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

This work was supported in part by the I-CORE program (grants No. 41/11), the Israel Science Foundation (grant No. 908/15), and the European Crohn&#39;s and Colitis Organization (ECCO).

Ethical approval for the study was granted by the Sheba Local Research Ethics Committee and all methods were performed in accordance with the relevant guidelines and regulations. The protocol received a patient consent exception from the local Ethical Review Board, since the fecal material that were used were already submitted to the microbiology core as part of clinical workup and without identifiable patient information other than age, gender, and microbial results. Written, informed consent was obtained from healthy v...

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16S rRNA-amplicon and metagenomics shotgun sequencing have gained popularity in clinical microbiology applications21,22,23. These techniques are advantageous in their increased ability to capture culturable and non-culturable taxa, providing data about the relative abundance of the pathogenic inoculum, and their ability to identify more precisely a polymicrobial infectious fingerprint24. The advances in t...

Concentrated efforts have been made to better understand microbiome diversity and abundance, as another aspect of capturing difference and similarities between individuals in healthy and pathological conditions. Age2,3, geography4, lifestyle5,6, and illness5 were shown to be associated with the composition of the gut microbiome, but many conditions and populations have not yet been fully characterized. Recently it has been reported that the microbiome can be modified for therapeutic applications7,8,9. Therefore, additional insight into the relationship between various physiological conditions and the microbial composition is the first step toward optimization of potential future modifications.
The traditional microbial culture methods are limited by low yields10,11, and are conceptualized as a binary state where a bacteria is either present in the gut or not. High-throughput DNA-based sequencing has revolutionized microbial ecology, enabling the capture of all members of the microbial community. However, sequence read length and quality remain significant barriers to accurate taxonomy assignment12. Furthermore, high-throughput based experiments may suffer from batch effects, where measurements are affected by non-biological or non-scientific variables13. In recent years, several programs have been established to study the human microbiome, including the American Gut project, the United States (US) Human Microbiome Project, and the United Kingdom (UK) MetaHIT project. These initiatives have generated vast amounts of data that are not easily comparable due to a lack of consistency in their approaches. A variety of international projects such as the International Human Microbiome Consortium, the International Human Microbiome Standards project, and the National Institute of Standards and Technology (NIST) attempted to address some of these issues14, and developed standards for microbiome measurements which should enable the achievement of reliable reproductive results. Described here is an integrated protocol of several broadly used methods15,16 for 16S rRNA high-throughput sequencing (16S-seq) starting from fecal sample collection thru data analyses. The protocol describes a column-free PCR approach, originally designed for direct extraction of plant DNA16, to enable the simultaneous handling of large numbers of fecal samples in a relatively short time with high quality amplified DNA for targeted sequencing of the microbial variable V4 region on a common sequencing platform. This protocol aims to guide scientists interested in initiating the use of 16S rRNA-amplicon sequencing in a robust, reproductive, easy to use, detailed way, using important controls. Having a guided and detailed step-by step protocol may minimize batch effect and thus will allow more comparable sequencing results between labs.

<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

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. Deviations from what is considered a healthy microbiota composition (dysbiosis) may impair vital functions leading to pathologic conditions. Recent and ongoing research efforts have been directed toward the characterization of associations between microbial composition and human health and disease.
Advances in high-throughput sequencing technologies enable characterization of the gut microbial composition. These methods include 16S rRNA-amplicon sequencing and shotgun sequencing. 16S rRNA-amplicon sequencing is used to profile taxonomical composition, while shotgun sequencing provides additional information about gene predictions and functional annotation. An advantage in using a targeted sequencing method of the 16S rRNA gene variable region is its substantially lower cost compared to shotgun sequencing. Sequence differences in the 16S rRNA gene are used as a microbial fingerprint to identify and quantify different taxa within an individual sample.
Major international efforts have enlisted standards for 16S rRNA-amplicon sequencing. However, several studies report a common source of variation caused by batch effect. To minimize this effect, uniformed protocols for sample collection, processing, and sequencing must be implemented. This protocol proposes the integration of broadly used protocols starting from fecal sample collection to data analyses. This protocol includes a column-free, direct-PCR approach that enables simultaneous handling and DNA extraction of large numbers of fecal samples, along with PCR amplification of the V4 region. In addition, the protocol describes the analysis pipeline and provides a script using the latest version of QIIME (QIIME 2 version 2017.7.0 and DADA2). This step-by-step protocol is aimed to guide those interested in initiating the use of 16S rRNA-amplicon sequencing in a robust, reproductive, easy to use, detailed way.

A schematic illustration of the protocol is shown in Figure 1.
We have prospectively collected stool samples from hospitalized patients with suspected infectious diarrhea. Those samples were submitted to the Clinical Microbiology Lab at the Sheba Medical Center between February and May 2015, as was previously described1. Stool samples were subjected to conventional microbiolo...

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

This manuscript describes a detailed standardized protocol of high-throughput 16S rRNA-amplicon sequencing. The protocol introduces an integrated, uniformed, feasible, and inexpensive protocol starting from fecal sample collection through data analyses. This protocol enables analysis of large numbers of samples with rigorous standards and several controls.

The human intestinal microbiome plays a central role in protecting cells from injury, in processing energy and nutrients, and in promoting immunity. ...

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