Phyloseq Tutorial

Phyloseq is an R package designed for the object-oriented representation and analysis of microbiome census data. It is a powerful tool that integrates different types of data with methods from various fields such as ecology, genetics, phylogenetics, multivariate statistics, visualization, and testing. The package is particularly useful for researchers dealing with the complexities of microbial community analysis through DNA sequencing.

The main goal of Phyloseq is to provide a standardized framework for handling and analyzing high-throughput microbiome census data. It allows for the import of data from common formats and supports a wide range of analysis techniques. These include calibration, filtering, subsetting, agglomeration, multi-table comparisons, diversity analysis, parallelized Fast UniFrac, ordination methods, and the creation of publication-quality graphics.

One of the key features of Phyloseq is its ability to facilitate reproducible research. This is achieved by making it easy to document, share, and modify analyses. The package is open-source and freely available on the web from both GitHub and Bioconductor, encouraging collaborative development and use.

Phyloseq emphasizes preprocessing tools to reduce the opacity and idiosyncrasies often encountered by investigators in this field. By providing a unified and integrated representation of data, Phyloseq enables the use of a large number of open-source analysis techniques available in R, making it easier for researchers to conduct robust and interactive analyses.


To install Phyloseq, you will need to have R installed on your system. Phyloseq is available through Bioconductor, which is a repository for R packages that are used in bioinformatics. To install Phyloseq, you can use the following commands in your R console:

if (!requireNamespace("BiocManager", quietly = TRUE))


This will install Phyloseq along with its dependencies, which include other R packages like ade4, ape, Biostrings, foreach, ggplot2, igraph, multtest, picante, plyr, reshape, RJSONIO, scales, and vegan.

Quick Start

Once Phyloseq is installed, you can quickly start by loading the package into your R session:


You can then import your microbiome census data from various formats supported by Phyloseq. The package provides functions to read data from popular OTU clustering pipelines such as QIIME, mothur, the RDP-pipeline, Pyrotagger, and the biom-format.

Code Examples Of Popular Commands

Here are five popular commands that you can use with Phyloseq to analyze your microbiome data:

  1. Importing Data:
    To import data from a QIIME pipeline, you can use the import_qiime function:

    qiime_data <- import_qiime(OTUfile = "otu_table.txt", mapfile = "mapping_file.txt")
  2. Filtering Data:
    You can filter your data based on sample data or OTU abundance using the prune_samples and prune_taxa functions:

    filtered_data <- prune_samples(sample_sums(qiime_data) > 1000, qiime_data)
  3. Diversity Analysis:
    Phyloseq allows you to calculate various diversity indices. For example, to calculate the Shannon diversity index, you can use:

    shannon_diversity <- estimate_richness(filtered_data, measures = "Shannon")
  4. Ordination Methods:
    You can perform ordination methods such as Principal Coordinates Analysis (PCoA) using the ordinate function:

    pcoa_results <- ordinate(filtered_data, method = "PCoA", distance = "Jaccard")
  5. Creating Graphics:
    Phyloseq integrates with ggplot2 to create publication-quality graphics. For instance, to plot the PCoA results, you can use:

    plot_pcoa <- plot_ordination(filtered_data, pcoa_results, color = "SampleType") + geom_point(size=3)

These commands are just the tip of the iceberg when it comes to the capabilities of Phyloseq. The package includes extensive documentation and examples that can help you explore its full potential for your specific research needs.

In conclusion, Phyloseq is a comprehensive tool for the analysis of microbiome census data within the R environment. Its emphasis on reproducibility, combined with its powerful analysis and graphics capabilities, makes it an invaluable resource for researchers in the field of microbial community analysis.