1. Genome Annotation and Databases Genomic DNA sequence Genomic annotation BIO520 BioinformaticsJim Lund Reading Ch 9, Ch10

2. Genome Annotation Find known repeats Search for new repeated sequences Predict Genes –BLASTX –Genewise, Fgenes, Genscan… Integrate other data sources. Accuracy highest in “high homology” class

3. Genome annotation servers Integrate information from several maps –DNA sequence (contigs, quality). –Physical (cytogenetic, STS content). –Genes (show gene annotations and evidence). Several prediction programs. Expressed sequence tags (ESTs, Unigene clusters) Evidence (Predicted, confirmed) Non-coding RNA (ncRNA) transcripts. –Variation (e.g., SNPs) –Regions of shared synteny.

4. Data Release Human genome sequence released under 1996 Bermuda rules –Assembled sequence greater than 1000bp long is deposited in public database (GenBank/EMBL/DDBJ) every 24 hours –No patents are filed Bermuda principles reaffirmed at January 2003 WT/NIH meeting –Pre-release of data for all “community projects” –Nature 421, 875 (2003) –NHGRI: http://www.genome.gov/page.cfm?pageID=10506376 –WT: http://www.wellcome.ac.uk/About-us/Policy/Policy-and-position- statements/WTD002751.htm Benefits of Open Data Access supported by OECD report –http://dataaccess.ucsd.edu

5. Accessing the Genome Genomes sequences are becoming available very rapidly –Large and difficult to handle computationally –Everyone expects to be able to access them immediately Bench Biologists –Has my gene been sequenced? –What are the genes in this region? –Where are all the GPCRs –Connect the genome to other resources. Research Bioinformatics –Give me a dataset of human genomic DNA. –Give me a protein dataset.

6. Getting information out Search/browse to find the gene or region. Export formats: –Screen shot –FASTA seq. –Genbank file with features annotated –Feature list (Gff, tab-delimited text) –Pip (plot of sequence identity between organisms).

7. Challenges Scale and data flow –Presentation, ease of use. –Engineering problems. –User interface design. Algorithmic –Partly engineering (pre-compute hard computations, etc.) –Partly research.

8. NCBI sequence assembly (sequence chromosome) Remove contaminants Bin by chromosome arms Sequence Layout Sequence Building Place on chromosomes http://www.ncbi.nlm.nih.gov/genome/guide/build.shtml

9. NCBI sequence assembly - a modified greedy approach Sequence Layout Curated Finished Regions Curated assembly instructions MegaBLAST hits Consider clone order BAC chromosome assignment annotation STS markers personal communication Remove conflicting overlaps, redundant BACs Sequence Building Consider fragment:fragment sequence overlaps for each BAC pair in layout Meld overlapping sequence Order and Orient (o+o ): alignments (mRNA, EST) BAC annotation paired plasmid reads BAC Sequence Fragments Assemble Order NCBI Contig

10. NCBI Genome Build Process Contig Build & Release Assembly Input Data: Sequences Curated NTs TPF BLAST hits Resource Updates STS Clones Annotation LocusLink RefSeq Collaboration Curation FTP BLAST Input Resources Map Viewer Update: Links gi’s Prepare for release Sequences (contig mRNA protein) Analysis & Review Corrections for next build Freeze LocusLink GenBank GenomeScan dbSNP Public Release Exclude Problem accessions

11. What is being annotated? Genes: By alignment, by prediction Markers: By ePCR Clones/Cytogenetic location: By alignment (BAC ends) Variation: By alignment Phenotype (MIM): Via Gene identification, associated markers Cytogenetic Position: By annotated BAC-END sequenced clones By FISH-mapped clones used in assembly Feature Method

12. RefSeq: a reagent for Contig Annotation GenomeScan ESTs TBLASTN RPSBLAST RefSeq mRNAs GenBank mRNAs RefSeq Advantages: Separate Gene Families Not Partial Means to correct problem sequences RefSeq process results in excluding problem GenBank sequences from annotation pipeline Potential Problems With ESTs: Gene Families Partial Chimeric Intron read-through Linker Vector Wrong organism genome

13. NCBI: Products of annotation RefSeqs (transcripts, proteins) Gene id (LocusID) features in chromosome coordinates features in contig (NT accession) coordinates Available in: Map Viewer –Graphical display –Tabular display –Sequence downloads FTP –RefSeqs (contigs, transcripts, proteins) –Mapping Data –LocusLink & Other resources

14. NCBI Map Viewer

15. NCBI Map Viewer: Tabular report

16. Genes in regions of conserved synteny Anchored by human gene order Anchored by mouse gene order

17. Chromosomal segments in dog conserved with human and mouse Dog: 38 autosomes + sex chr

18. Query by sequence: Review the alignment A click away: Alignments (BLAST hit) Gene Description (LocusLink) Report of all features in the region Contig sequence Sequence in the region other mRNAs aligning in the region Define your own gene model based on alignments in the region

19. Quality Control - Genome review Is the sequence correct? Is the feature correctly placed? Is there a feature that should be placed? Are the attributes of the feature correct? Approaches: In-house analysis & review (manual curation) Shared information (UCSC/Ensembl) Solicited review by experts in local regions

20. Ensembl Annotation pipeline Set of high quality gene predictions –From known human mRNAs aligned against genome –From similar protein and mRNAs aligned against genome –From Genscan predictions confirmed via BLAST of Protein, cDNA, ESTs databases. Initial functional annotation from Interpro Integration with external resources (SNPs, SAGE, OMIM) Comparative analysis between mouse/human –DNA sequence alignment –Protein orthologs

21. Ensembl gene prediction pipeline RepeatMasker Genscan Blast genscan peptides v Protein,unigene,est,vert mrna Pmatch all human Proteins and cdnas MiniGenewise MiniEst2genome Genes DNA

22. Genome Annotation The generic structure of an automatic genome annotation pipeline and delivery system

26. Detailed View Genes, ESTs, CpG etc. 100kb Overview Genes and Markers 1Mb Chromosome Configuration

27. Useful genomic annotation and browser URLs EBI/Sanger Institute Ensembl Project: http://www.ensembl.org/Homo_sapiens/ NCBI Human Genome Browser: http://www.ncbi.nlm.nih.gov/mapview/map_search.cgi?chr=hum_chr.inf&query The Oak Ridge National Laboratories Genome Channel: http://compbio.ornl.gov/channel/ UCSC Human Genome Browser: http://genome.ucsc.edu/cgi-bin/hgGateway The Institute for Genomic Research (TIGR): http://www.tigr.org/

28. Genome annotation -things still being worked out- Annotation servers. Pro: make genomics information accessible to biologists without expert bioinformatics skills. Con: makes it difficult to perform large-scale data mining. Solution: enable more experienced users to retrieve the data they require and to run analyses locally. Open annotation systems. Biologists need to have access to annotations available in the community and to share their own contributions with the community. A common protocol between systems that enables genome data to be freely exchanged AGAVE (Architecture for Genomic Annotation, Visualization and Exchange) Distributed Annotation System (DAS) projects

29. Genome annotation servers Several ways to find information: –Search by clone, gene, EST, marker. –Browse sequence. –BLAST searches. –Homology, start in one organism, jump to the syntenic region of another.

30. UCSC Genome Browser http://genome.ucsc.edu/cgi-bin/hgGateway