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Normalization is a term that is often used but rarely defined and poorly understood. The number of choices of normalization procedure is large-some are inappropriate or inadmissible-and all are narrowly relevant to a specific analysis that depends on both the nature of the data and the question being asked. This chapter describes key definitions of normalization as they apply in metagenomics, mainly for taxonomic profiling data; while also demonstrating specific, reproducible examples of normalization procedures in the context of analysis techniques for which they were intended. The analysis and graphics code is distributed as a supplemental companion to this chapter so that the motivated reader can re-use it on new data.

Hydrogen (H(2)) release from photosynthetic microbial mats has contributed to the chemical evolution of Earth and could potentially be a source of renewable H(2) in the future. However, the taxonomy of H(2)-producing microorganisms (hydrogenogens) in these mats has not been previously determined. With combined biogeochemical and molecular studies of microbial mats collected from Elkhorn Slough, Monterey Bay, California, we characterized the mechanisms of H(2) production and identified a dominant hydrogenogen. Net production of H(2) was observed within the upper photosynthetic layer (0-2 mm) of the mats under dark and anoxic conditions. Pyrosequencing of rRNA gene libraries generated from this layer demonstrated the presence of 64 phyla, with Bacteriodetes, Cyanobacteria and Proteobacteria dominating the sequences. Sequencing of rRNA transcripts obtained from this layer demonstrated that Cyanobacteria dominated rRNA transcript pyrotag libraries. An OTU affiliated to Microcoleus spp. was the most abundant OTU in both rRNA gene and transcript libraries. Depriving mats of sunlight resulted in an order of magnitude decrease in subsequent nighttime H(2) production, suggesting that newly fixed carbon is critical to H(2) production. Suppression of nitrogen (N(2))-fixation in the mats did not suppress H(2) production, which indicates that co-metabolic production of H(2) during N(2)-fixation is not an important contributor to H(2) production. Concomitant production of organic acids is consistent with fermentation of recently produced photosynthate as the dominant mode of H(2) production. Analysis of rRNA % transcript:% gene ratios and H(2)-evolving bidirectional [NiFe] hydrogenase % transcript:% gene ratios indicated that Microcoelus spp. are dominant hydrogenogens in the Elkhorn Slough mats.

Summary: We have created a Shiny-based Web application, called Shiny-phyloseq , for dynamic interaction with microbiome data that runs on any modern Web browser and requires no programming, increasing the accessibility and decreasing the entrance requirement to using phyloseq and related R tools. Along with a data- and context-aware dynamic interface for exploring the effects of parameter and method choices, Shiny-phyloseq also records the complete user input and subsequent graphical results of a user’s session, allowing the user to archive, share and reproduce the sequence of steps that created their result—without writing any new code themselves. Availability and implementation : Shiny-phyloseq is implemented entirely in the R language. It can be hosted/launched by any system with R installed, including Windows, Mac OS and most Linux distributions. Information technology administrators can also host Shiny-phyloseq from a remote server, in which case users need only have a Web browser installed. Shiny-phyloseq is provided free of charge under a GPL-3 open-source license through GitHub at http://joey711.github.io/shiny-phyloseq/ . Contact : mcmurdie@alumni.stanford.edu .

High-throughput DNA sequencing technologies, coupled with advanced bioinformatics tools, have enabled rapid advances in microbial ecology and our understanding of the human microbiome. QIIME (Quantitative Insights Into Microbial Ecology) is an open-source bioinformatics software package designed for microbial community analysis based on DNA sequence data, which provides a single analysis framework for analysis of raw sequence data through publication-quality statistical analyses and interactive visualizations. In this chapter, we demonstrate the use of the QIIME pipeline to analyze microbial communities obtained from several sites on the bodies of transgenic and wild-type mice, as assessed using 16S rRNA gene sequences generated on the Illumina MiSeq platform. We present our recommended pipeline for performing microbial community analysis and provide guidelines for making critical choices in the process. We present examples of some of the types of analyses that are enabled by QIIME and discuss how other tools, such as phyloseq and R, can be applied to expand upon these analyses.

This article compares different methods for combining abundance data, phylogenetic trees and clinical covariates in a nonparametric setting. In particular we study the output from the principal coordinates analysis on UNIFRAC and WEIGHTED UNIFRAC distances and the output from a double principal coordinate analyses DPCOA using distances computed on the phylogenetic tree. We also present power comparisons for some of the standard tests of phylogenetic signal between different types of samples. These methods are compared both on simulated and real data sets. Our study shows that DPCoA is less robust to outliers, and more robust to small noisy fluctuations around zero.

We present a detailed description of a new Bioconductor package, phyloseq, for integrated data and analysis of taxonomically-clustered phylogenetic sequencing data in conjunction with related data types. The phyloseq package integrates abundance data, phylogenetic information and covariates so that exploratory transformations, plots, and confirmatory testing and diagnostic plots can be carried out seamlessly. The package is built following the S4 object-oriented framework of the R language so that once the data have been input the user can easily transform, plot and analyze the data. We present some examples that highlight the methods and the ease with which we can leverage existing packages.

Vinyl chloride (VC) is a human carcinogen and widespread priority pollutant. Here we report the first, to our knowledge, complete genome sequences of microorganisms able to respire VC, Dehalococcoides sp. strains VS and BAV1. Notably, the respective VC reductase encoding genes, vcrAB and bvcAB, were found embedded in distinct genomic islands (GEIs) with different predicted integration sites, suggesting that these genes were acquired horizontally and independently by distinct mechanisms. A comparative analysis that included two previously sequenced Dehalococcoides genomes revealed a contextually conserved core that is interrupted by two high plasticity regions (HPRs) near the Ori. These HPRs contain the majority of GEIs and strain-specific genes identified in the four Dehalococcoides genomes, an elevated number of repeated elements including insertion sequences (IS), as well as 91 of 96 rdhAB, genes that putatively encode terminal reductases in organohalide respiration. Only three core rdhA orthologous groups were identified, and only one of these groups is supported by synteny. The low number of core rdhAB, contrasted with the high rdhAB numbers per genome (up to 36 in strain VS), as well as their colocalization with GEIs and other signatures for horizontal transfer, suggests that niche adaptation via organohalide respiration is a fundamental ecological strategy in Dehalococccoides. This adaptation has been exacted through multiple mechanisms of recombination that are mainly confined within HPRs of an otherwise remarkably stable, syntenic, streamlined genome among the smallest of any free-living microorganism., Author Summary Dehalococcoides are free-living sediment and subsurface bacteria with remarkably small, streamlined genomes and an unusual degree of niche specialization. These strictly anaerobic bacteria gain metabolic energy exclusively through a novel type of respiration that results in reductive elimination of chlorides from organochlorines, many of which are priority pollutants. In this article, we compare the first complete genome sequences of Dehalococcoides strains that grow via respiration of vinyl chloride (VC), a human carcinogen and abundant groundwater pollutant. Our work provides novel insights into Dehalococcoides chromosome organization and evolution, identifies specific positions in the chromosomes where new genes—like the genes responsible for growth on VC—are integrated, and generates clues how these dechlorinating bacteria adapt to anthropogenic contamination. This information sheds new light on Dehalococcoides biology and ecology, with implications for enhanced bioremediation to protect dwindling drinking water reservoirs.

Background The analysis of microbial communities through DNA sequencing brings many challenges: the integration of different types of data with methods from ecology, genetics, phylogenetics, multivariate statistics, visualization and testing. With the increased breadth of experimental designs now being pursued, project-specific statistical analyses are often needed, and these analyses are often difficult (or impossible) for peer researchers to independently reproduce. The vast majority of the requisite tools for performing these analyses reproducibly are already implemented in R and its extensions (packages), but with limited support for high throughput microbiome census data. Results Here we describe a software project, phyloseq, dedicated to the object-oriented representation and analysis of microbiome census data in R. It supports importing data from a variety of common formats, as well as many analysis techniques. These include calibration, filtering, subsetting, agglomeration, multi-table comparisons, diversity analysis, parallelized Fast UniFrac, ordination methods, and production of publication-quality graphics; all in a manner that is easy to document, share, and modify. We show how to apply functions from other R packages to phyloseq-represented data, illustrating the availability of a large number of open source analysis techniques. We discuss the use of phyloseq with tools for reproducible research, a practice common in other fields but still rare in the analysis of highly parallel microbiome census data. We have made available all of the materials necessary to completely reproduce the analysis and figures included in this article, an example of best practices for reproducible research. Conclusions The phyloseq project for R is a new open-source software package, freely available on the web from both GitHub and Bioconductor.

Background Vinyl chloride is a widespread groundwater pollutant and Group 1 carcinogen. A previous comparative genomic analysis revealed that the vinyl chloride reductase operon, vcrABC, of Dehalococcoides sp. strain VS is embedded in a horizontally-acquired genomic island that integrated at the single-copy tmRNA gene, ssrA. Results We targeted conserved positions in available genomic islands to amplify and sequence four additional vcrABC -containing genomic islands from previously-unsequenced vinyl chloride respiring Dehalococcoides enrichments. We identified a total of 31 ssrA-specific genomic islands from Dehalococcoides genomic data, accounting for 47 reductive dehalogenase homologous genes and many other non-core genes. Sixteen of these genomic islands contain a syntenic module of integration-associated genes located adjacent to the predicted site of integration, and among these islands, eight contain vcrABC as genetic 'cargo'. These eight vcrABC -containing genomic islands are syntenic across their ~12 kbp length, but have two phylogenetically discordant segments that unambiguously differentiate the integration module from the vcrABC cargo. Using available Dehalococcoides phylogenomic data we estimate that these ssrA-specific genomic islands are at least as old as the Dehalococcoides group itself, which in turn is much older than human civilization. Conclusions The vcrABC -containing genomic islands are a recently-acquired subset of a diverse collection of ssrA-specific mobile elements that are a major contributor to strain-level diversity in Dehalococcoides, and may have been throughout its evolution. The high similarity between vcrABC sequences is quantitatively consistent with recent horizontal acquisition driven by ~100 years of industrial pollution with chlorinated ethenes.