1. Bioinformatics Resources and Tools on the Web: A Primer

2. Outline Introduction: What is bioinformatics? The basics –The five sites that all biologists should know Some examples –Using the tools in a somewhat less-than-naïve manner Questions/comments are welcome at all points Much of this material comes from the Boston University course: BF527 Bioinformatic Applications (http://matrix.bu.edu/BF527/)http://matrix.bu.edu/BF527/

3. What is bioinformatics?

4. Examples of Bioinformatics Database interfaces –Genbank/EMBL/DDBJ, Medline, SwissProt, PDB, … Sequence alignment –BLAST, FASTA Multiple sequence alignment –Clustal, MultAlin, DiAlign Gene finding –Genscan, GenomeScan, GeneMark, GRAIL Protein Domain analysis and identification –pfam, BLOCKS, ProDom, Pattern Identification/Characterization –Gibbs Sampler, AlignACE, MEME Protein Folding prediction –PredictProtein, SwissModeler

5. Things to know and remember about using web server-based tools You are using someone else’s computer You are (probably) getting a reduced set of options or capacity Servers are great for sporadic or proof-of- principle work, but for intensive work, the software should be obtained and run locally

6. Five websites that all biologists should know NCBI (The National Center for Biotechnology Information; –http://www.ncbi.nlm.nih.gov/http://www.ncbi.nlm.nih.gov/ EBI (The European Bioinformatics Institute) –http://www.ebi.ac.uk/http://www.ebi.ac.uk/ The Canadian Bioinformatics Resource –http://www.cbr.nrc.ca/http://www.cbr.nrc.ca/ SwissProt/ExPASy (Swiss Bioinformatics Resource) –http://expasy.cbr.nrc.ca/sprot/http://expasy.cbr.nrc.ca/sprot/ PDB (The Protein Databank) –http://www.rcsb.org/PDB/http://www.rcsb.org/PDB/

7. NCBI (http://www.ncbi.nlm.nih.gov/)http://www.ncbi.nlm.nih.gov/ Entrez interface to databases –Medline/OMIM –Genbank/Genpept/Structures BLAST server(s) –Five-plus flavors of blast Draft Human Genome Much, much more…

8. EBI (http://www.ebi.ac.uk/)http://www.ebi.ac.uk/ SRS database interface –EMBL, SwissProt, and many more Many server-based tools –ClustalW, DALI, …

9. SwissProt (http://expasy.cbr.nrc.ca/sprot/)http://expasy.cbr.nrc.ca/sprot/ Curation!!! –Error rate in the information is greatly reduced in comparison to most other databases. Extensive cross-linking to other data sources SwissProt is the ‘gold-standard’ by which other databases can be measured, and is the best place to start if you have a specific protein to investigate

10. A few more resources to be aware of Human Genome Working Draft –http://genome.ucsc.edu/http://genome.ucsc.edu/ TIGR (The Institute for Genomics Research) –http://www.tigr.org/http://www.tigr.org/ Celera –http://www.celera.com/http://www.celera.com/ (Model) Organism specific information: –Yeast: http://genome-www.stanford.edu/Saccharomyces/http://genome-www.stanford.edu/Saccharomyces/ –Arabidopis: http://www.tair.org/http://www.tair.org/ –Mouse: http://www.jax.org/http://www.jax.org/ –Fruitfly: http://www.fruitfly.org/http://www.fruitfly.org/ –Nematode: http://www.wormbase.org/http://www.wormbase.org/ Nucleic Acids Research Database Issue –http://nar.oupjournals.org/ (First issue every year)http://nar.oupjournals.org/

11. Example 1: Searching a new genome for a specific protein Specific problem: We want to find the closest match in C. elegans of D. melanogaster protein NTF1, a transcription factor First- understanding the different forms of blast

12. The different versions of BLAST

13. 1 st Step: Search the proteins blastp is used to search for C. elegans proteins that are similar to NTF1 Two reasonable hits are found, but the hits have suspicious characteristics –besides the fact that they weren’t included in the complete genome !

14. 2 nd Step: Search the nucleotides tblastn is used to search for translations of C. elegans nucleotide that are similar to NTF1 Now we have only one hit –How are they related?

15. Conclusion: Incorrect gene prediction/annotation The two predicted proteins have essentially identical annotation The protein-protein alignments are disjoint and consecutive on the protein The protein-nucleotide alignment includes both protein-protein alignments in the proper order Why/how does this happen?

16. Final(?) Check: Gene prediction Genscan is the best available ab initio gene predictor –http://genes.mit.edu/GENSCAN.htmlhttp://genes.mit.edu/GENSCAN.html Genscan’s prediction spans both protein- protein alignments, reinforcing our conclusion of a bad prediction

17. Ab initio vs. similarity vs. hybrid models for gene finding Ab initio: The gene looks like the average of many genes –Genscan, GeneMark, GRAIL… Similarity: The gene looks like a specific known gene –Procrustes,… Hybrid: A combination of both –Genomescan (http://genes.mit.edu/genomescan/)http://genes.mit.edu/genomescan/

18. A similar example: Fruitfly homolog of mRNA localization protein VERA Similar procedure as just described –Tblastn search with BLOSUM45 produces an unexpected exon Conclusion: Incomplete (as opposed to incorrect) annotation –We have verified the existence of the rare isoform through RT- PCR

19. Another example: Find all genes with pdz domains Multiple methods are possible The ‘best’ method will depend on many things –How much do you know about the domain? –Do you know the exact extent of the domain? –How many examples do you expect to find?

20. Some possible methods if the domain is a known domain: SwissProt –text search capabilities –good annotation of known domains –crosslinks to other databases (domains) Databases of known domains: –BLOCKS (http://blocks.fhcrc.org/)http://blocks.fhcrc.org/ –Pfam (http://pfam.wustl.edu/)http://pfam.wustl.edu/ –Others (ProDom, ProSite, DOMO,…)

21. Determination of the nature of conservation in a domain For new domains, multiple alignment is your best option –Global: clustalw –Local: DiAlign –Hidden Markov Model: HMMER For known domains, this work has largely been done for you –BLOCKS –Pfam

22. If you have a protein, and want to search it to known domains Search/Analysis tools –Pfam –BLOCKS –PredictProtein ( http://cubic.bioc.columbia.edu/predictprotein/predictprotein.html ) http://cubic.bioc.columbia.edu/predictprotein/predictprotein.html

23. Different representations of conserved domains BLOCKS –Gapless regions –Often multiple blocks for one domain PFAM –Statistical model, based on HMM –Since gaps are allowed, most domains have only one pfam model

24. Conclusions We have only touched small parts of the elephant Trial and error (intelligently) is often your best tool Keep up with the main five sites, and you’ll have a pretty good idea of what is happening and available