Extending the ITS2-workbench with DNA barcoding capabilities

Background
The internal transcribed spacer 2 (ITS2) is a well-established marker for DNA barcoding, especially for plants and fungi.
Yet so far, no specific reference database exists dedicated to this purpose.
Extracting ITS2 sequences for a taxonomic group from GenBank is tedious manual work, which is prone to errors due to misannotations.
The same challenges also apply for phylogenetic analyses, for which we have already developed the ITS2-workbench.
It provides structure-based annotations and quality control for sequences extracted from GenBank.
Further, it offers tools for Hidden Markov Models (HMM) annotation, secondary structure prediction, alignment, and tree calculation.
However, it lacked the ability to taxonomically classify barcode sequences with confidence estimations.

Results
Here, we present an extension of the ITS2-workbench for the application in DNA barcoding.
It is based on the underlying data of 700,000 quality-controlled sequences from more than 175,000 species (2015 update).
This includes approximately 80,000 distinct species for plants and fungi, each.
Additionally, other eukaryotic groups that have been so far less represented in barcoding studies are covered (e.g., the class Bacillariophyceae).
The workbench uses UTAX as the classifier, which allows for barcoding of user-provided sequences and provides confidence values for each taxonomic rank.
For smaller data sets the results can be immediately viewed on the website.
For large data sets (e.g., next-generation sequencing amplicon data), the user is notified by email as soon as the download of the classification results is ready.

SignificanceAnkenbrand, Markus J. et al.. (2015). Extending the ITS2-workbench with DNA barcoding capabilities. Zenodo. 10.5281/zenodo.30543
The combination of a huge amount of quality-controlled ITS2 sequences with the powerful classification algorithm UTAX facilitates the broad usage of ITS2 as a DNA barcode.
The integration into the ITS2-workbench provides the means to also use the rich phylogenetic methods and supplementary reference sequences for further analyses.
The ITS2-workbench is freely accessible online at http://its2.bioapps.biozentrum.uni-wuerzburg.de

Cite as: Ankenbrand, Markus J. et al. (2015). Extending the ITS2-workbench with DNA barcoding capabilities. Zenodo. 10.5281/zenodo.30543 (http://dx.doi.org/10.5281/zenodo.30543)

Transcript

1. Extending the ITS2-workbench with DNA barcoding capabilities Markus J. Ankenbrand,1 Alexander Keller,1 Christian Koetschan,2 Matthias Wolf,2 J¨ org Schultz,2 Frank F¨ orster2 markus.ankenbrand@uni-wuerzburg.de 1Department of Animal Ecology and Tropical Biology (Molecular Biodiversity Group) - University of W¨ urzburg 2Department of Bioinformatics - University of W¨ urzburg August 20, 2015 MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 1 / 9
2. ITS2 Barcode 18S 5.8S 28S ITS1 ITS2 MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 2 / 9
3. ITS2 Barcode 18S 5.8S 28S ITS1 ITS2 MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 2 / 9
4. ITS2 Barcode 18S 5.8S 28S ITS1 ITS2 Barcode for fungia plantsb animalsc aSchoch et al. 2012, PNAS. bChen et al. 2010, PLoS ONE. cYao et al. 2010, PLoS ONE. MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 2 / 9
5. ITS2 Data Sources NCBI1 lots of data general inaccurate annotation not curated 1 NCBI Resource Coordinators 2015, Nucleic Acids Research. MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 3 / 9
6. ITS2 Data Sources NCBI1 lots of data general inaccurate annotation not curated BOLD Systems2 high quality specialized fungi only 1 NCBI Resource Coordinators 2015, Nucleic Acids Research. 2 Ratnasingham and Hebert 2007, Molecular ecology notes. MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 3 / 9
7. ITS2 Data Sources ITS2 Internal Transcribed Spacer 2 Ribosomal RNA Database NCBI1 lots of data general inaccurate annotation not curated BOLD Systems2 high quality specialized fungi only ITS2-database3,4 quality controlled data all eukaryotes contains 72% of plant species in USA over 700 000 sequences 1 NCBI Resource Coordinators 2015, Nucleic Acids Research. 2 Ratnasingham and Hebert 2007, Molecular ecology notes. 3 Schultz et al. 2006, Nucleic Acids Research. 4 Ankenbrand et al. 2015, Molecular Biology and Evolution. MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 3 / 9
8. ITS2-database Barcoding by Wipeter (https://commons.wikimedia.org/wiki/File:Diatom2.jpg) MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 4 / 9
9. ITS2-database Barcoding by Wipeter (https://commons.wikimedia.org/wiki/File:Diatom2.jpg) MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 4 / 9
10. ITS2-database Barcoding by Wipeter (https://commons.wikimedia.org/wiki/File:Diatom2.jpg) MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 4 / 9
11. ITS2-database Barcoding by Wipeter (https://commons.wikimedia.org/wiki/File:Diatom2.jpg) MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 4 / 9
12. ITS2-database Barcoding by Wipeter (https://commons.wikimedia.org/wiki/File:Diatom2.jpg) MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 4 / 9
13. ITS2 Barcoding - BLAST5 ITS2 Internal Transcribed Spacer 2 Ribosomal RNA Database 5 Altschul et al. 1990, Journal of Molecular Biology. MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 5 / 9
14. ITS2 Barcoding - BLAST5 ITS2 Internal Transcribed Spacer 2 Ribosomal RNA Database 5 Altschul et al. 1990, Journal of Molecular Biology. MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 5 / 9
15. ITS2 Barcoding - BLAST5 ITS2 Internal Transcribed Spacer 2 Ribosomal RNA Database UTAX Fast algorithm for taxonomy assignment, implemented in usearch6(v8) 5 Altschul et al. 1990, Journal of Molecular Biology. 6 Edgar 2010, Bioinformatics. MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 5 / 9
16. ITS2-database Barcoding by Wipeter (https://commons.wikimedia.org/wiki/File:Diatom2.jpg) MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 6 / 9
17. ITS2-database Barcoding by Wipeter (https://commons.wikimedia.org/wiki/File:Diatom2.jpg) MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 6 / 9
18. ITS2-database Barcoding by Wipeter (https://commons.wikimedia.org/wiki/File:Diatom2.jpg) MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 6 / 9
19. ITS2-database Barcoding by Wipeter (https://commons.wikimedia.org/wiki/File:Diatom2.jpg) MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 6 / 9
20. ITS2-database Meta-barcoding by Wipeter (https://commons.wikimedia.org/wiki/File:Diatom2.jpg) MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 7 / 9
21. ITS2-database Meta-barcoding by Wipeter (https://commons.wikimedia.org/wiki/File:Diatom2.jpg) by F¨ orstner (https://commons.wikimedia.org/wiki/File:Illumina_MiSeq_sequencer.jpg) MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 7 / 9
22. ITS2-database Meta-barcoding 7 by Wipeter (https://commons.wikimedia.org/wiki/File:Diatom2.jpg) by F¨ orstner (https://commons.wikimedia.org/wiki/File:Illumina_MiSeq_sequencer.jpg) 7 Sickel et al. 2015, BMC Ecology. MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 7 / 9
23. ITS2-database Meta-barcoding by Wipeter (https://commons.wikimedia.org/wiki/File:Diatom2.jpg) by F¨ orstner (https://commons.wikimedia.org/wiki/File:Illumina_MiSeq_sequencer.jpg) 7 Sickel et al. 2015, BMC Ecology. MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 7 / 9
24. ITS2-database Meta-barcoding by Wipeter (https://commons.wikimedia.org/wiki/File:Diatom2.jpg) by F¨ orstner (https://commons.wikimedia.org/wiki/File:Illumina_MiSeq_sequencer.jpg) 7 Sickel et al. 2015, BMC Ecology. MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 7 / 9
25. ITS2-database Meta-barcoding Amphora 0.5% Achnanthales 0.4% + 0.1% Bacillariophyceae Bacillariaceae Pseudo-nitzschia P. delicatissim a 29% P. pungens 19% 4% P. calliantha 3% P. australis 2% P. multistriata 2% P. cuspidata 1% P. fraudulenta 1% P. m ultiseries 1% P. dolorosa 1% P. brasiliana 0.8% P. pseudodelicatissim a 0.8% P. seriata 18 more Fragilariopsis 2% F. sp. NL-2010 1% F. sp. DITS004-08 1% F. nana 3 more 3 more Naviculales Navicula 4% N. phyllepta 2% N. cryptocephala 0.8% N. trivialis 6 m ore Sellaphora 3% S. pupula 3% S. capitata 2% P. tricornutum 6% E. bilunaris 8 by F¨ orstner (https://commons.wikimedia.org/wiki/File:Illumina_MiSeq_sequencer.jpg) 7 Sickel et al. 2015, BMC Ecology. 8 Ondov, Bergman, and Phillippy 2011, BMC bioinformatics. MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 7 / 9
26. Integration Barcoding and Phylogeny MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 8 / 9
27. Integration Barcoding and Phylogeny MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 8 / 9
28. Integration Barcoding and Phylogeny MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 8 / 9
29. Integration Barcoding and Phylogeny MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 8 / 9
30. Integration Barcoding and Phylogeny MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 8 / 9
31. Integration Barcoding and Phylogeny MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 8 / 9
32. Conclusion Soon: http://its2.bioapps.biozentrum.uni-wuerzburg.de Beta: http://barcode.its2database.info MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 9 / 9
33. Conclusion Soon: http://its2.bioapps.biozentrum.uni-wuerzburg.de Beta: http://barcode.its2database.info Further development P-values instead of raw-scores Custom reference sets Dynamic taxonomic levels MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 9 / 9
34. Conclusion Soon: http://its2.bioapps.biozentrum.uni-wuerzburg.de Beta: http://barcode.its2database.info Further development P-values instead of raw-scores Custom reference sets Dynamic taxonomic levels Acknowledgements Dept. of Animal Ecology and Tropical Biology Dept. of Bioinformatics for funding MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 9 / 9
35. Conclusion Soon: http://its2.bioapps.biozentrum.uni-wuerzburg.de Beta: http://barcode.its2database.info Further development P-values instead of raw-scores Custom reference sets Dynamic taxonomic levels Acknowledgements Dept. of Animal Ecology and Tropical Biology Dept. of Bioinformatics for funding Slides are available at https://speakerdeck.com/iimog/. This work is licensed under http://creativecommons.org/licenses/by/4.0/ except logos or where otherwise noted. MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 9 / 9
36. References I Altschul, S. F. et al. (1990). “Basic local alignment search tool”. eng. In: Journal of Molecular Biology 215.3, pp. 403–410. doi: 10.1016/S0022-2836(05)80360-2. Ankenbrand, Markus J. et al. (2015). “ITS2 database V: Twice as much”. en. In: Molecular Biology and Evolution, msv174. doi: 10.1093/molbev/msv174. Chen, Shilin et al. (2010). “Validation of the ITS2 Region as a Novel DNA Barcode for Identifying Medicinal Plant Species”. In: PLoS ONE 5.1. doi: 10.1371/journal.pone.0008613. Edgar, Robert C. (2010). “Search and clustering orders of magnitude faster than BLAST”. en. In: Bioinformatics 26.19, pp. 2460–2461. doi: 10.1093/bioinformatics/btq461. NCBI Resource Coordinators (2015). “Database resources of the National Center for Biotechnology Information”. In: Nucleic Acids Research 43.Database issue, pp. D6–D17. doi: 10.1093/nar/gku1130. MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 1 / 3
37. References II Ondov, Brian D, Nicholas H Bergman, and Adam M Phillippy (2011). “Interactive metagenomic visualization in a Web browser.” In: BMC bioinformatics 12.1, p. 385. doi: 10.1186/1471-2105-12-385. Ratnasingham, Sujeevan and Paul DN Hebert (2007). “BOLD: The Barcode of Life Data System (http://www.barcodinglife.org)”. In: Molecular ecology notes 7.3, pp. 355–364. Schoch, Conrad L. et al. (2012). “Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi”. In: PNAS 109.16, pp. 6241–6246. doi: 10.1073/pnas.1117018109. Schultz, J¨ org et al. (2006). “The internal transcribed spacer 2 database—a web server for (not only) low level phylogenetic analyses”. en. In: Nucleic Acids Research 34.suppl 2, W704–W707. doi: 10.1093/nar/gkl129. Sickel, Wiebke et al. (2015). “Increased efficiency in identifying mixed pollen samples by meta-barcoding with a dual-indexing approach”. en. In: BMC Ecology 15.1, p. 20. doi: 10.1186/s12898-015-0051-y. MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 2 / 3
38. References III Yao, Hui et al. (2010). “Use of ITS2 Region as the Universal DNA Barcode for Plants and Animals”. In: PLoS ONE 5.10, e13102. doi: 10.1371/journal.pone.0013102. MJ Ankenbrand (MolBioDiv W¨ urzburg) ITS2-workbench barcoding August 20, 2015 3 / 3