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J Vis Exp. 2012 Mar 12;(61). pii: 3806. doi: 10.3791/3806.

The ITS2 Database.

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Department of Bioinformatics, Biocenter, University of Würzburg, Germany.


The internal transcribed spacer 2 (ITS2) has been used as a phylogenetic marker for more than two decades. As ITS2 research mainly focused on the very variable ITS2 sequence, it confined this marker to low-level phylogenetics only. However, the combination of the ITS2 sequence and its highly conserved secondary structure improves the phylogenetic resolution(1) and allows phylogenetic inference at multiple taxonomic ranks, including species delimitation(2-8). The ITS2 Database(9) presents an exhaustive dataset of internal transcribed spacer 2 sequences from NCBI GenBank(11) accurately reannotated(10). Following an annotation by profile Hidden Markov Models (HMMs), the secondary structure of each sequence is predicted. First, it is tested whether a minimum energy based fold(12) (direct fold) results in a correct, four helix conformation. If this is not the case, the structure is predicted by homology modeling(13). In homology modeling, an already known secondary structure is transferred to another ITS2 sequence, whose secondary structure was not able to fold correctly in a direct fold. The ITS2 Database is not only a database for storage and retrieval of ITS2 sequence-structures. It also provides several tools to process your own ITS2 sequences, including annotation, structural prediction, motif detection and BLAST(14) search on the combined sequence-structure information. Moreover, it integrates trimmed versions of 4SALE(15,16) and ProfDistS(17) for multiple sequence-structure alignment calculation and Neighbor Joining(18) tree reconstruction. Together they form a coherent analysis pipeline from an initial set of sequences to a phylogeny based on sequence and secondary structure. In a nutshell, this workbench simplifies first phylogenetic analyses to only a few mouse-clicks, while additionally providing tools and data for comprehensive large-scale analyses.

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