Paul J. McMurdie and Susan Holmes
If you find phyloseq and/or its tutorials useful, please acknowledge and cite phyloseq in your publications:
Introduction
The analysis of microbiological communities brings many challenges: the integration of many different types of data with methods from ecology, genetics, phylogenetics, network analysis, visualization and testing. The data itself may originate from widely different sources, such as the microbiomes of humans, soils, surface and ocean waters, wastewater treatment plants, industrial facilities, and so on; and as a result, these varied sample types may have very different forms and scales of related data that is extremely dependent upon the experiment and its question(s). The phyloseq package is a tool to import, store, analyze, and graphically display complex phylogenetic sequencing data that has already been clustered into Operational Taxonomic Units (OTUs), especially when there is associated sample data, phylogenetic tree, and/or taxonomic assignment of the OTUs. This package leverages many of the tools available in R for ecology and phylogenetic analysis (vegan, ade4, ape, picante), while also using advanced/flexible graphic systems (ggplot2) to easily produce publication-quality graphics of complex phylogenetic data. phyloseq uses a specialized system of S4 classes to store all related phylogenetic sequencing data as single experiment-level object, making it easier to share data and reproduce analyses. In general, phyloseq seeks to facilitate the use of R for efficient interactive and reproducible analysis of OTU-clustered high-throughput phylogenetic sequencing data.
About this vignette
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Other links and tutorials
An overview of phyloseq’s intended functionality, goals, and design is provided in the following free and open access article:
McMurdie and Holmes (2013). phyloseq: An R package for reproducible interactive analysis and graphics of microbiome census data. PLoS ONE e61217.
The most updated examples are posted in our online tutorials from the phyloseq home page
A separate vignette describes analysis tools included in phyloseq along with various examples using included example data. A quick way to load it is:
vignette("phyloseq_analysis")
By contrast, this vignette is intended to provide functional examples of the basic data import and manipulation infrastructure included in phyloseq. This includes example code for importing OTU-clustered data from different clustering pipelines, as well as performing clear and reproducible filtering tasks that can be altered later and checked for robustness. The motivation for including tools like this in phyloseq is to save time, and also to build-in a structure that requires consistency across related data tables from the same experiment.This not only reduces code repetition, but also decreases the likelihood of mistakes during data filtering and analysis. For example, it is intentionally difficult in phyloseq to create an experiment-level object in which a component tree and OTU table have different OTU names. The import functions, trimming tools, as well as the main tool for creating an experiment-level object, phyloseq, all automatically trim the OTUs and samples indices to their intersection, such that these component data types are exactly coherent.
phyloseq classes
The class structure in the phyloseq package follows the inheritance diagram shown in the figure below. Currently, phyloseq uses 4 core data classes. They are (1) the OTU abundance table (otu_table), a table of sample data (sample_data); (2) a table of taxonomic descriptors (taxonomyTable); and (3) a phylogenetic tree ("phylo"-class, ape package.
The otu_table class can be considered the central data type, as it directly represents the number and type of sequences observed in each sample. otu_table extends the numeric matrix class in the R base, and has a few additonal feature slots. The most important of these feature slots is the taxa_are_rows slot, which holds a single logical that indicates whether the table is oriented with taxa as rows (as in the genefilter package in Bioconductor or with taxa as columns (as in vegan and picante packages). In phyloseq methods, as well as its extensions of methods in other packages, the taxa_are_rows value is checked to ensure proper orientation of the otu_table. A phyloseq user is only required to specify the otu_table orientation during initialization, following which all handling is internal.
The sample_data class directly inherits R’s data.frame class, and thus effectively stores both categorical and numerical data about each sample. The orientation of a data.frame in this context requires that samples/trials are rows, and variables are columns (consistent with vegan and other packages). The taxonomyTable class directly inherits the matrix class, and is oriented such that rows are taxa/OTUs and columns are taxonomic levels (e.g. Phylum).
The phyloseq-class can be considered an “experiment-level class” and should contain two or more of the previously-described core data classes. We assume that phyloseq users will be interested in analyses that utilize their abundance counts derived from the phylogenetic sequencing data, and so the phyloseq() constructor will stop with an error if the arguments do not include an otu_table. There are a number of common methods that require either an otu_table and sample_data combination, or an otu_table and phylogenetic tree combination. These methods can operate on instances of the phyloseq-class, and will stop with an error if the required component data is missing.
Classes and inheritance in the phyloseq package. The class name and its slots are shown with red- or blue-shaded text, respectively. Coercibility is indicated graphically by arrows with the coercion function shown. Lines without arrows indicate that the more complex class (``phyloseq“) contains a slot with the associated data class as its components.
Load phyloseq and import data
Now let’s get started by loading phyloseq, and describing some methods for importing data.
Load phyloseq
To use phyloseq in a new R session, it will have to be loaded. This can be done in your package manager, or at the command line using the library() command:
library("phyloseq")
Import data
An important feature of phyloseq are methods for importing phylogenetic sequencing data from common taxonomic clustering pipelines. These methods take file pathnames as input, read and parse those files, and return a single object that contains all of the data.
Some additional background details are provided below. The best reproducible examples on importing data with phyloseq can be found on the official data import tutorial page:
http://joey711.github.com/phyloseq/import-data
Import from QIIME (Modern)
The default output from modern versions of QIIME is a BIOM-format file (among others). This is suppored in phyloseq.
Sample data from QIIME
Sometimes inaccurately referred to as metadata, additional observations on samples provided as mapping file to QIIME have not typically been output in the BIOM files, even though BIOM format supports it. This failure to support the full capability of the BIOM format means that you’ll have to provide sample observations as a separate file. There are many ways to do this, but the QIIME sample map is supported.
Output
- Before Merging - Before merging with
merge_phyloseq, the output from these import activities is the three separate objects listed in the previous table.
- After Merging - After merging you have a single self-consistent phyloseq object that contains an OTU table, taxonomy table, sample-data, and a phylogenetic tree.
QIIME Example Tutorial
QIIME’s “Moving Pictures” example tutorial output is a little too large to include within the phyloseq package (and thus is not directly included in this vignette). However, the phyloseq home page includes a full reproducible example of the import procedure described above:
Link HERE
For reference, or if you want to try yourself, the following is the relative paths within the QIIME tutorial directory for each of the files you will need.
- BIOM file, originally at: moving_pictures_tutorial-1.9.0/illumina/precomputed-output/otus/otu_table_mc2_w_tax_no_pynast_failures.biom
- Tree file, originally at: moving_pictures_tutorial-1.9.0/illumina/precomputed-output/otus/rep_set.tre
- Map File, originally at: moving_pictures_tutorial-1.9.0/illumina/map.tsv
Import from QIIME Legacy
QIIME is a free, open-source OTU clustering and analysis pipeline written for Unix (mostly Linux). It is distributed in a number of different forms (including a pre-installed virtual machine). See the QIIME home page for details.
Output
The class of the object returned by import_qiime() depends upon which filenames are provided. The most comprehensive class is chosen automatically, based on the input files listed as arguments. At least one argument needs to be provided.
Import from mothur
The open-source, platform-independent, locally-installed software package, mothur, can also process barcoded amplicon sequences and perform OTU-clustering. It is extensively documented on the mothur wiki
Output
The output from import_mothur() depends on which file types are provided. If all three file types are provided, an instance of the phyloseq-class is returned that contains both an OTU abundance table and its associated phylogenetic tree.
Import from PyroTagger
PyroTagger is an OTU-clustering pipeline for barcoded 16S rRNA amplicon sequences, served and maintained by the Department of Energy’s (DOE’s) Joint Genome Institute (JGI). It can be used through a straightforward web interface at the PyroTagger home page
PyroTagger takes as input the untrimmed sequence (.fasta) and sequence-quality (.qual) files, as well as a sample mapping file that contains the bar code sequence for each sample and its name. It uses a 97% identity threshold for defining OTU clusters (approximately species-level of taxonomic distinction), and provides no options for specifying otherwise. It does allow users to modify the threshold setting for low-quality bases.
Output
import_RDP_tab() returns an instance of the phyloseq-class that contains the OTU abundance table and taxonomy table. To my knowledge, PyroTagger does not calculate a tree of the representative sequences from each OTU cluster, nor a distance object, so analyses like tip_glom() and UniFrac are not applicable.
Import from RDP pipeline
The Ribosomal Database Project (RDP) provides a web-based barcoded 16S rRNA amplicon sequence processing pipeline called the RDP Pyrosequencing Pipeline. A user must run all three of the “Data Processing” steps sequentially through the web interface in order to acquire the output from Complete Linkage Clustering, the approach to OTU clustering used by the RDP Pipeline. Note that this import function assumes that the sequence names in the resulting cluster file follow a particular naming convention with underscore delimiter (see below).
Output
This importer returns an otu_table object.
Expected Naming Convention
The RDP cluster pipeline (specifically, the output of the complete linkage clustering step) has no formal documentation for the “.clust” file structure or its apparent sequence naming convention.
The cluster file itself contains the names of all sequences contained in the input alignment. If the upstream barcode and aligment processing steps are also done with the RDP pipeline, then the sequence names follow a predictable naming convention wherein each sequence is named by its sample and sequence ID, separated by a "_" as delimiter:
sampleName_sequenceIDnumber
This import function assumes that the sequence names in the cluster file follow this convention, and that the sample name does not contain any "_". It is unlikely to work if this is not the case. It is likely to work if you used the upstream steps in the RDP pipeline to process your raw (barcoded, untrimmed) fasta/fastq data.
Example Data (included)
There are multiple example data sets included in phyloseq. Many are from published investigations and include documentation with a summary and references, as well as some example code representing some aspect of analysis available in phyloseq. In the package index, go to the names beginning with “data-” to see the documentation of currently available example datasets.
To load example data into the working environment, use the data() command:
data(GlobalPatterns)
data(esophagus)
data(enterotype)
data(soilrep)
Similarly, entering ?enterotype will reveal the documentation for the so-called “enterotype” dataset. For details examples, see the Example Data tutorial
phyloseq Object Summaries
In small font, the following is the summary of the GlobalPatterns dataset that prints to the terminal. These summaries are consistent among all phyloseq-class objects. Although the components of GlobalPatterns have many thousands of elements, the command-line returns only a short summary of each component. This encourages you to check that an object is still what you expect, without needing to let thousands of elements scroll across the terminal. In the cases in which you do want to see more of a particular component, use an accessor function (see table below).
data(GlobalPatterns)
GlobalPatterns
## phyloseq-class experiment-level object
## otu_table() OTU Table: [ 19216 taxa and 26 samples ]
## sample_data() Sample Data: [ 26 samples by 7 sample variables ]
## tax_table() Taxonomy Table: [ 19216 taxa by 7 taxonomic ranks ]
## phy_tree() Phylogenetic Tree: [ 19216 tips and 19215 internal nodes ]
Convert raw data to phyloseq components
Suppose you have already imported raw data from an experiment into R, and their indices are labeled correctly. How do you get phyloseq to recognize these tables as the appropriate class of data? And further combine them together? Table Table of Component Constructor Functions lists key functions for converting these core data formats into specific component data objects recognized by phyloseq. These will also
Table of component constructor functions for building component data objects
phyloseq constructors: functions for building/merging phyloseq objects.
The following example illustrates using the constructor methods for component data tables.
otu1 <- otu_table(raw_abundance_matrix, taxa_are_rows=FALSE)
sam1 <- sample_data(raw_sample_data.frame)
tax1 <- tax_table(raw_taxonomy_matrix)
tre1 <- read_tree(my_tree_file)
phyloseq() function: building complex phyloseq objects
Once you’ve converted the data tables to their appropriate class, combining them into one object requires only one additional function call, phyloseq():
ex1b <- phyloseq(my_otu_table, my_sample_data, my_taxonomyTable, my_tree)
You do not need to have all four data types in the example above in order to combine them into one validity-checked experiment-level phyloseq-class object. The phyloseq() method will detect which component data classes are present, and build accordingly. Downstream analysis methods will access the required components using phyloseq’s accessors, and throw an error if something is missing. For most downstream methods you will only need to supply the combined, phyloseq-class object (the output of phyloseq() ), usually as the first argument.
ex1c <- phyloseq(my_otu_table, my_sample_data)
Whenever an instance of the phyloseq-class is created by phyloseq — for example, when we use the import_qiime() function to import data, or combine manually imported tables using phyloseq() — the row and column indices representing taxa or samples are internally checked/trimmed for compatibility, such that all component data describe exactly (and only) the same OTUs and samples.
Merge
The phyloseq project includes support for two complete different categories of merging.
- Merging the OTUs or samples in a phyloseq object, based upon a taxonomic or sample variable:
merge_samples(), merge_taxa()
- Merging two or more data objects that come from the same experiment, so that their data becomes part of the same phyloseq object:
merge_phyloseq()
For further details, see the reproducible online tutorial at:
http://joey711.github.com/phyloseq/merge
Trimming, subsetting, filtering phyloseq data
Trimming: prune_taxa()
Trimming high-throughput phylogenetic sequencing data can be useful, or even necessary, for certain types of analyses. However, it is important that the original data always be available for reference and reproducibility; and that the methods used for trimming be transparent to others, so they can perform the same trimming or filtering steps on the same or related data. To facilitate this, phyloseq contains many ways to trim/filter the data from a phylogenetic sequencing project. Because matching indices for taxa and samples is strictly enforced, subsetting one of the data components automatically subsets the corresponding indices from the others. Variables holding trimmed versions of your original data can be declared, and further trimmed, without losing track of the original data.
In general, most trimming should be accomplished using the S4 methods prune_taxa() or prune_samples().
Simple filtering example
For example, lets make a new object that only holds the most abundant 20 taxa in the experiment. To accomplish this, we will use the prune_taxa() function.
data(GlobalPatterns)
most_abundant_taxa <- sort(taxa_sums(GlobalPatterns), TRUE)[1:topN]
ex2 <- prune_taxa(names(most_abundant_taxa), GlobalPatterns)
Now we can ask the question, “what taxonomic Family are these OTUs?” (Subsetting still returns a taxonomyTable object, which is summarized. We will need to convert to a vector)
topFamilies <- tax_table(ex2)[, "Family"]
as(topFamilies, "vector")
## [1] NA "ACK-M1" "ACK-M1"
## [4] "Bifidobacteriaceae" NA NA
## [7] "Nostocaceae" NA "Neisseriaceae"
## [10] "Neisseriaceae" "Pasteurellaceae" "Enterobacteriaceae"
## [13] "Bacteroidaceae" "Bacteroidaceae" "Bacteroidaceae"
## [16] "Clostridiaceae" "Ruminococcaceae" "Ruminococcaceae"
## [19] "Ruminococcaceae" "Streptococcaceae"
Arbitrarily complex abundance filtering
The previous example was a relatively simple filtering in which we kept only the most abundant 20 in the whole experiment. But what if we wanted to keep the most abundant 20 taxa of each sample? And of those, keep only the taxa that are also found in at least one-third of our samples? What if we wanted to keep only those taxa that met some across-sample criteria?
genefilter_sample(): Filter by Within-Sample Criteria
For this more complicated filtering phyloseq contains a function, genefilter_sample, that takes as an argument a phyloseq object, as well as a list of one or more filtering functions that will be applied to each sample in the abundance matrix (otu_table), as well as an integer argument, A, that specifies for how many samples the filtering function must return TRUE for a particular taxa to avoid removal from the object. A supporting function filterfun_sample is also included in phyloseq to facilitate creating a properly formatted function (enclosure) if more than one function is going to be applied simultaneously. genefilter_sample returns a logical vector suitable for sending directly to prune_taxa for the actual trimming.
Here is an example on a completely fabricated otu_table called testOTU.
testOTU <- otu_table(matrix(sample(1:50, 25, replace=TRUE), 5, 5), taxa_are_rows=FALSE)
f1<- filterfun_sample(topk(2))
wh1 <- genefilter_sample(testOTU, f1, A=2)
wh2 <- c(T, T, T, F, F)
prune_taxa(wh1, testOTU)
prune_taxa(wh2, testOTU)
Here is a second example using the included dataset, GlobalPatterns. The most abundant taxa are kept only if they are in the most abundant 10% of taxa in at least half of the samples in dataset GlobalPatterns. Note that it is not necessary to subset GlobalPatterns in order to do this filtering. The S4 method prune_taxa subsets each of the relavent component objects, and returns the complex object back.
data(GlobalPatterns)
f1<- filterfun_sample(topp(0.1))
wh1 <- genefilter_sample(GlobalPatterns, f1, A=(1/2*nsamples(GlobalPatterns)))
sum(wh1)
## [1] 795
ex2 <- prune_taxa(wh1, GlobalPatterns)
print(ex2)
## phyloseq-class experiment-level object
## otu_table() OTU Table: [ 795 taxa and 26 samples ]
## sample_data() Sample Data: [ 26 samples by 7 sample variables ]
## tax_table() Taxonomy Table: [ 795 taxa by 7 taxonomic ranks ]
## phy_tree() Phylogenetic Tree: [ 795 tips and 794 internal nodes ]
If instead of the most abundant fraction of taxa, you are interested in the most abundant fraction of individuals (aka sequences, observations), then the topf function is appropriate. For steep rank-abundance curves, topf will seem to be much more conservative (trim more taxa) because it is based on the cumulative sum of relative abundance. It does not guarantee that a certain number or fraction of total taxa (richness) will be retained.
data(GlobalPatterns)
f1<- filterfun_sample(topf(0.9))
wh1 <- genefilter_sample(GlobalPatterns, f1, A=(1/3*nsamples(GlobalPatterns)))
sum(wh1)
prune_taxa(wh1, GlobalPatterns)
filter_taxa(): Filter by Across-Sample Criteria
The filter_taxa function is directly analogous to the genefilter function for microarray filtering, but is used for filtering OTUs from phyloseq objects. It applies an arbitrary set of functions – as a function list, for instance, created by genefilter::filterfun – as across-sample criteria, one OTU at a time. It can be thought of as an extension of the genefilter-package (from the Bioconductor repository) for phyloseq objects. It takes as input a phyloseq object, and returns a logical vector indicating whether or not each OTU passed the criteria. Alternatively, if the prune option is set to FALSE, it returns the already-trimmed version of the phyloseq object.
Inspect the following example. Note that the functions genefilter and kOverA are from the genefilter package.
data("enterotype")
library("genefilter")
flist<- filterfun(kOverA(5, 2e-05))
ent.logi <- filter_taxa(enterotype, flist)
ent.trim <- filter_taxa(enterotype, flist, TRUE)
identical(ent.trim, prune_taxa(ent.logi, enterotype))
## [1] TRUE
identical(sum(ent.logi), ntaxa(ent.trim))
## [1] TRUE
filter_taxa(enterotype, flist, TRUE)
## phyloseq-class experiment-level object
## otu_table() OTU Table: [ 416 taxa and 280 samples ]
## sample_data() Sample Data: [ 280 samples by 9 sample variables ]
## tax_table() Taxonomy Table: [ 416 taxa by 1 taxonomic ranks ]
subset_samples(): Subset by Sample Variables
It is possible to subset the samples in a phyloseq object based on the sample variables using the subset_samples() function. For example to subset GlobalPatterns such that only certain environments are retained, the following line is needed (the related tables are subsetted automatically as well):
ex3 <- subset_samples(GlobalPatterns, SampleType%in%c("Freshwater", "Ocean", "Freshwater (creek)"))
ex3
## phyloseq-class experiment-level object
## otu_table() OTU Table: [ 19216 taxa and 8 samples ]
## sample_data() Sample Data: [ 8 samples by 7 sample variables ]
## tax_table() Taxonomy Table: [ 19216 taxa by 7 taxonomic ranks ]
## phy_tree() Phylogenetic Tree: [ 19216 tips and 19215 internal nodes ]
For this example only a categorical variable is shown, but in principle a continuous variable could be specified and a logical expression provided just as for the subset function. In fact, because sample_data component objects are an extension of the data.frame class, they can also be subsetted with the subset function:
subset(sample_data(GlobalPatterns), SampleType%in%c("Freshwater", "Ocean", "Freshwater (creek)"))
## Sample Data: [8 samples by 7 sample variables]:
## X.SampleID Primer Final_Barcode Barcode_truncated_plus_T
## LMEpi24M LMEpi24M ILBC_13 ACACTG CAGTGT
## SLEpi20M SLEpi20M ILBC_15 ACAGAG CTCTGT
## AQC1cm AQC1cm ILBC_16 ACAGCA TGCTGT
## AQC4cm AQC4cm ILBC_17 ACAGCT AGCTGT
## AQC7cm AQC7cm ILBC_18 ACAGTG CACTGT
## NP2 NP2 ILBC_19 ACAGTT AACTGT
## NP3 NP3 ILBC_20 ACATCA TGATGT
## NP5 NP5 ILBC_21 ACATGA TCATGT
## Barcode_full_length SampleType
## LMEpi24M CATGAACAGTG Freshwater
## SLEpi20M AGCCGACTCTG Freshwater
## AQC1cm GACCACTGCTG Freshwater (creek)
## AQC4cm CAAGCTAGCTG Freshwater (creek)
## AQC7cm ATGAAGCACTG Freshwater (creek)
## NP2 TCGCGCAACTG Ocean
## NP3 GCTAAGTGATG Ocean
## NP5 GAACGATCATG Ocean
## Description
## LMEpi24M Lake Mendota Minnesota, 24 meter epilimnion
## SLEpi20M Sparkling Lake Wisconsin, 20 meter eplimnion
## AQC1cm Allequash Creek, 0-1cm depth
## AQC4cm Allequash Creek, 3-4 cm depth
## AQC7cm Allequash Creek, 6-7 cm depth
## NP2 Newport Pier, CA surface water, Time 1
## NP3 Newport Pier, CA surface water, Time 2
## NP5 Newport Pier, CA surface water, Time 3
subset_taxa(): subset by taxonomic categories
It is possible to subset by specific taxonomic category using the subset_taxa() function. For example, if we wanted to subset GlobalPatterns so that it only contains data regarding the phylum Firmicutes:
ex4 <- subset_taxa(GlobalPatterns, Phylum=="Firmicutes")
ex4
## phyloseq-class experiment-level object
## otu_table() OTU Table: [ 4356 taxa and 26 samples ]
## sample_data() Sample Data: [ 26 samples by 7 sample variables ]
## tax_table() Taxonomy Table: [ 4356 taxa by 7 taxonomic ranks ]
## phy_tree() Phylogenetic Tree: [ 4356 tips and 4355 internal nodes ]
random subsample abundance data
Can also randomly subset, for example a random subset of 100 taxa from the full dataset.
randomSpecies100 <- sample(taxa_names(GlobalPatterns), 100, replace=FALSE)
ex5 <- prune_taxa(randomSpecies100, GlobalPatterns)
Phylogenetic smoothing
tax_glom()
Suppose we are skeptical about the importance of OTU-level distinctions in our dataset. For this scenario, phyloseq includes a taxonomic-agglommeration method,tax_glom(), which merges taxa of the same taxonomic category for a user-specified taxonomic level. In the following code, we merge all taxa of the same Genus, and store that new object as ex6.
ex6 <- tax_glom(GlobalPatterns, taxlevel="Genus")
tip_glom()
Similarly, our original example object (GlobalPatterns) also contains a phlyogenetic tree corresponding to each OTU, which we could also use as a means to merge taxa in our dataset that are closely related. In this case, we specify a threshold patristic distance. Taxa more closely related than this threshold are merged. This is especially useful when a dataset has many taxa that lack a taxonomic assignment at the level you want to investigate, a problem when using tax_glom(). Note that for datasets with a large number of taxa, tax_glom will be noticeably faster than tip_glom. Also, keep in mind that tip_glom requires that its first argument be an object that contains a tree, while tax_glom instead requires a taxonomyTable (See phyloseq classes).
ex7 <- tip_glom(GlobalPatterns, speciationMinLength = 0.05)
Command output not provided here to save time during compilation of the vignette. The user is encouraged to try this out on your dataset, or even this example, if interested. It may take a while to run on the full, untrimmed data.
References
Robert C Gentleman, Vincent J. Carey, Douglas M. Bates, et al. Bioconductor: Open software development for computational biology and bioinformatics. Genome Biology 5:R80, 2004.
J Gregory Caporaso, Justin Kuczynski, Jesse Stombaugh, Kyle Bittinger, Frederic D Bushman QIIME allows analysis of high-throughput community sequencing data. Nature Methods 7(5):335-336, 2010.
P D Schloss, S L Westcott, T Ryabin, J R Hall, M Hartmann, et al. Introducing mothur: Open-Source, Platform-Independent, Community-Supported Software for Describing and Comparing Microbial Communities. Applied and Environmental Microbiology 75(23):7537-7541, 2009.
J R Cole, Q Wang, E Cardenas, J Fish, B Chai et al. The Ribosomal Database Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Research 37(Database issue):D141-5, 2009.
A typical QIIME output directory. The two output files suitable for import by phyloseq are highlighted. A third file describing the samples, their barcodes and covariates, is created by the user and required as input to QIIME. It is a good idea to import this file, as it can be converted directly to a