Journal article
Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing
Journal of visualized experiments : JoVE, Vol.152, e59980
10/15/2019
DOI: 10.3791/59980
PMCID: PMC6945761
PMID: 31680682
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.
Details
- Title: Subtitle
- Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing
- Creators
- Shailesh K Shahi - Department of Pathology, University of IowaKasra Zarei - Medical Scientist Training Program, University of IowaNatalya V Guseva - Department of Pathology, University of IowaAshutosh K Mangalam - Department of Pathology, University of Iowa; Medical Scientist Training Program, University of Iowa; Graduate Program in Immunology, University of Iowa; Graduate Program in Molecular Medicine, University of Iowa; ashutosh-mangalam@uiowa.edu
- Resource Type
- Journal article
- Publication Details
- Journal of visualized experiments : JoVE, Vol.152, e59980
- DOI
- 10.3791/59980
- PMID
- 31680682
- PMCID
- PMC6945761
- NLM abbreviation
- J Vis Exp
- ISSN
- 1940-087X
- eISSN
- 1940-087X
- Publisher
- United States
- Grant note
- R01 AI137075 / NIAID NIH HHS P30 ES005605 / NIEHS NIH HHS
- Language
- English
- Date published
- 10/15/2019
- Academic Unit
- Pathology; Iowa Neuroscience Institute
- Record Identifier
- 9984070221002771
Metrics
44 Record Views