This work was funded by Genome BC, Genome Canada, and Coastal Genomics. The authors thank Kirby Cronin and Michael Chan for their help in sample collection and processing. We also would like to acknowledge Thea Van Rossum and Dr. Fiona Brinkman for bioinformatics assistance.

1.水收集和过滤<ol><li>收集来自各部位（ 图1）的淡水样品。通过一系列的过滤器通过样品：1微米的过滤器，0.2微米的过滤器，和30 kDa的截止切向流过滤器，以系统地分开真核生物，细菌和病毒大小的颗粒，分别。 注意：只有在纯化和回收在0.2微米的过滤器（自由生活的细菌）的材料的分析在本报告中进行了说明，但类似的方法可用于从过滤器回收的其它颗粒。 </li></ol> 2.细菌浓度，DNA提取和纯化<ol><li>除去从水过滤系统（示意图， 图2）的0.2微米的膜过滤器（多个）。折叠为0.2μm盘式过滤器在一半并将其放入50ml管与打开的一面朝上。加入5 ml的1×磷酸缓冲液（PBS）及0.01％吐温，pH 7.4中进管。如果一个以上的过滤器的过程中使用的采用每一个过滤器的不同管中。商店管（多个）带过滤器在4℃下直到处理。否则，继续执行步骤2.2。 </li><li>删除50毫升管，用无菌镊子0.2微米的滤膜过滤。在一个培养皿的地方过滤器（多个）（离开PBS缓冲液在管中）。 <ol><li>用无菌镊子和无菌剪刀，剪去滤波器成小条带（1毫升×1厘米）。通过浸泡在70％的异丙醇，用DNA表面去污剂擦拭和与第一类型不同的样品之间的超纯水漂洗消毒镊子和剪刀。 </li></ol></li><li>放置切成条状的过滤器放回同样的50毫升管。加入10 mL的1×PBS和0.01％吐温，pH 7.4中的过滤器，使总共15 ml缓冲液中的50ml管中有。 </li><li>添加6洁净钨珠（直径3）每次50毫升管，覆盖管封口膜和涡大力为20分钟（使用50ml试管涡适配器）。如果MULTIPLE过滤器从一个样品，转移和池中的所有匀浆处理成清洁的50ml管中。离心管在3300 XG为15分钟，在4℃。 </li><li>悬浮颗粒和等份的细菌细胞（〜1ml等份）插入1.7毫升离心管。需要注意的是将有每个样品5微量离心管中。 </li><li>降速的离心管以10,000rpm离心10分钟，并移除大部分上清液降到〜200微升标记。 </li><li>存储所述样品在-80℃下，或进行通过使用市售的DNA分离试剂盒按照制造商的说明提取细菌的DNA。在细菌的DNA提取使用PBS或水作为阴性提取控制和E.大肠杆菌作为阳性对照提取。 </li><li>因为从多个管的DNA被提取，汇集平行子等份从同一样品放入1个2毫升管。然后通过使用包括0.1的溶液进行O / N沉淀体积的3M乙酸钠，2体积的100％乙醇和5微升5微克/微升的线性丙烯酰胺。搅拌均匀，将标本在-80℃。 </li><li>以最大速度离心（17000×g离心）处理30分钟，在4℃。用1ml冰冷的70％乙醇，空气干燥在生物安全柜中于不超过5分钟，重悬DNA沉淀在34微升10毫摩尔Tris-CL，pH值8.5洗沉淀。 注：线性丙烯酰胺有利于O / N沉淀后可视化DNA沉淀。 </li><li>确定使用高灵敏度荧光核酸定量测定和一个微量分光光度计，分别按照制造商的说明（见表材料）的DNA的数量和质量。 注意：如果多个样品制备的时候，可以用于定量双链DNA和板为基础的荧光计/分光光度计的超灵敏荧光核酸测定法来代替。 </li></ol> 3. DNA库准备<ol><li&gt;正常化的基因组DNA的细菌从水样提取到0.2纳克/微升〜浓度。 </li><li>细菌DNA的使用转座子为基础的方法（tagmentation）酶促片段1纳克。添加适配器和索引序列到片段化DNA按照制造商的说明进行操作。受试者tagmented样品的PCR 12个循环，在制造商的说明，在该点的测序索引被并入表示。 </li></ol> 4.自动凝胶尺寸选择<ol><li>跳过在制造商的说明书中所述的标准PCR后清理步骤（见表材料）。大小选择PCR后的样品直接用自动基于凝胶的大小选择平台。 <ol><li>加入3.5微升加样缓冲液到每个样品。 </li><li>根据制造商的协议中，加载样品到电泳工作站，指定的500-800碱基对的大小选择目标，在一个300μl的提取液离子量，使用的是预铸1.5％琼脂糖盒。 </li><li>集中的原始输出材料（300微升）下降到通过乙醇沉淀25微升。 注：另外，用电泳工作站自动浓度的样品通过过滤板和真空歧管更大的数字。 <ol><li>沉淀用0.1倍体积的3M乙酸钠，2体积的100％乙醇和5微升5微克/微升的线性丙烯酰胺的原料的洗脱体积。拌匀，放置样品在-80°CO / N。离心样品在17000×g离心30分钟。 </li><li>丢弃上清液，然后继续用1ml冰冷的70％乙醇洗涤DNA沉淀。 </li><li>离心样品再次在17000×g离心30分钟，弃去上清液，空气干燥，最后重悬DNA沉淀在25微升10毫摩尔Tris-CL，pH值8.5。 </li></ol></li></ol></li><li> （可选）使用1微升的DNA库的大小与自动化凝胶大小选择平台选择做一个五倍稀释使用的Tris-EDTA缓冲溶液，pH为8.0。分析使用基于芯片的毛细管电泳仪，根据生产商的说明来分析双链DNA质量库。 </li></ol> 5.图书馆规范化和测序<ol><li>进行到正常化使用如制造商的指导制备描述库正常化珠粒（包含转座子为基础的文库制备试剂盒）的样品。 <ol><li>可替代地，使用高灵敏度荧光核酸定量测定，接着汇集于等摩尔比，以达到为2nM的终双链DNA浓度测量双链DNA文库。继续以变性，用10微升汇集库和10μl的0.2N的氢氧化钠库，孵育5分钟，在室温。用稀释杂交缓冲液（包含转座子为基础制备试剂盒），以1毫升的最终体积的下午11时05分和浓度变性库。 </li></ol></li><li>加载样品在下一代测序平台S按以下所述制造商的标准测序方案。 </li></ol>

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贾里德R.斯洛博丹·马太J.内斯比特均持有沿海基因组学，是一家私人拥有的不列颠哥伦比亚省公司提供游侠科技股。

适配器二聚体和小尺寸插入优先被测序在现有平台的集群的存在代表了可用率和利用不足的设备的能力15的下降。使用珠击方法结合座子为基础的方法来制备文库可以导致更多的DNA剪切相比萃取4,5的其他方法。然而，提取和文库制备的所有方法可能引入偏倚测序的分布读取8,16。使用自动核酸提取平台，结合一个连接基法的实验观察，似乎没有去除过量的从鸟枪文库适配器?...

Metagenomics involves the sequencing of all the genetic material in a sample to characterize the microbial communities present. It is a complex and expensive process which involves the conversion of extracted nucleic acids into DNA libraries followed by next-generation sequencing. High quality libraries are essential for maximal data output and accurate metagenomics analysis. Environmental samples, such as water samples, often pose significant challenges to generating high quality libraries, due to low amounts of DNA that may also be degraded1-3 and the presence of inhibitors of PCR4-6. 
High quality libraries ideally consist of longer segments of DNA within a narrow range of lengths. In order to maximize the amount of useful data generated per sequencing run, the length of the DNA in the library should be at least as long as the maximum read length of the sequencing method being used. When using a sequencing-by-synthesis technology such as the Illumina MiSeq, the size of the DNA fragments affects the efficiency at which clusters are generated on the flow cell. For instance, when a library contains both shorter and longer DNA fragments, the shorter ones will be over-represented in the sequencing data7,8. In contrast, a library with similarly sized DNA fragments will be proportionally represented in the sequencing data. Many library preparation kits use ligation-based methods to add adapters to the DNA fragments and size selection is necessary to remove adapter dimers that do not contain an insert9,10. There are numerous methods11,12 to achieve this but the one technique that gives the most consistent results is the electrophoretic separation of DNA followed by the recovery of the desired lengths of DNA13,14. This process can be performed manually for a small number of samples, but when faced with processing hundreds of samples, automated solutions are required. The currently available platforms for automated gel size selection are low throughput and new platforms are needed to process large numbers of samples for sequencing. The Ranger Technology can be integrated with existing liquid handling workstations to enable the use of agarose gel electrophoresis for size selection and analytical purposes on a scale that satisfies today&#8217;s high throughput environment.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Peristaltic pump Masterflex P/S 1400 Series</td>
			<td>Thermo Scientific&#160;</td>
			<td>1400-1620</td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 &#181;m Supor Membrane</td>
			<td>VWR</td>
			<td>CA28143-969</td>
			<td>Pall Corporation, Ann Harbor, MI</td>
		</tr>
		<tr>
			<td>Tungsten carbide beads 3 mm (200)</td>
			<td>Qiagen</td>
			<td>69997</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropyl alcohol 70%</td>
			<td>Jedmon Products</td>
			<td>825751</td>
			<td>Healthcare Plus</td>
		</tr>
		<tr>
			<td>DNA away</td>
			<td>VWR</td>
			<td>7010</td>
			<td>Molecular BioProducts, Inc. San Diego, CA</td>
		</tr>
		<tr>
			<td>Milli-Q water purification system</td>
			<td>Fisher Scientific</td>
			<td>ZMQS6VF0Y</td>
			<td>Merck Millipore. This system has been discontinued.</td>
		</tr>
		<tr>
			<td>20 X PBS pH 7.5</td>
			<td>VWR</td>
			<td>E703-1L</td>
			<td>Amresco, Inc., Solon, OH</td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher Scientific</td>
			<td>BP337-100</td>
			<td>Fisher chemicals</td>
		</tr>
		<tr>
			<td>Vortex adapter for 2 (50 ml) tubes</td>
			<td>VWR</td>
			<td>13000-V1-50</td>
			<td>MoBio, Carlsbad, CA</td>
		</tr>
		<tr>
			<td>Vortex-Genie 2, 120V (Model G560)</td>
			<td>VWR</td>
			<td>SI-0236</td>
			<td>Scientific Industries, Inc.</td>
		</tr>
		<tr>
			<td>Beckman Centrifuge</td>
			<td>Raeyco Lab Equipment Systems Management Ltd</td>
			<td>Model J-6B</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerLyzer Powersoil DNA isolation kit</td>
			<td>VWR</td>
			<td>12855-100</td>
			<td>MoBio, Carlsbad, CA</td>
		</tr>
		<tr>
			<td>Vortex adapter for 24 (1.5-2 ml) tubes</td>
			<td>VWR</td>
			<td>13000-V1-24</td>
			<td>MoBio, Carlsbad, CA</td>
		</tr>
		<tr>
			<td>Microfuge 18 centrifuge</td>
			<td>Beckman Coulter</td>
			<td>367160</td>
			<td></td>
		</tr>
		<tr>
			<td>Nimbus Select workstation with Ranger Technology</td>
			<td>Hamilton Robotics</td>
			<td>92720-01</td>
			<td>Includes the liquid handling workstation and integrated Ranger Tech (electrophoresis hardware)</td>
		</tr>
		<tr>
			<td>Ranger reagent kit</td>
			<td>Coastal Genomics</td>
			<td>CG-10600-150-12-21</td>
			<td>Includes loading buffer and cassettes</td>
		</tr>
		<tr>
			<td>Ethyl alcohol (anhydrous)</td>
			<td>Commercial Alcohols</td>
			<td>P016EAAN</td>
			<td>Greenfield Ethanol</td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>Sigma-Aldrich</td>
			<td>S2889-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear acrylamide (5 mg/ml)</td>
			<td>Life Technologies</td>
			<td>AM9520</td>
			<td>Ambion</td>
		</tr>
		<tr>
			<td>Eppendorf refrigerated centrifuge</td>
			<td>Raeyco Lab Equipment Systems Management Ltd.</td>
			<td>5417R</td>
			<td></td>
		</tr>
		<tr>
			<td>Buffer EB (250 ml)</td>
			<td>Qiagen</td>
			<td>19086</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 1000 Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>ND-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit fluorometer</td>
			<td>Life Technologies</td>
			<td>Q32857</td>
			<td>Invitrogen. This product has been discontinued.</td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS assay kit</td>
			<td>Life Technologies</td>
			<td>Q32854</td>
			<td>Invitrogen</td>
		</tr>
		<tr>
			<td>High sensitivity DNA reagent</td>
			<td>Agilent Technologies</td>
			<td>5067-4626</td>
			<td></td>
		</tr>
		<tr>
			<td>High sensitivity DNA chips</td>
			<td>Agilent Technologies</td>
			<td>5067-4626</td>
			<td></td>
		</tr>
		<tr>
			<td>Agilent 2011 Bioanalyzer</td>
			<td>Agilent Technologies</td>
			<td>G2938B</td>
			<td></td>
		</tr>
		<tr>
			<td>Nextera XT DNA sample preparation kit</td>
			<td>Illumina</td>
			<td>FC-131-1024</td>
			<td></td>
		</tr>
		<tr>
			<td>Nextera XT index kit</td>
			<td>Illumina</td>
			<td>FC-131-1001</td>
			<td></td>
		</tr>
		<tr>
			<td>MiSeq reagent kit v2 (500-cycles)</td>
			<td>Illumina</td>
			<td>MS-102-2003</td>
			<td></td>
		</tr>
		<tr>
			<td>Miseq system</td>
			<td>Illumina</td>
			<td>SY-410-1003</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

DNA浓度和质量评估 细菌的DNA浓度范围为0.01至0.11纳克/毫升％的不同流域部位的水样品（ 见表1）。 DNA细菌部分分离了一个260/280和A 260/230比例为1.4〜1.8和0.3〜1.6，分别为。虽然一些甲二百三十〇分之二百六十比率相对较低，没有明显的抑制作用，观察在制备的文库和其它下游应用。这些应用包括PCR和qPCR测试靶向16S rRNA和CPN<...

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

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 ...

automated-gel-size-selection-to-improve-quality-next-generation

Research

JoVE Journal

Environment

10.4K 次观看.  The University of British Columbia. 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. 