1. Alastair Kerr, Ph.D. WTCCB Bioinformatics Core An introduction to DNA and Protein Sequence Databases

2. Questions to address What are the main sequence databases? Which one to use for: Looking up a gene name/identifier from a paper Identifiers What should I use and why? Coordinate based systems Annotation Protein domains Gene Ontology

3. Database Varieties Sequence Warehouses “everything under one roof” Genome Databases Containing single genome dataset(s) Reference Sets Often human curated, the 'standard' for a particular gene or protein from which variants are defined Specialist Short reads from next generation sequencing (Short read archive) [EST] Expressed sequence tags and [GSS] Genome survey sequence

4. NCBI GenBank EMBL DDBJ Sharing primary data

5. NCBI Warehouse GenBank NR dataset : NR = non redundant (but is is not..) Reference Dataset RefSeq Genome Datasets NCBI Genomes

6. EMBL Warehouse EMBL Historically Protein set was call translated EMBL (trEMBL) Gold standard reference set was called SwissProt Reference set = Uniprot UniProtKB/Swiss-Prot Manually annotated and reviewed UniProtKB/TrEMBL automatically annotated and not reviewed Genome database Ensembl

7. Live Demo Search GenBank for human adh4 How many are there? How many should there be? Why are some different to those found in Uniprot? Are there better databases to use? Which identifier should you use in your lab book?

8. We should now be able to answer these: What are the main sequence databases? Which one to use for: Looking up a gene identifier from a paper Searching for a gene name Searching for an orthologus genes from another species

9. Identifiers Or what to write in your lab book

10. How to identify a feature Gene/protein name Common name Standardised Name Database identifier Unique for each database Some have revision numbers Position in genome Dependant on Genome build Position in a Gene/Protein Protein Domains

11. Never use common names Example of EPHB2

12. Consortia identifiers Most key species have a consortia / group / community that provides the key identifiers in the field Humans Was HUGO (HUman Genome Organisation) now the HGNC (Human Genome Nomenclature Committee)

13. Database Identifiers Every dataset has their own system of identifying gene/protein Example: Human ADH4 Ensembl ENSG00000198099 ENST00000423445 ENSP00000397939 SwissProt ADH4_HUMAN P08319 RefSeq NM_000670.3NP_000661.2 GenBank gi|71565152|ref|NP_000661.2|

14. Keeping Track of Changes Gene models can change Will the id you used yesterday still get the same sequence today? Or: How to you get the latest version of a sequence?

15. Keeping Track of Changes Genbank: GI or “genbank identifier” Gi number changes each time, often removed when it gets superseded SwissProt: Accession and ID Accession changes each time (P08319) but the ID remains constant (ADH4_HUMAN) RefSeq and Ensembl Revision based ids NM_000670.3 ENSG00000198099.1 XXX.number XXX always retrieve latest XXX.number retrieves the version

16. Demo: Retrieving old data

17. Definining: Chromosome coordinates Demo: Ensembl

18. Chromosome Positions Features identified by Chromosome & position File formats: BED, WIG, gff.. All major genome databases store features as coordinates Ubiquitous in deep sequencing studies Note: coordinates change depending on the assembly Always note the build number of the genome assembly if you are using coordinates

19. Coordinates New concept of PATCH This is an assembly update without changing the primary sequence However additional 'improved' contigs map to the reference These will be in the net assembly: you may wish to use them Genome assembly names can differ by institution but are the same underlying sequence: GenBank/UCSC DEMO liftOver

20. Protein Domains: Protein Positions

21. Protein Domains Interpro Site that stores information on known protein domains from different projects Covered by Interpro Similarities between proteins Conserved region in an alignment Conserved protein folds Not Covered by Interpro Predicted features on primary protein sequence Trans-membrane regions Low complexity regions Phosphorylation sites

22. Domain Complexity Many different types of domains Vast amounts of domain based data Many different projects identifying them x =

23. Old way of interacting with a database Request information Retrieve information From single source

24. Distributed Annotation

25. DAS clients Different type of software can have a DAS client build-in Genome Browsers: ensembl, IGB, IGV.. Multiple Alignment editors: Jalview, STRAP 3D Structures: Spice 3D electron microscopy data: PeppeR Demo

26. Annotation

27. Problem: Many ways to name a gene Reductase = oxidase = dehydrogenase Gene Ontology Consortium [GO] GO terms standardise naming Note that errors may still occur in the assignment of terms Found in RefSeq, UniProt and most genome databases GO browsers e.g. AmiGO

28. Gene Ontology all [535063 gene products] GO:0008150 : biological_process [404412 gene products] GO:0005575 : cellular_component [372379 gene products] GO:0003674 : molecular_function [436597 gene products]

29. Gene Ontology: acyclical Tree

30. Evidence Codes Experimental # EXP: Inferred from Experiment # IDA: Inferred from Direct Assay # IPI: Inferred from Physical Interaction # IMP: Inferred from Mutant Phenotype # IGI: Inferred from Genetic Interaction # IEP: Inferred from Expression Pattern Computational # ISS: Inferred from Sequence or Structural Similarity # ISO: Inferred from Sequence Orthology# ISA: Inferred from Sequence Alignment # ISM: Inferred from Sequence Model# IGC: Inferred from Genomic Context # RCA: inferred from Reviewed Computational Analysis Author Statement # TAS: Traceable Author Statement# NAS: Non-traceable Author Statement # Curator Statement Evidence Codes# IC: Inferred by Curator # ND: No biological Data available Automatically-assigned # IEA: Inferred from Electronic Annotation

31. Best annotation? Use DAS clients to get more information on genomic, gene or protein features Protein Domains are especially useful The Gene Ontology is useful for general classification BUT be aware from where the annotation was derived