The internal transcribed spacer two ribosomal gene or I TS two is one of the most important markers in molecular systematics. Research of the last 20 years has mainly focused on exploiting the I Ts two sequences for phylogenetic reconstruction. However, the secondary structure of I TS two is increasingly being used in the field of phylogenetic inference due to several reasons.
Secondary structure analysis extends the taxonomic applicability of this marker, whereas the rapidly evolving and therefore very variable I TS two sequence is mainly suited for phylogenetic analysis at species level or lower. Adding structural information permits analysis at higher taxonomic rank simultaneously due to the strongly conserved I ts two secondary structure consisting of its four characteristic ess. Thus, the results from secondary structure analysis can improve the phylogenetic resolution obtained from the primary sequence.
Welcome, dear colleagues, my name is Matthias Wolf. I'm working in the I ts two proof for about eight years now. I'm working here at the Department of Bioinformatics at the bio center at the University of Wurzburg in Germany.
Today we would like to present you a little movie on how to use the I ts two database. Hi, I'm y Chu. The reason for that movie is that the I Ts two database is in fact not only a database for storage, but we also provide a lot of tools which allow you to improve your phylogenetic analysis.
In this movie, we want to show you how to work with these tools and how to connect all these tools together.Correct. Delineation of the I Ts two sequence can be crucial for structure prediction. Hence, a web-based interface for the hidden markoff model-based annotation has been implemented.
To achieve a correct annotation of your I Ts two sequences, you can paste them into the sequence editor at the top of the website. This is demonstrated by loading the example sequences of plans. The sequence editor itself checks whether your I TS two sequences are valid or not, and outputs error messages.
For example, when you use non PAC characters After choosing the correct HMM, you can start the process by clicking annotate each 5.8 s and 28 SRNA That could be identified by the program is removed, leaving the correctly annotated I ts two sequences, which were located in between. As a result, delimited I TS two sequences are shown as well as the predicted hybrid of 5.8 s and 28 S-R-R-N-A. As a confirmation of the HMM annotations accuracy after annotation of newly retained I ts two sequences, secondary structures may be determined by two means.
First prediction may be accomplished by homology modeling with the complete set of sequences and structures of the database serving as templates. To predict the structure of your annotated, its two sequence click predict structure. If your right two sequence can fold directly into the typical its two secondary structure, the result is depicted immediately.
You can now choose between several options to further process your sequence structure, including adding it to your data pool. If your right here, two sequence cannot fold directly, a blast search against the database is performed. The resulting page shows the best blast hits, including the homology modeling for each plastic as template.
With a click on the plus sign details are shown, you can select the sequence structure pairs of your choice and add them to the data pool, either by track and drop or using the context menu with the right click. A second approach is to manually enter one or multiple sequences, including their secondary structures as templates to transfer the most suitable secondary structures to your query sequences. This is demonstrated by loading the example file for multiple templates.
By clicking Predict best templates, you perform the homology modeling with default settings. The Homology modeling program will calculate all against all structures and display the best query template combinations. In the resulting table, you can see which templates successfully could use its secondary structure to model one or several query sequences.
Again, details of the results can be opened with a click on the plus sign as the complete homology modeling approach is independent of the I TS two. It may be used to predict the secondary structure of any RNA given a homologous molecule with a known structure. Also, resulting sequence structure pairs can be added to the data pool per drag and tr or through the context menu, which is opened with the right click.
In addition to the overall structure, conserved motives like A-U-G-G-U sequence preceding the apex of the third helix and a perine pyramid in mismatch in the second helix have been described for the I TS two. This UU mismatch is surrounded by two motives, one to the left of helix two and one to the right between helix two and three with an additional triple A nucleotide. Having transformed these sequence motives into hms, we now provide identification of these motives in sequences of interest.
To search for these motives, enter your query sequence into the search field and choose the correct model. Again, this is demonstrated by loading the example file for plans. To start the calculations, click on motive search.
Found I ts two motives are annotated and highlighted in your query sequences. One possibility to retrieve I ts two sequence structure pairs from the I Ts two database is the search function. It allows the user to search for sequence structures either by taxon name or by gen bank identifier.
To search for a sequence structure by GenBank identifier, type the GI into the search field and click search. A list of hits appears in a new tab. You can view the details of a sequence structure by clicking on show details.
Also, you can show the sequence S directs of all hits within the tab by clicking on show sequence structure per drag and drop, or through the context menu with a right click, the sequence structures of choice can be added to the data pool. Beside the search for sequence structure pairs by gi, you can type a taxon name into the search field, which is supported by an appearing live search box. After the new tab appears listing all the hits, you can access the same functions as before.
For instance, adding selection to the data pool per drag and drop. The second possibility to retrieve sequence structure pairs from the database is the browse function. Here, the text are assorted.
According to the NCBI taxonomy database, the data is accessible through the tree-like structure. On the left of the website. With the click on the plus sign, you can show the texta one level beneath.
With a click on a taxon name, you open a new tab containing each sequence structure, pair of the taxon. In addition to a search for sequences and structures with CEM bank identifiers or species information, the I Ts two database also provides a blast based search. However, standout blast procedures are frequently not able to identify distantly related.
I ts two sequences because of the high sequence divergence. To overcome this hindrance, we have implemented a sequence and structure-based blast search that includes information about the highly conserved structure for the homology search. This is done by translating the sequence structure into a 12 letter pseudo protein sequence.
Thus species sampling that starts with any sequence of interest and covers. Broad taxonomic ranges has become as simple as a blast search. To perform a blast search, you can type your query sequences into the sequence editor.
If your query sequence is a plain nucleotide sequence without structure, a common blast n algorithm is used for sequences including their secondary structures. The web interface uses the I Ts two blast algorithm. Start the process by clicking blast.
After a few moments, your blast hits are listed in a new tap. Here you see one tap for each query sequence. With the click on show alignments, you can scroll through plaster alignments As before you can select the sequence structure pairs of your choice and put them into the data pool.
During your work on the ITS two database, your data pool may have filled with several sequence structures. You can access your data pool anytime at all and show its content. The next step of your analysis is the multiple sequence structure alignment.
Click on analyze dataset, and then sequence and structure to perform the sequence structure alignment. Now, you are asked to select the graphic mode of your alignment. For a large set of sequences, it is recommended to select the slim version.
Since our data pool only contains a few sequence structures, we choose the full graphic mode. After the calculation is complete, the alignment is added to your data pool. By highlighting one side of a base pair, you automatically highlight its counterpart.
Finally, the alignment can be safe with a click on safe alignment. Once you are satisfied with the quality of your sequence structure alignment, you can calculate a phylogenetic neighbor joining tree by clicking analyze data set, and then neighbor. Joining the resulting tree appears in a new tab.
You can scale your tree freely using the scroll bar, reroute your tree by clicking on an arbitrary node or leaf of the tree and then reroute at this node. If you want to remove a taxon from your data pool, click on the leaf and choose. Remove this node from pool.
Now you can recalculate your alignment and tree with a reduced taxon sampling with a click on safe tree. You can save your phylogenetic tree in the NU format. Click on about this website and then tools to find additional information about the standalone tools for sale and pro s.
Beside the alignment at Neighbor joining function as also provided by the I Ts two database web interface, you can now access several new functions, for example, species Del Limitation based on compensatory based changes. Within the last minutes, we wanted to show you a few tricks how you can improve your phylogenetic analysis. We showed you how to gather your data from the I Ts two database, align them with four cell, and finally calculate your trees with Pro S.In all of these steps, we did not only consider sequence, but sequence and structure.Sure.
We hope you enjoyed our little movie and heavy tree building. Good night.
