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A QIIME2 plugin to trim ITS regions using ITSxpress

Project description

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Authors

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

  • Kyle C. Weber, US Department of Agriculture, Agricultural Research Service

Introduction

The internally transcribed spacer (ITS) is a region between the small subunit and large subunit rRNA genes. In is a commonly used phylogenetic marker for Fungi and other Eukaryotes. The ITS contains the 5.8s gene 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 a software package ITSx to do this.

Q2_ITSxpress extends this work by rapidly trimming FASTQ sequences within Qiime2. Q2_ITSxpress is the Qiime2 plugin version of the stand alone command line utility ITSxpress. Q2_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 models from ITSx so results are nearly identical.

Requirements/Dependencies

Q2_itsxpress Installation

  1. Activate the Qiime2 conda environment

source activate qiime2-2018.6
  1. Install Q2_itsxpress using BioConda. Be sure to install Q2_itsxpres in the Qiime2 environment.

conda config --add channels bioconda

      conda install q2-itsxpress
  1. In your Qiime2 environment, refresh the plugins.

qiime dev refresh-cache
  1. Check to see if the ITSxpress plugin is installed. You should see an output similar to the image below.

qiime itsxpress
https://i.gyazo.com/2216236a43c75a92174185b4d81a2eb5.png

Usage

Within Qiime2 you can trim paired end or single end reads either of these commands

      qiime itsxpress trim-single

qiime itsxpress trim-paired
  1. qiime itsxpress trim-single

Command-requirement

Description

–i-per-sample-sequences

  • The artifact that contains the sequence file(s).

  • Either Joined Paired or just a single fastq.

  • One file sequence in the qza data folder.

–p-region

  • The regions ITS2, ITS1, and ALL.

–p-taxa

  • Select the taxonomic group sequenced: A, B, C, D, E, F, G, H, I, L, M, O, P, Q, R, S, T, U, V, ALL.

–p-threads

  • The amount of threads to use.

–o-trimmed

  • The resulting trimmed sequences from ITSxpress in a qza format.

–cluster-id

  • The percent identity for clustering reads, set to 1 for exact dereplication.

  1. qiime itsxpress trim-pair

Command-requirement

Description

–i-per-sample-sequences

  • The artifact that contains the sequence file.

  • Only paired will work.

  • Two file sequences in the qza data folder.

–p-region

  • The regions ITS2, ITS1, and ALL.

–p-taxa

  • Select the taxonomic group sequenced: A, B, C, D, E, F, G, H, I, L, M, O, P, Q, R, S, T, U, V, ALL.

–p-threads

  • The amount of threads to use.

–o-trimmed

  • The resulting trimmed sequences from ITSxpress in a qza format.

–cluster-id

  • The percent identity for clustering reads, set to 1 for exact dereplication.

Taxa Key

A

Alveolata

B

Bryophyta

C

Bacillariophyta

D

Amoebozoa

E

Euglenozoa

F

Fungi

G

Chlorophyta (green algae)

H

Rhodophyta (red algae)

I

Phaeophyceae (brown algae)

L

Marchantiophyta (liverworts)

M

Metazoa

O

Oomycota

P

Haptophyceae (prymnesiophytes)

Q

Raphidophyceae

R

Rhizaria

S

Synurophyceae

T

Tracheophyta (higher plants)

U

Eustigmatophyceae

ALL

All

Example

Use case: Trimming the ITS2 region from a fungal amplicon sequencing dataset with a PairedSequencesWithQuailty qza using two cpu threads. The example file used is in the Tests folder under paired.qza.

qiime itsxpress trim-pair --i-per-sample-sequences ~/parired.qza --p-region
ITS2 \ --p-taxa F --p-threads 2 --o-trimmed ~/Desktop/out.qza

License information

This software is a work of the United States Department of Agriculture, Agricultural Research Service. 17 U.S.C. Section 105 states that “Copyright protection under this title is not available for any work of the United States Government”. While I anticipate that this work will be released under a CC0 public domain attribution, only the USDA ARS Office of Technology transfer has the authority to make that determination.

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