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>

<ol>
	<li>Siuda, W., Chrost, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concentration+and+susceptibility+of+dissolved+DNA+for+enzyme+degradation+in+lake+water+-+some+methodological+remarks.">Concentration and susceptibility of dissolved DNA for enzyme degradation in lake water - some methodological remarks.</a> Aquatic Microbial Ecology. 21, 195-201 (2000).</li><li>Ficetola, G. F., Miaud, C., Pompanon, F., Taberlet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species+detection+using+environmental+DNA+from+water+samples.">Species detection using environmental DNA from water samples.</a> Biology letters. 4, 423-425 (2008).</li><li>Liles, M. R., 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=Recovery,+purification,+and+cloning+of+high-molecular-weight+DNA+from+soil+microorganisms.">Recovery, purification, and cloning of high-molecular-weight DNA from soil microorganisms.</a> Applied and environmental microbiology. 74, 3302-3305 (2008).</li><li>Bey, B. S., Fichot, E. B., Dayama, G., Decho, A. W., Norman, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+high+molecular+weight+DNA+from+microbial+mats.">Extraction of high molecular weight DNA from microbial mats.</a> BioTechniques. 49, 631-640 (2010).</li><li>Tebbe, C. C., Vahjen, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interference+of+humic+acids+and+DNA+extracted+directly+from+soil+in+detection+and+transformation+of+recombinant+DNA+from+bacteria+and+a+yeast.">Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast.</a> Applied and environmental microbiology. 59, 2657-2665 (1993).</li><li>Solonenko, S. A., 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=Sequencing+platform+and+library+preparation+choices+impact+viral+metagenomes.">Sequencing platform and library preparation choices impact viral metagenomes.</a> BMC genomics. 14, 320 (2013).</li><li>Aird, 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=Analyzing+and+minimizing+PCR+amplification+bias+in+Illumina+sequencing+libraries.">Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries.</a> Genome biology. 12, R18 (2011).</li><li>Quail, M. A., Swerdlow, H., Turner, D. J., 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=Improved+protocols+for+the+illumina+genome+analyzer+sequencing+system.">Improved protocols for the illumina genome analyzer sequencing system.</a> Current protocols in human genetics / editorial board, Jonathan L. Haines .. [et al.]. 18, 18 (2009).</li><li>Head, S. R., 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=Library+construction+for+next-generation+sequencing:+overviews+and+challenges.">Library construction for next-generation sequencing: overviews and challenges.</a> BioTechniques. 56, 61-64, 66, 68 (2014).</li><li>Dijk, E. L., Jaszczyszyn, Y., Thermes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Library+preparation+methods+for+next-generation+sequencing:+tone+down+the+bias.">Library preparation methods for next-generation sequencing: tone down the bias.</a> Experimental cell research. 322, 12-20 (2014).</li><li>Grunenwald, H., Baas, B., Caruccio, N., Syed, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid,+high-throughput+library+preparation+for+next-generation+sequencing.">Rapid, high-throughput library preparation for next-generation sequencing.</a> Nature Methods. 7, (2010).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning : a laboratory manual. , (2001).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis+for+the+separation+of+DNA+fragments.">Agarose gel electrophoresis for the separation of DNA fragments.</a> Journal of visualized experiments. , (2012).</li><li>Quail, M. A., 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=A+large+genome+center's+improvements+to+the+Illumina+sequencing+system.">A large genome center's improvements to the Illumina sequencing system.</a> Nature methods. 5, 1005-1010 (2008).</li><li>Marine, R., 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=Evaluation+of+a+transposase+protocol+for+rapid+generation+of+shotgun+high-throughput+sequencing+libraries+from+nanogram+quantities+of+DNA.">Evaluation of a transposase protocol for rapid generation of shotgun high-throughput sequencing libraries from nanogram quantities of DNA.</a> Applied and environmental microbiology. 77, 8071-8079 (2011).</li><li>Rohland, N., Reich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cost-effective+high-throughput+DNA+sequencing+libraries+for+multiplexed+target+capture.">Cost-effective high-throughput DNA sequencing libraries for multiplexed target capture.</a> Genome research. 22, 939-946 (2012).</li><li>Lamble, S., 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=Improved+workflows+for+high+throughput+library+preparation+using+the+transposome-based+nextera+system.">Improved workflows for high throughput library preparation using the transposome-based nextera system.</a> Bmc Biotechnology. 13, (2013).</li><li>Picelli, S., 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=Tn5+transposase+and+tagmentation+procedures+for+massively+scaled+sequencing+projects.">Tn5 transposase and tagmentation procedures for massively scaled sequencing projects.</a> Genome research. , (2014).</li><li>Rhodes, J., Beale, M. A., Fisher, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illuminating+Choices+for+Library+Prep:+A+Comparison+of+Library+Preparation+Methods+for+Whole+Genome+Sequencing+of+Cryptococcus+neoformans+Using+Illumina+HiSeq.">Illuminating Choices for Library Prep: A Comparison of Library Preparation Methods for Whole Genome Sequencing of Cryptococcus neoformans Using Illumina HiSeq.</a> PloS One. 9, e113501 (2014).</li></ol>

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><tbody><tr><td>Peristaltic pump Masterflex P/S 1400 Series</td><td>Thermo Scientific&amp;nbsp;</td><td>1400-1620</td><td></td></tr><tr><td>0.2 &amp;micro;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>20x 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&amp;nbsp;ml) tubes</td><td>VWR</td><td>13000-V1-50</td><td>MoBio, Carlsbad, CA</td></tr><tr><td>Vortex-Genie 2, 120&amp;nbsp;V (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&amp;nbsp;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<...

Watch this Scientific Journal Video about Automated Gel Size Selection to Improve the Quality of Next-generation Sequencing Libraries Prepared from Environmental Water Samples at JoVE.com

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

Universidad San Sebastian

Research

JoVE Journal

Environment

10.5K 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. 

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

Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The technique is highly efficient with millions of sequencing reads being produced in a short time span and at relatively low cost. Specifically, targeted NGS is able to focus investigations to genomic regions of particular interest based on the disease of study. Not only does this further reduce costs and increase the speed of the process, but it lessens the computational burden that often accompanies NGS. Although targeted NGS is restricted to certain regions of the genome, preventing identification of potential novel loci of interest, it can be an excellent technique when faced with a phenotypically and genetically heterogeneous disease, for which there are previously known genetic associations. Because of the complex nature of the sequencing technique, it is important to closely adhere to protocols and methodologies in order to achieve sequencing reads of high coverage and quality. Further, once sequencing reads are obtained, a sophisticated bioinformatics workflow is utilized to accurately map reads to a reference genome, to call variants, and to ensure the variants pass quality metrics. Variants must also be annotated and curated based on their clinical significance, which can be standardized by applying the American College of Medical Genetics and Genomics Pathogenicity Guidelines. The methods presented herein will display the steps involved in generating and analyzing NGS data from a targeted sequencing panel, using the ONDRISeq neurodegenerative disease panel as a model, to identify variants that may be of clinical significance.

Targeted next-generation sequencing is a time- and cost-efficient approach that is becoming increasingly popular in both disease research and clinical diagnostics. The protocol described here presents the complex workflow required for sequencing and the bioinformatics process used to identify genetic variants that contribute to disease.

有针对性的下一代测序和生物信息学管道评价宪政疾病的遗传决定因素

Next-generation sequencing (NGS) is quickly revolutionizing how research into the genetic determinants of constitutional disease is performed. The ...

科研

遗传学

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are&#160;therefore potentially replication-competent. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5&#39; and 3&#39; LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required.

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

用于下一代测序的近全长 HIV-1 Proviruses 的扩增

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

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 ...

生物工程

In Vitro Selection of Aptamers to Differentiate Infectious from Non-Infectious Viruses

Virus infections have a major impact on society; most methods of detection have difficulties in determining whether a detected virus is infectious, causing delays in treatment and further spread of the virus. Developing new sensors that can inform on the infectability of clinical or environmental samples will meet this unmet challenge. However, very few methods can obtain sensing molecules that can recognize an intact infectious virus and differentiate it from the same virus that has been rendered non-infectious by disinfection methods. Here, we describe a protocol to select aptamers that can distinguish infectious viruses vs non-infectious viruses using systematic evolution of ligands by exponential enrichment (SELEX). We take advantage of two features of SELEX. First, SELEX can be tailor-made to remove competing targets, such as non-infectious viruses or other similar viruses, using counter selection. Additionally, the whole virus can be used as the target for SELEX, instead of, for example, a viral surface protein. Whole virus SELEX allows for the selection of aptamers that bind specifically to the native state of the virus, without the need to disrupt of the virus. This method thus allows recognition agents to be obtained based on functional differences in the surface of pathogens, which do not need to be known in advance.

We provide a protocol that can be generally applied to select aptamers that bind to infectious viruses only and not to viruses that have been rendered non-infectious by a disinfection method or to any other similar viruses. This opens the possibility of determining infectivity status in portable and rapid tests.

体外选择核酸适配体以区分感染性和非感染性病毒

Virus infections have a major impact on society; most methods of detection have difficulties in determining whether a detected virus is infectious, ...

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

的16S核糖体RNA基因的扩增子的新一代测序

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

生物学

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

增强的减少表示亚硫酸氢盐测序的DNA甲基化的评估，在碱基对决议

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

有针对性的DNA甲基化分析通过新一代测序

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

DamID-seq: Genome-wide Mapping of Protein-DNA Interactions by High Throughput Sequencing of Adenine-methylated DNA Fragments

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is an alternative approach to chromatin immunoprecipitation (ChIP). An expressed fusion protein consisting of the protein of interest and the E. coli DNA adenine methyltransferase can methylate the adenine base in GATC motifs near the sites of protein-DNA interactions. Adenine-methylated DNA fragments can then be specifically amplified and detected. The original DamID assay detects the genomic locations of methylated DNA fragments by hybridization to DNA microarrays, which is limited by the availability of microarrays and the density of predetermined probes. In this paper, we report the detailed protocol of integrating high throughput DNA sequencing into DamID (DamID-seq). The large number of short reads generated from DamID-seq enables detecting and localizing protein-DNA interactions genome-wide with high precision and sensitivity. We have used the DamID-seq assay to study genome-nuclear lamina (NL) interactions in mammalian cells, and have noticed that DamID-seq provides a high resolution and a wide dynamic range in detecting genome-NL interactions. The DamID-seq approach enables probing NL associations within gene structures and allows comparing genome-NL interaction maps with other functional genomic data, such as ChIP-seq and RNA-seq.

We describe herein an assay by coupling DNA adenine methyltransferase identification (DamID) to high throughput sequencing (DamID-seq). This improved method provides a higher resolution and a wider dynamic range, and allows analyzing DamID-seq data in conjunction with other high throughput sequencing data such as ChIP-seq, RNA-seq, etc.

DamID-SEQ:蛋白质-DNA相互作用的全基因组映射腺嘌呤甲基化的DNA片段通过高通量测序

The DNA adenine methyltransferase identification (DamID) assay is a powerful method to detect protein-DNA interactions both locally and genome-wide. It is ...

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

高效的核酸提取和16S rRNA基因序列的细菌群落特征

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...