Improved software detection and extraction of ITS1 and ITS2 from ribosomal ITS sequences of fungi and other eukaryotes for analysis of environmental sequencing data

Abstract

Summary1. The nuclear ribosomal internal transcribed spacer (ITS) region is the primary choice for molecular identification of fungi. Its two highly variable spacers (ITS1 and ITS2) are usually species specific, whereas the intercalary 5.8S gene is highly conserved. For sequence clustering and BLAST searches, it is often advantageous to rely on either one of the variable spacers but not the conserved 5.8S gene. To identify and extract ITS1 and ITS2 from large taxonomic and environmental data sets is, however, often difficult, and many ITS sequences are incorrectly delimited in the public sequence databases. 2. We introduce ITSx, a Perl-based software tool to extract ITS1, 5.8S and ITS2 -as well as full-length ITS sequences -from both Sanger and high-throughput sequencing data sets. ITSx uses hidden Markov models computed from large alignments of a total of 20 groups of eukaryotes, including fungi, metazoans and plants, and the sequence extraction is based on the predicted positions of the ribosomal genes in the sequences. 3. ITSx has a very high proportion of true-positive extractions and a low proportion of false-positive extractions. Additionally, process parallelization permits expedient analyses of very large data sets, such as a one million sequence amplicon pyrosequencing data set. ITSx is rich in features and written to be easily incorporated into automated sequence analysis pipelines. 4. ITSx paves the way for more sensitive BLAST searches and sequence clustering operations for the ITS region in eukaryotes. The software also permits elimination of non-ITS sequences from any data set. This is particularly useful for amplicon-based next-generation sequencing data sets, where insidious non-target sequences are often found among the target sequences. Such non-target sequences are difficult to find by other means and would contribute noise to diversity estimates if left in the data set.

Assembly of seed-associated microbial communities within and across successive plant generations

“…The variable ITS1 regions of the fungal internal transcribed spacer were extracted with the Perl-based software ITSx 1.0.4 (Bengtsson-Palme et al 2013). Reference-based clustering was performed with UCLUST 1.2.22 algorithm (Edgar 2010) at a 97% identity cut-off against the UNITE 7.1 database (Abarenkov et al 2010).…”

Stochastic distribution of small soil eukaryotes resulting from high dispersal and drift in a local environment

“…Chimeras were checked in UCHIME (Edgar et al, 2011). The full ITS2 region was extracted in the ITSx (Bengtsson-Palme et al, 2013). The extracted sequences were assembled into Operational Taxonomic Units (OTUs) based on 97% pairwise similarity using CD-hit 4.6.1 (Fu et al, 2012).…”

Ecological succession reveals potential signatures of marine–terrestrial transition in salt marsh fungal communities

“…Initial read quality filtering and dereplication were carried out using USEARCH v7 (Edgar, 2013) (USEARCH parameter: maxEE: remove reads with expected number of base call errors exceeding 0.5). We further used ITSx 1.0.11 (Bengtsson-Palme et al, 2013) to remove the flanking 5.8S and 18S rRNA gene sequences for optimal resolution of ITS1 clustering and removal of compromised and nonfungal target sequences. The extracted sequences were trimmed to equal lengths and further binned into operational taxonomic units (OTUs) at 97% identity using UPARSE (Edgar, 2013).…”