Name: guided protocol for fecal microbial characterization by 16s rrna-amplicon sequencing
Uploaded: 2018-03-19T13:30:00
Description: Watch this Scientific Journal Video about Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing at JoVE.com

这项工作的一部分是由核心项目 (赠款 41/11)、以色列科学基金会 (908/15 号赠款) 和欧洲克罗恩病组织 (ECCO) 支持。

该研究的伦理批准是由示巴地方研究伦理学委员会批准的, 所有方法都是按照相关的指导方针和条例进行的。该议定书收到了当地道德审查委员会的病人同意例外, 因为使用的粪便材料已经作为临床检查的一部分提交给微生物学核心, 而没有其他人的年龄以外的可识别的病人信息,性别和微生物结果。从健康志愿者那里获得书面、知情的同意, 机构审查委员会批准了这项研究。其中一些示例已包含在?...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+concepts+in+diagnostics+for+infectious+diarrhea.">New concepts in diagnostics for infectious diarrhea.</a> Mucosal Immunology. 6, 876-885 (2013).</li><li>Bokulich, N. 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=Quality-filtering+vastly+improves+diversity+estimates+from+Illumina+amplicon+sequencing.">Quality-filtering vastly improves diversity estimates from Illumina amplicon sequencing.</a> Nature Methods. 10, 57-59 (2013).</li><li>Leek, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tackling+the+widespread+and+critical+impact+of+batch+effects+in+high-throughput+data.">Tackling the widespread and critical impact of batch effects in high-throughput data.</a> Nature Reviews Genetics. 11, (2010).</li><li>Dubilier, N., McFall-Ngai, M., Zhao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Create+a+global+microbiome+effort.">Create a global microbiome effort.</a> Nature. 526, 631-634 (2015).</li><li>Caporaso, J. G., 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=Global+patterns+of+16S+rRNA+diversity+at+a+depth+of+millions+of+sequences+per+sample.">Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample.</a> PNAS. 108, 4516-4522 (2011).</li><li>Flores, G. E., Henley, J. B., Fierer, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+direct+PCR+approach+to+accelerate+analyses+of+human-associated+microbial+communities.">A direct PCR approach to accelerate analyses of human-associated microbial communities.</a> PloS One. 7, (2012).</li><li>Caporaso, J. G., 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=QIIME+allows+analysis+of+high-throughput+community+sequencing+data.">QIIME allows analysis of high-throughput community sequencing data.</a> Nature Methods. 7, 335-336 (2010).</li><li>Callahan, B. 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=DADA2:+High-resolution+sample+inference+from+Illumina+amplicon+data.">DADA2: High-resolution sample inference from Illumina amplicon data.</a> Nature Methods. 13, 581-583 (2016).</li><li>Lozupone, C., Knight, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UniFrac:+A+new+phylogenetic+method+for+comparing+microbial+communities.">UniFrac: A new phylogenetic method for comparing microbial communities.</a> Applied and Environmental Microbiology. 71, 8228-8235 (2005).</li><li>Lozupone, C., Lladser, M. E., Knights, D., Stombaugh, J., Knight, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UniFrac:+An+effective+distance+metric+for+microbial+community+comparison.">UniFrac: An effective distance metric for microbial community comparison.</a> The ISME Journal. 5, 169-172 (2011).</li><li>Srinivasan, 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=Use+of+16S+rRNA+gene+for+identification+of+a+broad+range+of+clinically+relevant+bacterial+pathogens.">Use of 16S rRNA gene for identification of a broad range of clinically relevant bacterial pathogens.</a> PloS One. 10, e0117617 (2015).</li><li>Hiergeist, A., Gl&#228;sner, J., Reischl, U., Gessner, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyses+of+intestinal+microbiota:+culture+versus+sequencing.">Analyses of intestinal microbiota: culture versus sequencing.</a> ILAR Journal. 56, 228-240 (2015).</li><li>Didelot, X., Bowden, R., Wilson, D. J., Peto, T. E. A., Crook, 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=Transforming+clinical+microbiology+with+bacterial+genome+sequencing.">Transforming clinical microbiology with bacterial genome sequencing.</a> Nature Reviews Genetics. 13, 601-612 (2012).</li><li>Salipante, S. 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=Rapid+16S+rRNA+next-generation+sequencing+of+polymicrobial+clinical+samples+for+diagnosis+of+complex+bacterial+infections.">Rapid 16S rRNA next-generation sequencing of polymicrobial clinical samples for diagnosis of complex bacterial infections.</a> PloS One. 8, e65226 (2013).</li><li>Caporaso, J. 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16S rRNA-扩增子和 metagenomics 猎枪测序在临床微生物学应用中得到了普及21, 2223.这些技术有利于提高捕获可培养和非可培养类群的能力, 提供有关致病接种的相对丰度的数据, 以及它们识别更精确的 polymicrobial 传染性的能力。指纹24。微生物研究领域的进展产生了大量的数据, 由于各种方法缺乏一致性, 因此难以比较。最近几年, 一些财团试...

为了更好地了解微生物的多样性和丰度, 已作出了集中努力, 作为在健康和病理条件下捕捉个体差异和相似性的另一个方面。年龄2,3, 地理4, 生活方式5,6, 疾病5被证明与肠道微生物组的组成有关, 但许多条件和人口尚未完全特点.最近据报道, 微生物菌群可以被修改为治疗性应用7,8,9。因此, 进一步了解各种生理条件和微生物组成之间的关系, 是迈向优化未来潜在的修改的第一步。
传统的微生物培养方法受低产量的限制10,11, 并被概念化为一个二进制状态, 其中细菌是存在于肠道或没有。高通量 DNA 测序已经彻底改变了微生物生态学, 使微生物群落的所有成员都能被捕获。但是, 序列读取长度和质量仍然是精确分类分配12的重要障碍。此外, 基于高通量的实验可能会受到批处理效果的影响, 其中测量受非生物或非科学变量13的影响。近年来, 已经建立了一些研究人类微生物菌群的方案, 包括美国肠道项目、美国 (美国) 人类微生物学项目和联合王国 (英国) MetaHIT 项目。这些倡议产生了大量的数据, 由于它们的做法缺乏连贯性, 因此难以比较。各种国际项目, 如国际人类微生物菌群联合会、国际人类微生物菌群标准项目以及国家标准和技术研究所 (NIST) 试图解决其中的一些问题14, 并制定了微生物菌群测量标准, 这将有助于实现可靠的生殖结果。这里描述的是一些广泛使用的方法15、16的集成协议, 用于 16S rRNA 高通量测序 (十六年代序列), 从粪便样本收集通过数据分析开始。该议定书描述了一种无柱 PCR 方法, 最初设计用于直接提取植物 DNA16, 以便在相对较短的时间内同时处理大量的粪便样本, 并以高质量的扩增 DNA 为靶向常规测序平台上微生物变量 V4 区域的测序。该议定书的目的是引导感兴趣的科学家开始使用 16S rRNA 扩增子测序, 在健壮, 生殖, 易于使用, 详细的方式, 使用重要的控制。具有引导和详细的分步协议可能会减少批处理效果, 从而允许在实验室之间进行更可比较的排序结果。

<table>
	<tbody>
		<tr>
			<td>Primers</td>
			<td>Integrated DNA Technologies (IDT)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Extraction solution</td>
			<td>Sigma-Aldrich</td>
			<td>E7526</td>
			<td></td>
		</tr>
		<tr>
			<td>Dilution solution</td>
			<td>Sigma-Aldrich</td>
			<td>D5688</td>
			<td></td>
		</tr>
		<tr>
			<td>Kapa HiFi HotStart ReadyMix PCR Kit</td>
			<td>KAPABIOSYSTEMS</td>
			<td>KK2601</td>
			<td>PCR Master mix</td>
		</tr>
		<tr>
			<td>Quant-iT PicoGreen dsDNA Reagent kit</td>
			<td>Invitrogen</td>
			<td>P7589</td>
			<td>dsDNA quantify reagent</td>
		</tr>
		<tr>
			<td>MinElute Gel extraction kit</td>
			<td>Qiagen</td>
			<td>28606</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Amresco</td>
			<td>0710-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra Pure Water Dnase and Rnase Free</td>
			<td>Biological Industries</td>
			<td>01-866-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit dsDNA HS assay kit</td>
			<td>Molecular probes</td>
			<td>Q32854</td>
			<td>dsDNA detecting kit</td>
		</tr>
		<tr>
			<td>High Sensitivity D1000</td>
			<td>Agilent Technologies</td>
			<td>Screen Tape 5067-5582</td>
			<td>separation and analysis</td>
		</tr>
		<tr>
			<td>Screen Tape Assay</td>
			<td>Agilent Technologies</td>
			<td>Reagents 5067-5583</td>
			<td>for DNA libraries</td>
		</tr>
		<tr>
			<td>PhiX Control v3</td>
			<td>Illumina</td>
			<td>15017666</td>
			<td>control library</td>
		</tr>
		<tr>
			<td>MiSeq Reagent Kit v2 (500 cycle)</td>
			<td>Illumina</td>
			<td>MS-102-2003</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>Amresco</td>
			<td>E406-10mL-TAM</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL collection tubes</td>
			<td>SARSTEDT</td>
			<td>72.695.400</td>
			<td>Safe Seal collection tubes</td>
		</tr>
		<tr>
			<td>Plastic stick swab in PP test tube</td>
			<td>STERILE INTERIOR</td>
			<td>23117</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PCR Machine</td>
			<td>Applied Biosystems</td>
			<td>2720 Thermal Cycler</td>
			<td></td>
		</tr>
		<tr>
			<td>Sequncing Machine</td>
			<td>Illumina</td>
			<td>Miseq</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR workstation</td>
			<td>Biosan</td>
			<td>UV-cleaner</td>
			<td></td>
		</tr>
		<tr>
			<td>scissors</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>vortexer</td>
			<td>Scientific Industries</td>
			<td>Vortex-Genie 2</td>
			<td></td>
		</tr>
	</tbody>
</table>

guided protocol for fecal microbial characterization by 16s rrna-amplicon sequencing

人体肠道菌群在保护细胞免遭伤害、处理能量和养分以及促进免疫力方面起着重要作用。从被认为是健康的微生物组成 (dysbiosis) 的偏差可能会损害导致病理状况的重要功能。最近和正在进行的研究工作已被用于描述微生物组成与人类健康和疾病之间的关联。
高通量测序技术的进展使肠道微生物成分的特征得以表征。这些方法包括 16S rRNA 扩增子测序和猎枪测序。16S rRNA 扩增子测序用于分析分类成分, 而猎枪测序则提供有关基因预测和功能注释的附加信息。使用 16S rRNA 基因可变区的目标测序方法的优点是它的成本与猎枪测序相比大大降低。在 16S rRNA 基因中的序列差异被用作微生物指纹, 用来识别和量化个体样本中不同的分类群。
主要的国际努力已登记了 16S rRNA 扩增子测序标准。然而, 一些研究报告了一个常见的变异来源, 由间歇效应引起。为了最小化这种效果, 必须实现示例收集、处理和排序的统一协议。该协议提出了从粪便样本采集开始到数据分析的广泛应用协议的集成。该协议包括无列的直接 pcr 方法, 能够同时处理和 DNA 提取大量粪便样本, 以及 V4 区域的 PCR 扩增。此外, 该协议描述分析管道, 并提供一个脚本使用最新版本的 QIIME (QIIME 2 版 2017.7. 0 和 DADA2)。这一步步协议的目的是引导那些感兴趣的人开始使用 16S rRNA 扩增子测序的健壮, 繁殖, 易于使用, 详细的方式。

该协议的示意图说明显示在图 1中。
我们有前瞻性收集的粪便样本, 从住院病人疑似传染性腹泻。这些样本是在2月至2015年5月期间提交给示巴医疗中心的临床微生物学实验室的, 如前所述的1。粪便标本在临床微生物学实验室进行常规微生物培养, 并在十六年代平行的范围内进行广泛的高?...

Watch this Scientific Journal Video about Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing at JoVE.com

Guided Protocol for Fecal Microbial Characterization by 16S rRNA-Amplicon Sequencing

这篇手稿描述了一个详细的标准化协议高通量 16S rRNA 扩增子测序。该协议引入了一个集成的, 统一的, 可行的, 廉价的协议, 从粪便样本收集, 通过数据分析。该协议允许分析大量的样本, 严格的标准和几个控制。

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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 ...

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

经过一蛋白饱和诱变图书馆的功能评价规范利用高通量测序

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...

Interactome-Seq: A Protocol for Domainome Library Construction, Validation and Selection by Phage Display and Next Generation Sequencing

Interactome: Domainome 图书馆建设、噬菌体展示和下一代测序的验证与选择协议

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to ...

Tick Microbiome Characterization by Next-Generation 16S rRNA Amplicon Sequencing

下一代 16S rRNA 扩增子测序的蜱菌群鉴定

In recent decades, vector-borne diseases have re-emerged and expanded at alarming rates, causing considerable morbidity and mortality worldwide. Effective ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

使用两步PCR和下一代16S rRNA基因测序进行微生物群分析

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...