*Supported by the NSF Plant Genome Research and REU Programs

1. Tutorial of bioinformatics and tree generation at the Cell Wall Genomics website Bryan Penning *Supported by the NSF Plant Genome Research and REU Programs

2. Bioinformatics Goals • We currently have a wealth of Arabidopsis thaliana cell wall gene information on the website, we wanted to: • Add family information about rice and maize Type II cell walls to compare to A. thaliana Type I cell walls • Add links to outside information on rice genes like we have for A. thaliana • Include annotated composite trees of A. thaliana, rice and maize gene families • Add links to sites used to generate the data • Add source protein sequence used for our family trees so other researchers can make their own adding their genes of interest • Generate a tutorial on how researchers can make use of the bioinformatics data on our site

3. Homologous rice genes Draw rice dendrogram A thaliana & rice genes Genes from A. thaliana Good tree? Publish to website Diagram of our bioinformatics approach Too few genes, Blast other sites N Blast TIGR Choose genes Make tree Y Too many genes, tighten criteria N Diagram of the process used to find the genes and draw family trees for cell wall related rice genes. The same approach is used for maize.

4. Diagram of our bioinformatics approach A. thaliana genes Draw tree with all family members Annotate Publish to web Rice genes Diagram of the process used to integrate cell wall related genes from all three family trees into a composite tree. Maize genes

5. BLASTing genes • To be considerate of the bioinformatics community with the number of BLASTs to be performed and to speed the process, we downloaded the text or “flat file” of the TIGR rice protein sequences (available at: http://www.tigr.org/tdb/e2k1/osa1/data_download.shtml) and performed local blasts using blastall from NCBI (available at: http://www.ncbi.nlm.nih.gov/BLAST/download.shtml) • Direction for use of these tools is available at the above sites and is beyond the scope of this tutorial • For a small number of BLASTs, you can use web-based methods and common programs such as Word and Excel plus any of a number of downloadable tree drawing programs to make these kinds of trees on your own if you are not familiar with programming languages such as Perl to automate the process. Although web searches can be more time consuming, they work just as well for a few sequences

6. Web BLASTing • For smaller numbers of BLASTs to the rice genome, TIGR provides an excellent Web BLAST at: http://tigrblast.tigr.org/euk-blast/index.cgi?project=osa1 • You can also use the new BLAST tool at Gramene: http://www.gramene.org/multi/blastview for most cereal sequences • Note: gene model versions sometimes differ between Gramene and TIGR as one site may update to the latest model before the other

7. Web BLASTing • Downloading the protein sequence for Arabidopsis SUD1 (At3g46440) from TIGR, you can BLAST it against the TIGR Rice Pseudomolecules – Protein database using BLASTp

8. Web BLASTing • You get a series of “hits” to the gene of interest • A higher score and smaller probability is a better match to the original gene • This procedure is followed for all of the genes in a family to gather the best possible hits, sort the hits to remove duplicates and choose the best rice matches to the Arabidopsis families • You can use NCBI’s blastall tool for multiple simultaneous blasts as we do for this step

9. Organizing BLASTs • This is a word document generated by BLASTing SUD1 and SUD2 of Arabidopsis against the TIGR Rice Protein database • The hits were copied into word and set to the font Courier New, 9 pt and saved as a text only document (to remove the HTML code) • The file was reloaded in Word and converted to a table (table menu) using other and the character | (shift \) to separate the columns

10. Organizing BLASTs • The Word file is copied into Excel and the Data – Sort menu is used to sort by the first column • This brings all of the same named genes together (the two highlighted lines for example) • Duplicate genes are removed from the spreadsheet and the far right column only (LOC_Osxxgxxxxxx) tags can be copied back to word

11. Organizing BLASTs • You can use the table menu to convert table to text (Paragraph Marks) to generate a list of genes • These genes can be searched through a downloaded database using the NCBI fastacmd (included in the BLAST download tools) or you can search them one at a time using a web-based database such as the locus search name on TIGR: (http://www.tigr.org/tdb/e2k1/osa1/LocusNameSearch.shtml)

12. Generating a tree • Once you have found all of your sequences, check that each sequence name has a < in front of it (denoting a new sequence name) and the sequence starts on a new line • Copy and paste all of your sequences into an alignment program like ClustalW (we use: http://align.genome.jp/ from the Kyoto University Bioinformatics center, but any ClustalW program will work)

13. Generating a tree • For our trees we use: Slow/Accurate pair-wise comparisons and Gonnet for our Weight Matrix (two spots on the website) • Click execute alignment to get your sequence alignment • At the end of the alignment page will be the information needed for tree drawing programs • You can click on clustal.dnd for a quick tree or take the information after it – A Newick format tree and copy it into a new Word file, saving it as a text file (include all parenthesis)

14. Creating a tree • We use the program TreeDyn to generate our trees (available at: http://www.treedyn.org/) • This is an example of the Arabidopsis and rice 1.1 family • The tree text file was loaded into TreeDyn and the frame enlarged • The red text for Arabidopsis sequences was done by changing the font color to red and using the find panel to find all At* sequences (which turn red) • The scale at the bottom was added by right clicking on that space and choosing the tree name, annotation, and scale sub-menus • This square tree is useful to see associations of genes for different species

15. Square tree example • This is part of the family 1.1 square dendrogram of Arabidopsis, rice and maize from our website • The red names are Arabidopsis sequence, the black names are rice, and the green names are maize • Regions alternate between grey shaded and white backgrounds (added with Photoshop) to indicate clades of similar sequence genes which may relate function (such as AUD/SUD or GME, etc)

16. Radial dendrograms • TreeDyn can also draw radial dendrograms such as the one shown for rice family 1.1 • This can be done by right clicking on the tree area to bring up the grey box in TreeDyn, choosing your tree, then Conformation- Radial • Treedyn allows you to resize, rotate, and flip clades around (see http://www.treedyn.org/ for detailed tutorials on these processes) • For our site, we export the radial trees as jpeg images

17. Finishing a radial dendrogram The TreeDyn tree jpeg is finished as a FLASH file where the ovals and family names are added (Rice family 1.1 shown) Each individual clade of a family tree is also prepared in TreeDyn and link buttons added later in FLASH (AUD/SUD-like shown)

18. Viewing your gene of interest • We provide protein sequence information you can download and add in your own sequence of interest for comparison to these three species • Under each tree (family 1.1 shown) is the link “View the protein sequence file” • Right click and choose Save Target as… to download the sequencewith a filename and location you will remember • You can do this for each Arabidopsis, rice, and maize family

19. Viewing your gene of interest • You may have a sequence you think is related to a particular family such as nucleotide interconversion pathway (family 1.1) • For example, the wheat EST CV523101 from Genebank: http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=CV523101 might be related to the TIGR rice gene: Os05g29990 in the AUD/SUD clade of family 1.1 according to information from Gramene

20. Viewing your gene of interest • You can take the nucleotide sequence and covert it to protein sequence using a program such as Genemark: (http://opal.biology.gatech.edu/GeneMark/eukhmm.cgi) • Protein sequence returned: >CV523101_wheat IARIFNTYGPRMCIDDGRVVSNFVAQALRKEPLTVYGDGKQTRSFQYVSDLVEGLMRLMEGDHIGPFNLGNPGEFTMLELAKVVQDTIDPNARIEFRENTQDDPHKRKPDITKAKEQLGWEPKIALRDGLPLMVTDFRKRIFGDQDSAATATEG

21. Viewing your gene of interest • Paste all of the sequences for family 1.1 (Arabidopsis, rice, and maize) plus the Wheat EST, CV523101_wheat, converted to protein into a ClustalW program such as: http://align.genome.jp/ from the Kyoto University Bioinformatics center • Perform the multiple alignment, copy the Newick tree data generated into a new word file, and save a text file as previously shown

22. Viewing your gene of interest • Taking the Newick tree from clustalW into TreeDyn as previously shown will allow you to visualize the tree • The AUD/SUD clade of the tree generated by TreeDyn shows that the wheat EST (in blue) is most closely related to the rice gene Os05g29990 in the AUD clade The AUD/SUD clade of the family 1.1 tree for Arabidopsis (red), Rice (black), Maize (green), and a wheat EST (blue) added to demonstrate how you can visualize relatedness of your own genes using our protein sequences

23. Bioinformatics sites used • General • Multiple alignment for trees, ClustalW (http://align.genome.jp/) • Making trees, TreeDyn (http://www.treedyn.org/) • BLASTing NCBI (http://www.ncbi.nlm.nih.gov/BLAST/) • Proteins translated by GeneMark (http://opal.biology.gatech.edu/GeneMark/eukhmm.cgi) • Rice • Sequence BLAST using TIGR (http://www.tigr.org/tdb/e2k1/osa1/) • Downloading rice protein sequences from TIGR (http://www.tigr.org/tdb/e2k1/osa1/LocusNameSearch.shtml) • Maize • Sequence BLAST using TIGR ZmGI (http://www.tigr.org/tigr-scripts/tgi/T_index.cgi?species=maize) • Sequence BLAST using Gramene (http://www.gramene.org/multi/blastview)