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. ...

The overall goal of this high-throughput 16S rRNA-Amplicon Sequencing is to characterize the global microbiome composition starting from fecal samples. This method can help answer key questions about global microbial composition associated with many field of interests such as metabolism, digestion, and the gut and systemic inflammation. The main advantage of this technique is that it provides a census of aerobic and anaerobic microbial communities present in the fecal sample including those bacteria that are difficult to culture.This method can simultaneously handle large numbers of fecal samples thanks to rapid colon-free processing pump line and it resulted in robust and reproducible 16S rRNA-Amplicon Sequencing. For this protocol, have frozen feces samples that are roughly the size of a pencil eraser. The samples should be stored in two milliliter tubes with the trimmed off tip of the collection swab.Include negative controls consisting of swabs with no feces and internal duplicates controls and have sample duplicates from the sample original stool to serve as an internal control. Begin with thawing the extraction and dilution solutions at room temperature along with the fecal samples and control samples. Once thawed, add 250 microliters of extraction solution to each sample tube and mix the feces into solution using a vortex.Introduce to buffer-only negative controls at this point. Next, heat the samples for 10 minutes in a boiling water bath. After the incubation in boiling water, add 250 microliters of dilution solution to each sample and vortex the samples to mix.Then store the sample tubes at four degrees Celsius until PCR can be performed. Set up the PCRs at a pre-PCR workstation that has been cleansed of potential DNA contaminants like templates and Amplicons. Label the primers according to their bar code and dilute them in double distilled water to a concentration of 50 micromolar.Store the stocks of diluted primer at minus 20 degrees Celsius and thaw them completely before use. For each sample, prepare a 96-well plate for 32 reactions in triplicate. After warming to room temperature, dilute aliquots of the forward primer and the 32 reverse primers to five micromolar for the PCR.Then prepare enough base PCR mix for 100 reactions. Combine 100 microliters of five micromolar forward primer with one milliliter of 2X PCR master mix and 400 microliters of double distilled water. Vortex this mixture and then load 15 microliters of this mixture into each of the 96 wells.Next, load sets of three wells each with one microliter of five micromolar reverse primer. Thus, load all 32 different reverse index into the plate. Then to every plate well, add four microliters of the extracted DNA sample.This is done on a clean bench. Now run the PCR with a three-minute initial denaturation followed by 35 cycles with one minute of denaturation and kneeling at 55 degrees Celsius and one minute of extension. Finish with a 10-minute final extension.After the reaction is completed, work in a post-PCR dedicated workbench. There, combine each set of triplicate PCR products into a single tube for 60 microliters per sample. Then run four microliters of each sample on an ethidium bromide stained 1%agarose gel.The positive Amplicons will appear between 375 and 425 base pairs. The negative controls are shown and are processed along with the study positive Amplicons. Quantify the DNA concentrations of the positive reaction products according to manufacturer instructions.Then combine 500 nanograms of DNA from each of the products into a single tube. Vortex this mixture and then run out 200 microliters on a 1%gel as before. Now extract the bands from the gel between 375 and 425 base pairs.Next, prepare the library for sequencing. Measure the concentration using a highly sensitive double-stranded DNA detecting kit according to manufacturer instructions followed by use of a highly sensitive separation and analysis kit. Then dilute the library to seven picomolar and combine it four to one with a control library.Now that the sample is ready for sequencing, transfer it to the sequencing machine. The described method for a library preparation from stool samples was used to analyze the microbiome of hospitalized patients with suspected infectious diarrhea. In parallel, stool samples were also subjected to traditional culturing in a microbiology lab.Stool samples from healthy adults were sequenced for comparison. Several negative control samples were included, PCR without template, PCR on clean and sterile swabs in extraction and dilution solution and PCR on mixed extraction and dilution solutions. Each of these had less than 118 total reads which were mainly Mycoplasm taxa.Standard samples averaged greater than 10, 000 reads with no identification of Mycoplasma after quality control. Samples consistency and variability was measured using 16 stool samples prepared with two different bar coded primers to hence construct two different libraries from each sample. Half of the samples were positive for Campylobacter salmonella or Shigella in traditional culturing technique.Using the 16S sequencing results, an area plot at the phylo level shows consistency between libraries made from the same samples independent of the traditional culturing results. From this pool of 16 paired samples, principal coordinate analysis revealed that the sample pairs are closely aligned and that the positive culture samples had a significantly different PC1 value from the negative culture samples. Further analysis of the samples revealed that high levels of Proteobacteria were only found in the hospitalized patients.After watching this video, you should have a good understanding of how to simultaneously process a large number of fecal samples using a colon-free DNA extraction and PCR to generate bar coded sequencing libraries of the microbial variable V4 region. While attempting this procedure, it's important to remember to work in an organized and clean environment especially when preparing the libraries. Don't forget that working with human stool samples is hazardous and personal protection equipment should always be worn while performing this procedure.

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