微流控双合并装置.两步合并操作对条形码水滴与 tagmented 基因组在高吞吐量。首先生成一滴 PCR 混合物, 并将其与在黄色中使用盐水电极显示的条形码水滴合并。接下来, 一个包含微凝胶体的水滴在红色显示的区域中第二次被引入并合并。油入口允许精确控制重新注入水滴之间的间距。条形码回注室及其间隔油放置在较短的25µm 层上, 以蓝色着色。所有其他设备功能属于较厚的层, 总高度为45µm。<a href="/files/ftp_upload/57598/57598fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57598/57598fig6.jpg"> 图 6:10 细胞合成微生物群落的条码组度量.(A) 此面板显示条码组大小的分布。当组大小增加时, 给定大小的组数呈指数递减。每个组的最小阈值为 7.5 kbps 限制对具有足够信息量的组进行分析, 并删除 PCR 突变序列 "孤儿"。(B) 此面板显示条码组纯度的分布。绝大多数 (> 90%) 的群体是非常高纯度 (> 95%)。(C) 此面板显示了在读取和条码组级别计算的10种的相对丰度。2计数方法产生相似的结果, 表明条码组的大小是一致的不同物种。<a href="/files/ftp_upload/57598/57598fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57598/57598fig7.jpg"> 图 7: 聚合基因组覆盖率枯草芽孢杆菌条码组.将所有条码组的读数从映射到细菌枯草杆菌(N = 9398) 进行汇总并进行分析。圆形覆盖图说明了 SiC 序列读数的覆盖均匀性, 没有可观察到的辍学区域。绕圆周的虚线表示平均覆盖范围 (5.55x)。相对覆盖频率的嵌入直方图表明, 大部分的碱基覆盖在接近全基因组平均的深度, 由虚线表示。<a href="/files/ftp_upload/57598/57598fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>装置</td>
			<td>第一层高度 (µm)</td>
			<td>第一层旋转速度 (rpm)</td>
			<td>第二层高度 (µm)</td>
			<td>第二层旋转速度 (rpm)</td>
		</tr>
<tr>
<td>共流 dropmaker</td>
			<td>25</td>
			<td>4000</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
<tr>
<td>凝胶再 encapsulator</td>
			<td>25</td>
			<td>4000</td>
			<td>N/A</td>
			<td>N/A</td>
		</tr>
<tr>
<td>双重合并</td>
			<td>25</td>
			<td>4000</td>
			<td>20</td>
			<td>5000</td>
		</tr>
</tbody></table>表 1: 微流控设备制造参数.本表显示了在 SiC 序列工作流中使用的微流控器件的列表, 其所需的光刻胶自旋涂层的速度 (基于 SU-8 3025 的制造商规格)。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>标签</td>
			<td colspan="5">序列 (5 ' > 3 ')</td>
		</tr>
<tr>
<td>酒吧</td>
			<td colspan="5">GCAGCTGGCGTAATAGCGAGTACAATCTGCTCTGATGCCGCATAGNNNNNNNNNNNNNNNTAAGCCAGCCCCGACACT</td>
		</tr>
<tr>
<td>DNA_BAR</td>
			<td colspan="5">CTGTCTCTTATACACATCTCCGAGCCCACGAGACGTGTCGGGGCTGGCTTA</td>
		</tr>
<tr>
<td>P7_BAR</td>
			<td colspan="5">CAAGCAGAAGACGGCATACGAGATCAGCTGGCGTAATAGCG</td>
		</tr>
<tr>
<td>P5_DNA</td>
			<td colspan="5">AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTC</td>
		</tr>
<tr>
<td>I7_READ</td>
			<td colspan="5">GCCCACGAGACGTGTCGGGGCTGGCTTA</td>
		</tr>
</tbody></table>表 2: 底漆序列。
补充文件 1:<a href="/files/ftp_upload/57598/Suppl_File_1-SiC-seq_device_masks.dwg" target="_blank">请单击此处下载此文件.</a>
补充文件 2:<a href="/files/ftp_upload/57598/Suppl_File_2-centrifuge_syringe_holder_1mL.dwg" target="_blank">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing at JoVE.com

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

单细胞基因组测序的超高通量微流控平台

单细胞测序揭示了生物系统中的基因型异质性, 但目前的技术缺乏对群落组成和功能的深入分析所必需的吞吐量。在这里, 我们描述了一个微流控流程的序列 > 5万单细胞基因组从不同的细胞群。

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

an-ultrahigh-throughput-microfluidic-platform-for-single-cell-genome

Research

JoVE Journal

Bioengineering

17.5K 次观看.  University of California, San Francisco. 单细胞测序揭示了生物系统中的基因型异质性, 但目前的技术缺乏对群落组成和功能的深入分析所必需的吞吐量。在这里, 我们描述了一个微流控流程的序列 > 5万单细胞基因组从不同的细胞群。

视频: An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

High-throughput Protein Expression Generator Using a Microfluidic Platform

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and high-fidelity measurements of large systems. Microfluidics promises to fulfill many of these requirements, such as performing high-throughput screening experiments on-chip, encompassing biochemical, biophysical, and cell-based assays1. Since the early days of microfluidics devices, this field has drastically evolved, leading to the development of microfluidic large-scale integration2,3. This technology allows for the integration of thousands of micromechanical valves on a single device with a postage-sized footprint (Figure 1). We have developed a high-throughput microfluidic platform for generating in vitro expression of protein arrays (Figure 2) named PING (Protein Interaction Network Generator). These arrays can serve as a template for many experiments such as protein-protein 4, protein-RNA5 or protein-DNA6 interactions.
The device consist of thousands of reaction chambers, which are individually programmed using a microarrayer. Aligning of these printed microarrays to microfluidics devices programs each chamber with a single spot eliminating potential contamination or cross-reactivity Moreover, generating microarrays using standard microarray spotting techniques is also very modular, allowing for the arraying of proteins7, DNA8, small molecules, and even colloidal suspensions. The potential impact of microfluidics on biological sciences is significant. A number of microfluidics based assays have already provided novel insights into the structure and function of biological systems, and the field of microfluidics will continue to impact biology.

We present a microfluidic approach for the expression of protein arrays. The device consists of thousands of reaction chambers controlled by micro-mechanical valves. The microfluidic device is mated to a microarray-printed gene library. These genes are then transcribed and translated on-chip, resulting in a protein array ready for experimental use.

利用微流控平台的高通量蛋白质表达发生器

Rapidly increasing fields, such as systems biology, require the development and implementation of new technologies, enabling high-throughput and ...

科研

生物工程

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described in detail. The PLBR can be utilized for the analysis of single bacteria and microcolonies to investigate biotechnological and microbiological related questions concerning, e.g.&#160;cell growth, morphology, stress response, and metabolite or protein production on single-cell level. The device features continuous media flow enabling constant environmental conditions for perturbation studies, but in addition allows fast medium changes as well as oscillating conditions to mimic any desired environmental situation. To fabricate the single use devices, a silicon wafer containing sub micrometer sized SU-8 structures served as the replication mold for rapid polydimethylsiloxane casting. Chips were cut, assembled, connected, and set up onto a high resolution and fully automated microscope suited for time-lapse imaging, a powerful tool for spatio-temporal cell analysis. Here, the biotechnological platform organism Corynebacterium glutamicum&#160;was seeded into the PLBR and cell growth and intracellular fluorescence were followed over several hours unraveling time dependent population heterogeneity on single-cell level, not possible with conventional analysis methods such as flow cytometry. Besides insights into device fabrication, furthermore, the preparation of the preculture, loading, trapping of bacteria, and the PLBR cultivation of single cells and colonies is demonstrated. These devices will add a new dimension in microbiological research to analyze time dependent phenomena of single bacteria under tight environmental control. Due to the simple and relatively short fabrication process the technology can be easily adapted at any microfluidics lab and simply tailored towards specific needs.

In this protocol the fabrication, setup and basic operation of a microfluidic picoliter bioreactor (PLBR) for single-cell analysis of prokaryotic microorganisms is introduced. Industrially relevant microorganisms were analyzed as proof of principle allowing insights into growth rate, morphology, and phenotypic heterogeneity over certain time periods, hardly possible with conventional methods.

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

电池挤压作为一种强大的，微流控细胞内传送平台

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

Microfluidic Platform for Measuring Neutrophil Chemotaxis from Unprocessed Whole Blood

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher neutrophil counts in the blood are usually an indicator of ongoing infections, while low neutrophil counts are a warning sign for higher risks for infections. To accomplish their functions, neutrophils also have to be able to move effectively from the blood where they spend most of their life, into tissues, where infections occur. Consequently, any defects in the ability of neutrophils to migrate can increase the risks for infections, even when neutrophils are present in appropriate numbers in the blood. However, measuring neutrophil migration ability in the clinic is a challenging task, which is time consuming, requires large volume of blood, and expert knowledge. To address these limitations, we designed a robust microfluidic assays for neutrophil migration, which requires a single droplet of unprocessed blood, circumvents the need for neutrophil separation, and is easy to quantify on a simple microscope. In this assay, neutrophils migrate directly from the blood droplet, through small channels, towards the source of chemoattractant. To prevent the granular flow of red blood cells through the same channels, we implemented mechanical filters with right angle turns that selectively block the advance of red blood cells. We validated the assay by comparing neutrophil migration from blood droplets collected from finger prick and venous blood. We also compared these whole blood (WB) sources with neutrophil migration from samples of purified neutrophils and found consistent speed and directionality between the three sources. This microfluidic platform will enable the study of human neutrophil migration in the clinic and the research setting to help advance our understanding of neutrophil functions in health and disease.

This protocol details an assay designed to measure human neutrophil chemotaxis from one droplet of whole blood with robust reproducibility. This approach circumvents the need for neutrophil separation and requires only a few minutes of assay preparation time. The microfluidic chip enables the repeated measure of neutrophil chemotaxis over time in infants or small mammals, where sample volume is limited.

微流体平台从未经加工的全血测定中性粒细胞趋

Neutrophils play an essential role in protection against infections and their numbers in the blood are frequently measured in the clinic. Higher ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

多器官芯片 - 一种微流体平台的长期多组织共培养

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

高通量单细胞的分离和文化的微流体平台

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

与复用电子检测的粒子的空间跟踪微流体平台

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by their natural environment, root hair morphology and function are difficult to study and often excluded from plant research. In recent years, microfluidic platforms have offered a way to visualize root systems at high resolution without disturbing the roots during transfer to an imaging system. The microfluidic platform presented here builds on previous plant-on-a-chip research by incorporating a two-layer device to confine the Arabidopsis thaliana main root to the same optical plane as the root hairs. This design enables the quantification of root hairs on a cellular and organelle level and also prevents z-axis drifting during the addition of experimental treatments. We describe how to store the devices in a contained and hydrated environment, without the need for fluidic pumps, while maintaining a gnotobiotic environment for the seedling. After the optical imaging experiment, the device may be disassembled and used as a substrate for atomic force or scanning electron microscopy while keeping fine root structures intact.

Imaging the Root Hair Morphology of Arabidopsis Seedlings in a Two-layer Microfluidic Platform

This article demonstrates how to culture Arabidopsis thaliana seedlings in a two-layer microfluidic platform that confines the main root and root hairs to a single optical plane. This platform can be used for real-time optical imaging of fine root morphology as well as for high-resolution imaging by other means.

影像学在两层微流控平台拟南芥幼苗的根头发形态

Root hairs increase root surface area for better water uptake and nutrient absorption by the plant. Because they are small in size and often obscured by ...

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved useful as a convenient live imaging platform providing capabilities for precise control of experimental conditions in real time. In this article, we demonstrate live imaging of individual worms employing WormSpa, a previously-published custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes. To illustrate the capability, we performed proof-of-principle experiments in which worms were infected in the device with pathogenic bacteria, and the dynamics of expression of immune response genes and egg laying were monitored continuously in individual animals. The simple design and operation of this device make it suitable for users with no previous experience with microfluidic-based experiments. We propose that this approach will be useful for many researchers interested in longitudinal observations of biological processes under well-defined conditions.

A Microfluidic Platform for Longitudinal Imaging in Caenorhabditis elegans

In this article, we demonstrate live imaging of individual worms employing a custom microfluidic device. In the device, multiple worms are individually confined to separate chambers, allowing multiplexed longitudinal surveillance of various biological processes.

秀丽线虫纵向成像的微流控平台

In the last decade, microfluidic techniques have been applied to study small animals, including the nematode Caenorhabditis elegans, and have proved ...

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 ...