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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

Abstract

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome’s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Introduction

Microbiota refers to a collection of microorganisms (bacteria, viruses, archaea, bacteriophages, and fungi) living in a particular environment, and the microbiome refers to the collective genome of resident microorganisms. As bacteria are one of the most abundant microbes in humans and mice, this study is focused only on bacterial profiling. The human gut is colonized by trillions of bacteria and hundreds of bacterial strains1. The normal gut microbiota plays a vital role in maintaining a healthy state in the host by regulating functions (i.e., maintenance of an intact intestinal barrier, food metabolism, energy homeostasis, inhibition of colon....

Protocol

The protocol was approved by the Institutional Animal Care and Use Committee of the University of Iowa.

1. Fecal Sample Collection and Handling

  1. Sterilize the divider boxes (see Table of Materials, Supplementary Figure 1) with 70% ethanol.
  2. Pre-label microcentrifuge tubes (one per mouse) with the sample ID and treatment group (if applicable).
  3. Place the mice in sterilized divider boxes and allow them to defecate normally for up to .......

Representative Results

As MHC class II molecules (HLA in humans) are central players in the adaptive immune response and show strong associations with a predisposition to MS24,25,26, it was hypothesized that the HLA class II molecule would influence gut microbial composition. Therefore, mice lacking the MHC class II gene (AE.KO) or expressing human HLA-DQ8 gene (HLA-DQ8) were utilized to understand the importance of HLA class II molecules in shaping t.......

Discussion

The described protocol is simple, with easy-to-follow steps to perform microbiome profiling using 16S rRNA metagenomic sequencing from a large number of biospecimens of interest. Next-generation sequencing has transformed microbial ecology studies, especially in human and pre-clinical disease models31,32. The main advantage of this technique is its ability to successfully analyze complex microbial compositions (culturable and non-culturable microbes) in a given b.......

Acknowledgements

The authors acknowledge funding from the NIAID/NIH (1R01AI137075-01), the Carver Trust Medical Research Initiative Grant, and the University of Iowa Environmental Health Sciences Research Center, NIEHS/NIH (P30 ES005605).

....

Materials

NameCompanyCatalog NumberComments
1.5 ml Natural Microcentriguge TubeUSA, Scientific1615-5500Fecal collection
3M hand applicator squeegee PA1-G3M, MN, US7100038651Squeeger for proper sealing of PCR Plate
Agencourt AMPure XPBeckman Coulter, IN, USAA63880PCR Purification, NGS Clean-up, PCR clean-up
Agilent DNA 1000 REAGENTAgilent Technologies, CA, USA5067-1504DNA quantification and quality control
Bioanalyzer DNA 1000 chipAgilent Technologies, CA, USA5067-1504DNA quantification and quality control
Index Adopter Replacement CapsIllumina, Inc., CA, USA15026762New cap for Index 1 and 2 adopter primer
DNeasy PowerLyzer PowerSoil KitMoBio now part of QIAGEN, Valencia, CA, USA12855-100DNA isolation
KAPA HiFi HotStart ReadyMiX (2X)Kapa Biosystem, MA, USAKK2602PCR ready mix for Amplicon PCR1 and Indexed PCR2
Lewis Divider BoxesLewis Bins, WI, USND03080Fecal collection
Magnetic standNew England BioLabs, MA, USAS1509SFor PCR clean-up
MicroAmp Fast Optical 96-Well Reaction PlateApplied Biosystems, Thermo Fisher Scientific, CA, USA4346906PCR Plate
MicroAmp Optical Adhesive FilmApplied Biosystems, Thermo Fisher Scientific, CA, USA4311971PCR Plate Sealer
Microfuge 20 CentrifugeBeckman Coulter, IN, USAB30154Centrifuge used for DNA isolation
MiSeq Reagent Kit (600 cycles)v.3Illumina, Inc., CA, USAMS-102-3003For MiSeq Sequencing
Nextera XT DNA Library Preparation KitIllumina, Inc., CA, USAFC-131-100116S rRNA DNA Library Preparation
Reagent Reservoirs Multichannel TraysASI, FL,USARS71-1For Pooling of PCR2 Product
Plate CetrifugeThermo Fisher Scientific, CA, USA75004393For PCR reagent mixing and removing air bubble from Plate
PhiX ControlIllumina, Inc., CA, USAFC-110-3001For MiSeq Sequencing control
Microbiome DNA Purification KitThermo Fisher Scientific, CA, USAA29789For purification of PCR1 product
PowerLyzer 24 HomogenizerOmni International, GA, USA19-001Bead beater for DNA Isolation
Qubit dsDNA HS (High Sensitivity) assay kitThermo Fisher Scientific, CA, USAQ32854DNA quantification
TruSeq Index Plate FixtureIllumina, Inc., CA, USAFC-130-1005For Arranging of the index primers
Vertical Dividers (large)Lewis Bins, WI, USDV-2280Fecal collection
Vertical Dividers (small)Lewis Bins, WI, USDV-1780Fecal collection

References

  1. Lynch, S. V., Pedersen, O. The Human Intestinal Microbiome in Health and Disease. New England Journal of Medicine. 375 (24), 2369-2379 (2016).
  2. Blacher, E., Levy, M., Tatirovsky, E., Elinav, E. Microbiome-Modulated Metabolites at the....

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MicrobiomeMicrobiota16S RRNA Gene SequencingNext generation SequencingFecal SampleDNA ExtractionPCRGut MicrobiomeMicrobial Profiling

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