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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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. Age^2,3, geography⁴, lifestyle^5,6, 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 applications

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Protokół

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 volunteers and the Institutional Review Board approved the study. Some of those samples have already been included in a previous analysis

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Wyniki

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.......

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Dyskusje

16S rRNA-amplicon and metagenomics shotgun sequencing have gained popularity in clinical microbiology applications^21,22,23. 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.......

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Materiały

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