itsxpress 1.8.1

Rapidly trim sequences down to their Internally Transcribed Spacer ITS regions

Author

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

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

Citation

Rivers AR, Weber KC, Gardner TG et al. ITSxpress: Software to rapidly trim internally transcribed spacer sequences with quality scores for marker gene analysis [version 1]. F1000Research 2018, 7:1418 (doi: 10.12688/f1000research.15704.1)

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This is the end of life version 1 ITSxpress. The new version 2 of ITSxpress, has the Qiime2 plugin built in with the command line version of ITSxpress. See master branch of ITSxpress.

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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 ITS1, 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 available as a QIIME2 Plugin

Installation

This is the installation of the final iteration of ITSxpress version 1: (BBmap is no longer used in ITSxpress version 2):

• This version should primarily be used for reproducability with other datasets, which used ITSxpress =<1.8.1

• The new version 2 is compatible with the newer versions of Qiime2

• If you want to install this iteration of ITSxpress with Qiime2, then you you need to follow the install instructions here: QIIME2 Plugin

Since this version is no longer supported, you must create a new conda environment in order for the depenendencies to be compatible.

Example on how to install and create new conda environment for this version of ITSxpress. We are using mamba because it resolves packages better and faster, but conda can be substituted.

• Information on installing mamba or micromamba (either highly recommended) can be found here: mamba installation guide

mamba create -n ITSxpress_V1EOL python=3.8.13
mamba activate ITSxpress_V1EOL
#or
conda create -n ITSxpress_V1EOL python=3.8.13
conda activate ITSxpress_V1EOL

ITSxpress can be installed in 3 ways:

1. Bioconda: (preferred method because it handles dependencies):

mamba install -y -c bioconda itsxpress==1.8.1

2. Pip: https://pypi.org/project/itsxpress/:

   • If using Pip, you will need to specify the versions of the dependencies listed below before installing itsxpress

mamba install -y -c bioconda hmmer==3.1b2
mamba install -y -c bioconda bbmap==38.69
mamba install -y -c bioconda vsearch==2.21.1
pip install itsxpress

3. The Github repository: https://github.com/USDA-ARS-GBRU/itsxpress

git clone -branch 1.8.1-EOL https://github.com/USDA-ARS-GBRU/itsxpress.git

Dependencies

This software requires Vsearch=2.21.1, BBtools=38.69, Hmmer=3.1b2 and Biopython>=1.79. 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. Interleaved or not. 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, 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 gzipped or un-gzipped FASTQ files and it can write gzipped or un-gzipped FASTQ files. It expects FASTQ files to end in: .fq, .fastq, .fq.gz or fastq.gz.

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 gzipped or un-gzipped FASTQ files and it can write gzipped or un-gzipped FASTQ files. It expects FASTQ files to end in: .fq, .fastq, .fq.gz or fastq.gz.

Use case 3: Trimming the ITS2 region from a fungal amplicon sequencing dataset with an interleaved gzipped FASTQ files using two cpu threads. Return a single merged file for use in Deblur.

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

Use case 4: 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.

Use case 5: Trimming the ITS1 region from a Alveolata amplicon sequencing dataset with an interleaved gzipped FASTQ files using 8 cpu threads.

itsxpress --fastq interleaved.fastq.gz --region ITS1 --taxa Alveolata \
--log logfile.txt --outfile trimmed_reads.fastq.gz --threads 8

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.