Rsearch

Introduction

Rsearch is an R package designed for handling and analyzing targeted sequencing data. The package provides a user-friendly interface for core VSEARCH functions in addition to tools for visualization and parameter optimization.

The core idea behind Rsearch is to retain the output from VSEARCH within R’s generic data structures, rather than writing results to files as the original VSEARCH functions. By offering this option users can choose between working entirely within R and Rstudio or to export results to files as VSEARCH typically does. Keeping all results in R data structures allows users to leverage the power of standard data wrangling and visualization tools familiar to R users.

Another feature that enhances usability for R users is the consistent return format of the functions. All functions return a single table/data frame unless the user specifies that results should be written to a file. For functions that can return multiple results - such as those handling read pairs with forward and reverse reads - the secondary table is included as an attribute of the primary table. The same approach applies to tables containing statistics from function executions. By ensuring that all functions return only one table, navigating and managing results become more straightforward. Additionally, since all core functions return data frames or tibbles, they are compatible with piping using the %>% or |> operators.

More information about attributes in R can be found here and here.

Full documentation and tutorials with usage examples are available on the Rsearch website

Installation

• A short video on how to install on a Windows laptop
• A short video on how to install on a Mac laptop

Prerequisites

For the Rsearch package to function properly on your computer, VSEARCH must be installed as well (see below). Please ensure that you are using VSEARCH version 2.30.0 or newer.

Visit the VSEARCH GitHub site for learning more about VSEARCH.

Installing VSEARCH

You typically install VSEARCH by simply downloading a pre-compiled binary file to your computer (Windows or Mac). The latest release of VSEARCH, with corresponding binaries, for installation can be found under Releases. On a High Performance Computing (HPC) cluster we prefer to use an apptainer container for VSEARCH. These are freely available from many sites, e.g.  https://depot.galaxyproject.org/singularity/

After downloading the binary you may edit your PATH environment variable to tell your operating system where to find the VSEARCH binary. However, this is not required since the Rsearch package has a function set_vsearch_executable() where you specify where your VSEARCH binary file is found (see Set correct vsearch executable) below.

Installing Rsearch

Bioconductor dependency

Rsearch also relies on the Bioconductor package phyloseq. Please install it before installing Rsearch if you do not already have it installed:

if (!requireNamespace("BiocManager", quietly = TRUE)) {
    install.packages("BiocManager")
}
BiocManager::install("phyloseq")

You can install the development version of Rsearch from GitHub by using the devtools package from CRAN:

if (!requireNamespace("devtools", quietly = TRUE)) {
  install.packages("devtools")
}
devtools::install_github("CassandraHjo/Rsearch")

After installation, it is a good idea to restart your R session (in Rstudio: Session > Restart R) to make sure every thing is properly loaded.

Set correct VSEARCH executable

In order for most of the functions (those starting with vs_) in Rsearch to work, the command to invoke VSEARCH must be set correctly. The default command is simply vsearch, but this will only work if the file vsearch.exe is found in a folder that is included in the PATH environment variable.

If this is not the case, you must tell Rsearh explicitly where to find or how to invoke vsearch. The Rsearch function set_vsearch_executable() can be used to set the correct command to invoke VSEARCH on the computer like this:

# Windows example
Rsearch::set_vsearch_executable("C:/Documents/vsearch") # If the vsearch binary (vsearch.exe) is copied to C:/Documents/ on the computer

# Linux/macOS example
Rsearch::set_vsearch_executable("/usr/local/bin/vsearch") # If the vsearch binary (vsearch.exe) is copied to /usr/local/bin/ on the computer

This will store the path and use it in future sessions automatically.

Using an Apptainer/Singularity Container

Although Rsearch is primarily intended for local execution (as above), it is also possible to use vsearch packaged in an Apptainer or Singularity .sif container. However, since Rsearch expects a single executable path (not a full shell command), you must create a wrapper script to bridge the container invocation.

Step by step instructions:

1. Create a wrapper script (e.g., vsearch) with the following content:

#!/bin/bash
apptainer exec /path/to/vsearch_container.sif vsearch "$@"

2. Save it to a folder, for example:

/home/youruser/bin/vsearch

3. Make the script executable:

chmod +x /home/youruser/bin/vsearch

4. Point Rsearch to this wrapper script:

Rsearch::set_vsearch_executable("/home/youruser/bin/")

This will make Rsearch treat the containerized version of vsearch as a regular executable.

Test that it works

You may test if your executable is working properly by running the following command:

Rsearch::vsearch()

If everything is set up correctly you should see a message like this:

[1] "The VSEARCH executable is: /your/path/vsearch"
[1] "This is a valid command to invoke VSEARCH on this computer!"

Note: For large-scale analyses and computationally intensive workflows, calling vsearch directly from a shell script may be more efficient than using Rsearch through R or RStudio.

Documentation

Accessing help within R

Documentation can be accessed directly in the R console. Here are some methods to access help:

• Function-specific help: To get detailed information about a specific function, use the ? operator followed by the function name. For example, to access help for the vs_fastx_trim_filt function:

?vs_fastx_trim_filt

Alternatively, you can use the help() function:

help(vs_fastx_trim_filt)

• Package-wide help

To get an overview of the Rsearch package an its available functions, use:

# library(Rsearch)
help(package = "Rsearch")

Usage

Additional usage examples can be found in the documentation for each individual function and on the package website.

Example: Filter paired-end reads based on quality

library(Rsearch)

# Define input
fastx_input <- "R1_sample1.fq"
reverse <- "R2_sample1.fq"

# Execute filtering, with tibble as output
filt_seqs <- vs_fastx_trim_filt(fastx_input = fastx_input,
                                reverse = reverse)

# Extract tibbles with filtered sequences
R1_filt <- filt_seqs
R2_filt <- attr(filt_seqs, "reverse")

# Extract filtering statistics
statistics <- attr(filt_seqs, "statistics")

Contributors

The main contributors to Rsearch:

• Cassandra Hjortdahl cassandra.hjortdahl@nmbu.no (Coding, testing, documentation, maintaining)
• Lars Snipen lars.snipen@nmbu.no (Coding, documentation)
• Torbjørn Rognes torognes@ifi.uio.no (Coding, documentation)
• Hilde Vinje hilde.vinje@nmbu.no (Coding, documentation)

Citing Rsearch

Please cite the following publication if you use Rsearch:

xxx

Please note that citing any of the underlying algorithms, e.g. VSEARCH, may also be appropriate.

References

• Rognes T, Flouri T, Nichols B, Quince C, Mahé F (2016) VSEARCH: a versatile open source tool for metagenomics. PeerJ 4:e2584. doi: 10.7717/peerj.2584
• The subplot of the Rsearch logo is created in https://BioRender.com