title: Bioconductor Workflow for Microbiome Data Analysis: from raw reads to community analyses.
abstract: High-throughput sequencing of PCR-amplified taxonomic markers (like the 16S rRNA gene) has enabled a new level of analysis of complex bacterial communities known as microbiomes. Many tools exist to quantify and compare abundance levels or OTU composition of communities in different conditions. The sequencing reads have to be denoised and assigned to the closest taxa from a reference database. Common approaches use a notion of 97% similarity and normalize the data by subsampling to equalize library sizes. In this paper, we show that statistical models allow more accurate abundance estimates. By providing a complete workflow in R, we enable the user to do sophisticated downstream statistical analyses, whether parametric or nonparametric. We provide examples of using the R packages dada2, phyloseq, DESeq2, ggplot2 and vegan to filter, visualize and test microbiome data. We also provide examples of supervised analyses using random forests and nonparametric testing using community networks and the ggnetwork package.
authors.affiliation: Statistics Department, Stanford University, Stanford, CA, 94305, USA.
authors.forename: Ben J
authors.initials: BJ
authors.lastname: Callahan
authors.affiliation: Statistics Department, Stanford University, Stanford, CA, 94305, USA.
authors.forename: Kris
authors.initials: K
authors.lastname: Sankaran
authors.affiliation: Statistics Department, Stanford University, Stanford, CA, 94305, USA.
authors.forename: Julia A
authors.initials: JA
authors.lastname: Fukuyama
authors.affiliation: Whole Biome Inc., San Francisco, CA, 94107, USA.
authors.forename: Paul J
authors.initials: PJ
authors.lastname: McMurdie
authors.affiliation: Statistics Department, Stanford University, Stanford, CA, 94305, USA.
authors.forename: Susan P
authors.initials: SP
authors.lastname: Holmes
keywords: community analysis; microbiome; taxonomy