ITSx: Improved software detection and extraction of ITS1 and ITS2 from ribosomal ITS sequences of fungi and other eukaryotes for use in environmental sequencing
Johan Bengtsson-Palme, Vilmar Veldre, Martin Ryberg, Martin Hartmann, Sara Branco, Zheng Wang, Anna Godhe, Yann Bertrand, Pierre De Wit, Marisol Sanchez, Ingo Ebersberger, Kemal Sanli, Filipe de Souza, Erik Kristiansson, Kessy Abarenkov, K. Martin Eriksson, R. Henrik Nilsson
Methods in Ecology and Evolution, 4: 914-919, 2013
(DOI: 10.1111/2041-210X.12073)

ITSx is an open source software utility to extract the highly variable ITS1 and ITS2 subregions from ITS sequences, which is commonly used as a molecular barcode for e.g. fungi. As the inclusion of parts of the neighbouring, very conserved, ribosomal genes (SSU, 5S and LSU rRNA sequences) in the sequence identification process can lead to severely misleading results, ITSx identifies and extracts only the ITS regions themselves. The software is a complete re-write of the fungal ITS extractor, and among the new features included in ITSx are support for a wide range of additional phylums, and multicore support. A paper describing ITSx has been published in Methods in Ecology and Evolution.

ITSx is as far as we know bug free. Nevertheless, if you encounter any unexpected behavior, please contact me at the e-mail address below.

Download the ITSx package
Latest version (1.1.3)
You can read the manual here.

Please note that the “test.fasta” file indicated in the manual is accidentally missing in the 1.0.11 version of the software. It has returned in version 1.1.

ITSx is now available as a Bioconda package from here:

If you are interested in a third-party parallel ITSx implementation, allowing 2x to 10x speed increases on certain datasets (and maybe more if you have lots of CPU cores), please check out Andrew Krohn’s parallel ITSx implementation here: [Documentation]

Please e-mail me if you encounter bugs or have questions! If you do not find your answer there, then feel free to e-mail me. My e-mail address is firstname.lastname[a] (and my name is Johan Bengtsson-Palme).

Version history

  • 8th February 2021 – 1.1.3
  • 3rd October 2019 – 1.1.2
  • 29th January 2019 – 1.1.1
  • 30th June 2017 – 1.1b (public beta version)
  • 17th December 2014 – 1.0.11
  • 16th October 2014 – 1.0.10
  • 23rd June 2014 – 1.0.9
  • 24th May 2014 – 1.0.8
  • 2nd May 2014 – 1.0.7
  • 6th February 2014 – 1.0.6
  • 4th February 2014 – 1.0.5
  • 10th November 2013 – 1.0.4
  • 2nd August 2013 – 1.0.3
  • 9th July 2013 – 1.0.2
  • 10th May 2013 – 1.0.1 public beta
  • 2nd April 2013 – 1.0 public beta
  • 26th February 2013 – 1.0 pre-release version 6 – not a public release.
  • 29th January 2013 – 1.0 pre-release version 5 – not a public release.
  • 16th January 2013 – 1.0 pre-release version 4 – not a public release.
  • 6th December 2012 – 1.0 pre-release version 3 – not a public release.
  • 22nd November 2012 – 1.0 pre-release version 2 – not a public release.
  • 26th October 2012 – 1.0 pre-release version – not a public release.

If you for any reason would be interested in an older version of ITSx, please e-mail me at the address below.

Contact information

Johan Bengtsson-Palme (firstname.lastname [at]
University of Gothenburg
Department of Infectious Diseases