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# Exploring Taxonomy with R

## Overview

Teaching: 20 min
Exercises: 5 min
Questions
• How can I use my taxonomic assignment results to make analyses?

Objectives
• Comprehend which libraries are required for analysis of the taxonomy of metagenomes.

• Create and manage a Phyloseq object.

## Creating lineage and rank tables

In this episode we will use RStudio to analyze our microbial samples, you don’t have to install anything, you already have an instance on the cloud ready to be used.

Packages like Qiime2, MEGAN, Vegan, or Phyloseq in R allow us to analyze diversity and abundance by manipulating taxonomic assignment data. In this lesson, we will use Phyloseq. In order to do so, we need to generate an abundance matrix from the Kraken output files. One program widely used for this purpose is kraken-biom.

To do this we could go to our now familiar Bash terminal, but RStudio has an integrated terminal that uses the same language as the one we learned in the Command-line lessons, so let’s take advantage of it. Let’s open RStudio and go to the Terminal tab in the bottom left panel.

### Kraken-biom

Kraken-biom is a program that creates BIOM tables from the Kraken output.

In order to run Kraken-biom, we have to move to the folder where our taxonomic-data files are located:

$cd ~/dc_workshop/taxonomy  First, we will visualize the content of our directory by the ls command. $ ls

JC1A.kraken  JC1A.report  JP41.report  JP4D.kraken  JP4D.report  mags_taxonomy


The kraken-biom program is installed inside our metagenomics environment, so let’s activate it.

$conda activate metagenomics  Let’s take a look at the different flags that kraken-biom has: $ kraken-biom -h

usage: kraken-biom [-h] [--max {D,P,C,O,F,G,S}] [--min {D,P,C,O,F,G,S}]
[-o OUTPUT_FP] [--otu_fp OTU_FP] [--fmt {hdf5,json,tsv}]
[--gzip] [--version] [-v]
kraken_reports [kraken_reports ...]

Create BIOM-format tables (http://biom-format.org) from Kraken output
(http://ccb.jhu.edu/software/kraken/).
.
.
.


By a close look at the first output lines, it is noticeable that we need a specific output from Kraken: the .reports.

With the next command, we are going to create a table in Biom format called cuatroc.biom. We will include the two samples we have been working with (JC1A and JP4D) and a third one (JP41), to be able to perform certain analyses later on.

## Creating and manipulating Phyloseq objects

Phyloseq is a library with tools to analyze and plot the taxonomic assignment and abundance information of your metagenomics samples. Let’s install phyloseq (This instruction might not work on certain versions of R) and other libraries required for its execution:

> if (!requireNamespace("BiocManager", quietly = TRUE))
install.packages("BiocManager")

> BiocManager::install("phyloseq") # Install phyloseq

> install.packages(c("RColorBrewer", "patchwork")) #install patchwork to chart publication-quality plots and readr to read rectangular datasets.


Once the libraries are installed, we must make them available for this R session. Now load the libraries (a process needed every time we begin a new work session in R):

> library("phyloseq")
> library("ggplot2")
> library("RColorBrewer")
> library("patchwork")


### Creating the phyloseq object

First, we tell R in which directory we are working.

> setwd("~/dc_workshop/taxonomy/")


Let’s proceed to create the phyloseq object with the import_biom command:

> merged_metagenomes <- import_biom("cuatroc.biom")


Now, we can inspect the result by asking the class of the object created, and doing a close inspection of some of its content:

> class(merged_metagenomes)

[1] "phyloseq"
attr("package")
[1] "phyloseq"


The “class” command indicates that we already have our phyloseq object.

### Exploring the taxonomic labels

Let’s try to access the data that is stored inside our merged_metagenomes object. Since a phyloseq object is a special object in R, we need to use the operator @ to explore the subsections of data inside merged_metagenomes. If we type merged_metagenomes@ five options are displayed; tax_table and otu_table are the ones that we will use. After writing merged_metagenomes@otu_table or merged_metagenomes@tax_table, an option of .Data will be the one chosen in both cases. Let’s see what is inside of our tax_table:

> View(merged_metagenomes@tax_table@.Data)


Figure 1. Table of the taxonomic labels from our merged_metagenomes object.

Here we can see that the tax_table inside our phyloseq object stores all the taxonomic labels that correspond to each OTU. OTUs are identified by a number in the row names of the table.

Next, let’s get rid of some of the unnecessary characters in the OTUs id and put names to the taxonomic ranks:

To remove unnecessary characters in .Data (matrix), we are going to use the command substring(). This command is useful to extract or replace characters in a vector. To use the command, we have to indicate the vector (x) followed by the first element to replace or extract (first) and the last element to be replaced (last). For instance: substring (x, first, last). substring() is a “flexible” command, especially to select characters of different lengths as in our case. Therefore, it is not necessary to indicate “last”, so it will take the last position of the character by default. Considering that a matrix is an arrangement of vectors, we can use this command. Each character in .Data is preceded by 3 spaces occupied by a letter and two underscores, for example: o__Rhodobacterales. In this case, “Rodobacterales” starts at position 4 with an R. So to remove the unnecessary characters we will use the following code:

> merged_metagenomes@tax_table@.Data <- substring(merged_metagenomes@tax_table@.Data, 4)
> colnames(merged_metagenomes@tax_table@.Data)<- c("Kingdom", "Phylum", "Class", "Order", "Family", "Genus", "Species")


Figure 2. Table of the taxonomic labels from our merged_metagenomes object with corrections.

To explore how many phyla we have, we are going to use a command named unique(). Let’s see the result we obtain from the next code:

> unique(merged_metagenomes@tax_table@.Data[,"Phylum"])

 [1] "Proteobacteria"              "Actinobacteria"              "Firmicutes"
[4] "Cyanobacteria"               "Deinococcus-Thermus"         "Chloroflexi"
[13] "Lentisphaerae"               "Kiritimatiellaeota"          "Chlamydiae"
[16] "Acidobacteria"               "Spirochaetes"                "Synergistetes"
[19] "Nitrospirae"                 "Tenericutes"                 "Coprothermobacterota"
[22] "Ignavibacteriae"             "Candidatus Cloacimonetes"    "Fibrobacteres"
[25] "Fusobacteria"                "Thermotogae"                 "Aquificae"
[28] "Thermodesulfobacteria"       "Deferribacteres"             "Chrysiogenetes"
[31] "Calditrichaeota"             "Elusimicrobia"               "Caldiserica"
[34] "Candidatus Saccharibacteria" "Dictyoglomi"


This is useful, but what we need to do if we need to know how many of our OTUs have been assigned to the phylum Firmicutes?. Let´s use the command sum() to ask R:

> sum(merged_metagenomes@tax_table@.Data[,"Phylum"] == "Firmicutes")

[1] 580


Now, to know for that phylum in particular which taxa there are in a certain rank we can ask it to phyloseq as well.

> unique(merged_metagenomes@tax_table@.Data[merged_metagenomes@tax_table@.Data[,"Phylum"] == "Firmicutes", "Class"])

[1] "Bacilli"          "Clostridia"       "Negativicutes"    "Limnochordia"     "Erysipelotrichia" "Tissierellia"


### Exploring the abundance table

Until now we have looked at the part of the phyloseq object that stores the information about the taxonomy (at all the possible levels) of each OTU found in our samples. But there is also a part of the phyloseq object that stores the information about how many sequenced reads corresponding to a certain OTU are there in each sample. This table is the otu_table.

> View(merged_metagenomes@otu_table@.Data)


Figure 3. Table of the abundance of reads in the merged_metagenomes object.

We will take advantage of this information later on in our analyses.

## Phyloseq objects

Finally, we can review our object and see that all datasets (i.e. JC1A, JP4D, and JP41) are in the object. If you look at our Phyloseq object, you will see that there are more data types that we can use to build our object(?phyloseq()), such as a phylogenetic tree and metadata concerning our samples. These are optional, so we will use our basic phyloseq object, for now, composed of the abundances of specific OTUs and the names of those OTUs.

## Exercise 1: Explore a phylum

Go into groups and choose one phylum that is interesting for your group, and use the learned code to find out how many OTUs have been assigned to your chosen phylum and what are the unique names of the genera inside it. がんばれ! (ganbare; good luck/keep it up!):

## Solution

Change the name of a new phylum wherever it is needed and the name of the rank that we are asking for, to get the result. As an example, here is the solution for Proteobacteria:

sum(merged_metagenomes@tax_table@.Data[,"Phylum"] == "Proteobacteria")

unique(merged_metagenomes@tax_table@.Data[merged_metagenomes@tax_table@.Data[,"Phylum"] == "Proteobacteria", "Genus"])


## Exercise 2: Searching for the read counts

Using the information from both the tax_table and the otu_table, find how many reads there are for any species of your interest (one that can be found in the tax_table).
Hint: Remember that you can access the contents of a data frame with the ["row_name","column_name"] syntax.
がんばれ! (ganbare; good luck/keep it up!):

## Solution

Go to the tax_table:

> View(merged_metagenomes@tax_table@.Data)


Take note of the OTU number for some species: Figure 4. The row of the tax_table corresponding to the species Paracoccus zhejiangensis.

Search for the row of the otu_table that has the row name that you chose.

> merged_metagenomes@otu_table@.Data["1077935",]

JC1A JP4D JP41
42  782  257


## Key Points

• kraken-biom formats kraken output-files of several samples into the single .biom file that will be phyloseq input.

• The library phyloseq manages metagenomics objects and computes analyses.

• A phyloseq object stores a table with the taxonomic information of each OTU and a table with the abundance of each OTU.