ITSxpress: Software to rapidly trim the internal transcribed spacer (ITS) region of FASTQ files

Author

• Adam R. Rivers, US Department of Agriculture, Agricultural Research Service
• Sveinn V. Einarsson, US Department of Agriculture, Agricultural Research Service

Citations

Einarsson, SV and Rivers, AR. ITSxpress Version 2: Software to rapidly trim internal transcribed spacer sequences with quality scores for amplicon sequencing. Microbiology Spectrum. In press, 2024.

Rivers AR, Weber KC, Gardner TG, Liu, S, Armstrong, SD. ITSxpress: Software to rapidly trim internally transcribed spacer sequences with quality scores for marker gene analysis [version 1; referees: awaiting peer review]. F1000Research 2018, 7:1418 (doi: 10.12688/f1000research.15704.1)

Introduction

The internally transcribed spacer region is a region between highly conserved the small subunit (SSU) of rRNA and the large subunit (LSU) of the rRNA. In Eukaryotes it contains the 5.8s genes and two variable length spacer regions. In amplicon sequencing studies it is common practice to trim off the conserved (SSU, 5,8S or LSU) regions. Bengtsson-Palme et al. (2013) published software the software package ITSx to do this.

ITSxpress is designed to support the calling of exact sequence variants rather than OTUs. This newer method of sequence error-correction requires quality score data from each sequence, so each input sequence must be trimmed. ITSXpress makes this possible by taking FASTQ data, de-replicating the sequences then identifying the start and stop sites using HMMSearch. Results are parsed and the trimmed files are returned. The ITS 1, ITS2 or the entire ITS region including the 5.8s rRNA gene can be selected. ITSxpress uses the hmm model from ITSx so results are comparable.

ITSxpress is also a QIIME2 plugin. Starting from version 2.0.0 of ITSxpress, the QIIME2 plugin is included with the command line version of ITSxpress. The installation method will be slightly different depending on whether QIIME2 is being used.

Installing ITSxpress for use as a QIIME2 Plugin

To install ITSxpress as a plugin for QIIME 2 first install QIIME 2 as a separate Conda/Mamba environemnt using thier instructions https://docs.qiime2.org/2024.5/install/ then add ITSxress to the QIIME 2 Conda environment. The examples below are for QIIME2 2 version 2024.2 an so please update the commands if you want a newer release.

For Linux:

conda env create -n qiime2-amplicon-2024.5 --file https://data.qiime2.org/distro/amplicon/qiime2-amplicon-2024.5-py39-linux-conda.yml
conda activate qiime2-amplicon-2024.5
conda install -c bioconda -c conda-forge ITSxpress
qiime dev refresh-cache

For maxOS (Intel) and OS X:

conda env create -n qiime2-amplicon-2024.5 --file https://data.qiime2.org/distro/amplicon/qiime2-amplicon-2024.5-py39-osx-conda.yml
 conda activate qiime2-amplicon-2024.5
conda install -c bioconda -c conda-forge ITSxpress
qiime dev refresh-cache

For macOS (Arm / Apple Silicon):

Installing ITSxpress for standalone use

For Linux, maxOS (Intel), and OS X:

mamba create -n itsxpressenv -c bioconda -c conda-forge itsxpress
mamba activate itsxpressenv

For macOS (Arm/Apple Silicon):

CONDA_SUBDIR=osx-64 mamba create -n itsxpressenv -c bioconda -c conda-forge itsxpress
mamba activate itsxpressenv
conda  config --env --set subdir osx-64

Running ITSxpress as a Docker container

docker pull ghcr.io/usda-ars-gbru/itsxpress
docker run [Options...] itsxpress

Dependencies

The software requires Vsearch, Hmmer and Biopython. Bioconda takes care of this for you so it is the preferred installation method.

Usage

Option	Description
-h, --help	Show this help message and exit.
--fastq	A .fastq, .fq, .fastq.gz or .fq.gz file. Required.
--single_end	A flag to specify that the fastq file is single-ended (not paired). Default is false.
--fastq2	A .fastq, .fq, .fastq.gz or .fq.gz file representing read 2 if present, optional.
--outfile	The trimmed FASTQ file, if it ends in gz it will be gzipped.
--outfile2	The trimmed FASTQ read 2 file, if it ends in gz it will be gzipped. If used, reads will be retuned as unmerged pairs rather than than merged.
--tempdir	Specify the temp file directory. Default is None.
--keeptemp	Should intermediate files be kept? Default is false.
--region	Options : {ITS2, ITS1, ALL}
--taxa	Select the taxonomic group sequenced: {Alveolata, Bryophyta, Bacillariophyta, Amoebozoa, Euglenozoa, Fungi, Chlorophyta, Rhodophyta, Phaeophyceae, Marchantiophyta, Metazoa, Oomycota, Haptophyceae, Raphidophyceae, Rhizaria, Synurophyceae, Tracheophyta, Eustigmatophyceae, Parabasalia, All}. Default Fungi.
--cluster_id	The percent identity for clustering reads range [0.99-1.0], set to 1 for exact de-replication. Default 1.0.
--log	Log file. Default is ITSxpress.log.
--threads	Number of processor threads to use. Default is 1.
--reversed_primers	Primers are in reverse orientation as in Taylor et al. 2016, DOI:10.1128/AEM.02576-16. If selected ITSxpress returns trimmed reads flipped to the forward orientation
--allow_staggered_reads	Allow merging staggered reads with --fastq_allowmergestagger for Vsearch --fastq_mergepairs. See Vsearch documentation. (Optional) Default is true.

Examples

Use case 1: Trimming the ITS2 region from a fungal amplicon sequencing dataset with forward and reverse gzipped FASTQ files using two cpu threads. Return a single merged file for use in Deblur.

itsxpress --fastq r1.fastq.gz --fastq2 r2.fastq.gz --region ITS2 \
--taxa Fungi --log logfile.txt --outfile trimmed_reads.fastq.gz --threads 2

ITSxpress can take uncompressed, gzipped or zstd compressed FASTQ files and it can write uncompressed, gzipped or zstd compressed FASTQ files. It expects FASTQ files to end in: .fq, .fastq, .fq.gz, fastq.gz, .fq.zst or fastq.zst.

Use case 2: Trimming the ITS2 region from a fungal amplicon sequencing dataset with forward and reverse gzipped FASTQ files using two cpu threads. Return a forward and reverse read files for use in Dada2.

itsxpress --fastq r1.fastq.gz --fastq2 r2.fastq.gz --region ITS2 \
--taxa Fungi --log logfile.txt --outfile trimmed_reads.fastq.gz --threads 2

ITSxpress can take uncompressed, gzipped or zstd compressed FASTQ files and it can write uncompressed, gzipped or zstd compressed FASTQ files. It expects FASTQ files to end in: .fq, .fastq, .fq.gz, fastq.gz, .fq.zst or fastq.zst.

Use case 3: Trimming the ITS2 region from a fungal amplicon sequencing dataset with an single-ended gzipped FASTQ files using two cpu threads.

itsxpress --fastq single-end.fastq.gz --single_end --region ITS2 --taxa Fungi \
--log logfile.txt --outfile trimmed_reads.fastq.gz --threads 2

Single ended data is less common and may come from a dataset where the reads have already been merged.

License information

This software is a work of the United States Department of Agriculture, Agricultural Research Service and is released under a Creative Commons CC0 public domain attribution.