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

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 .......

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 models^31,32. The main advantage of this technique is its ability to successfully analyze complex microbial compositions (culturable and non-culturable microbes) in a given b.......

Materials

Name	Company	Catalog Number	Comments
1.5 ml Natural Microcentriguge Tube	USA, Scientific	1615-5500	Fecal collection
3M hand applicator squeegee PA1-G	3M, MN, US	7100038651	Squeeger for proper sealing of PCR Plate
Agencourt AMPure XP	Beckman Coulter, IN, USA	A63880	PCR Purification, NGS Clean-up, PCR clean-up
Agilent DNA 1000 REAGENT	Agilent Technologies, CA, USA	5067-1504	DNA quantification and quality control
Bioanalyzer DNA 1000 chip	Agilent Technologies, CA, USA	5067-1504	DNA quantification and quality control
Index Adopter Replacement Caps	Illumina, Inc., CA, USA	15026762	New cap for Index 1 and 2 adopter primer
DNeasy PowerLyzer PowerSoil Kit	MoBio now part of QIAGEN, Valencia, CA, USA	12855-100	DNA isolation
KAPA HiFi HotStart ReadyMiX (2X)	Kapa Biosystem, MA, USA	KK2602	PCR ready mix for Amplicon PCR1 and Indexed PCR2
Lewis Divider Boxes	Lewis Bins, WI, US	ND03080	Fecal collection
Magnetic stand	New England BioLabs, MA, USA	S1509S	For PCR clean-up
MicroAmp Fast Optical 96-Well Reaction Plate	Applied Biosystems, Thermo Fisher Scientific, CA, USA	4346906	PCR Plate
MicroAmp Optical Adhesive Film	Applied Biosystems, Thermo Fisher Scientific, CA, USA	4311971	PCR Plate Sealer
Microfuge 20 Centrifuge	Beckman Coulter, IN, USA	B30154	Centrifuge used for DNA isolation
MiSeq Reagent Kit (600 cycles)v.3	Illumina, Inc., CA, USA	MS-102-3003	For MiSeq Sequencing
Nextera XT DNA Library Preparation Kit	Illumina, Inc., CA, USA	FC-131-1001	16S rRNA DNA Library Preparation
Reagent Reservoirs Multichannel Trays	ASI, FL,USA	RS71-1	For Pooling of PCR2 Product
Plate Cetrifuge	Thermo Fisher Scientific, CA, USA	75004393	For PCR reagent mixing and removing air bubble from Plate
PhiX Control	Illumina, Inc., CA, USA	FC-110-3001	For MiSeq Sequencing control
Microbiome DNA Purification Kit	Thermo Fisher Scientific, CA, USA	A29789	For purification of PCR1 product
PowerLyzer 24 Homogenizer	Omni International, GA, USA	19-001	Bead beater for DNA Isolation
Qubit dsDNA HS (High Sensitivity) assay kit	Thermo Fisher Scientific, CA, USA	Q32854	DNA quantification
TruSeq Index Plate Fixture	Illumina, Inc., CA, USA	FC-130-1005	For Arranging of the index primers
Vertical Dividers (large)	Lewis Bins, WI, US	DV-2280	Fecal collection
Vertical Dividers (small)	Lewis Bins, WI, US	DV-1780	Fecal collection