Transcript Slide 1
Informatics for Molecular Biologists
Ansuman Chattopadhyay,PhD Head, Molecular Biology Information Service Falk Library, Health Sciences Library System University of Pittsburgh
Molecular Biology Information Service Falk Library of Health Sciences Health Sciences Library System University of Pittsburgh 200 Scaife Hall Desoto and Terrace Streets Pittsburgh, PA 15261
Topics
• Searching tools – Internet – PubMed • NCBI developed bioinformatics tools – Entrez Gene • Structure visualization tools – Cn3D • Genome Browsers – UCSC genome browsers – NCBI Map viewer
Information search space • Biomedical literature databases • Molecular databases • Organism whole genome sequences
Literature database
• NCBI PubMed – contains over 15 million citations dating back to the mid-1950's . Search: “apoptosis”: 130,476 “breast cancer”: 160,055 “p53”: 42,418
Molecular databases
600 500 400 300 200 100 0 1996 1997 1998 1999 2000 2001 2002 2003 2004 Articles Databases
Organisms whole genome sequences
http://www.genomesonline.org/
Internet for Biologists
• Google Vs Clusty – Google: Chronological list of search results – Clusty: Search results categorized into topical clusters Vivísimo's clustering technology creates topical categories on-the-fly from the search results, using terms in the title, snippet, and any other available textual description in the search results themselves
Google Vs Clusty
• Search Example: Pittsburgh – Google – Clusty
Clusty
Clusters help you see your search results by topic, so you can zero in on exactly what you’re looking for or discover unexpected relationships between items.
Search examples for Clusty
• • • SNP BLAST Lupus
Web 2.0
• Website bookmark and tagging tool – Del.icio.us a social bookmarking web service for storing, sharing, and discovering web bookmarks .
Web 2.0
• Connotea ; http://www.connotea.org/
Medline searching tool
• PubMed vs ClusterMed Search example : macular degeneration, cell cycle, p53
Molecular databases • • • • • • • • DNA Sequence Databases and Analysis Tools Enzymes and Pathways Gene Mutations, Genetic Variations and Diseases Genomics Databases and Analysis Tools Immunological Databases and Tools Microarray, SAGE, and other Gene Expression • • • • • Organelle Databases Other Databases and Tools (Literature Mining, Lab Protocols, Medical Topics, and others) Plant Databases Protein Sequence Databases and Analysis Tools Proteomics Resources RNA Databases and Analysis Tools Structure Databases and Analysis Tools
HSLS OBRC
• http://www.hsls.pitt.edu/guides/genetics/obrc/
Types of databases
– By level of curation: • Archival –GenBank, GenPept, ssSNP • Curated –Refseq, SwissProt, RefSNP
Types of databases
–
Archival data
• repository of information • redundant; might have many sequence records for the same gene, each from a different lab • submitters maintain editorial control over their records: what goes in is what comes out • no controlled vocabulary • variation in annotation of biological features Example: GenBank record
GenBank
• archival database of nucleotide sequences from >130,000 organisms • records annotated with coding region (CDS) features also include amino acid translations • each record represents the work of a single lab • redundant; can have many sequence records for a single gene
International Nucleotide Sequence Database Collaboration
Types of databases
Refseq
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Curated data
– non-redundant; one record for each gene, or each splice variant – each record is intended to present an encapsulation of the current understanding of a gene or protein, similar to a review article – records contain value-added information that have been added by an expert(s)
Refseq
• • • Database of reference sequences Curated Non-redundant; one record for each gene, or each splice variant, from each organism represented • • • A representative GenBank record is used as the source for a RefSeq record Value-added information is added by an expert(s) Each record is intended to present an encapsulation of the current understanding of a gene or protein, similar to a review article • • Variety of accession number prefixes (NM_ , NP_ , etc.) and status codes (provisional, reviewed, etc.). More about those in later slides.
RefSeq database includes genomic DNA, mRNA, and protein sequences, so organizes information according to the model of the central dogma of biology
RefSeq
Searching GenBank • Find messenger RNA sequence for Human epidermal growth factor (EGF) gene.
Databases developers
• NCBI • EBI
Neighbors and Hard Links Word weight
PubMed abstracts
Phylogeny
Taxonomy Genomes
VAST BLAST
Nucleotide sequences Protein sequences
BLAST
Source NCBI
NCBI Tools
Entrez Gene
NCBI’s database for gene centric information focuses on organisms genome • completely sequenced • an active research community to contribute gene-specific information • scheduled for intense sequence analysis – Total Taxa: 4246 ; Total Genes: 284,3587 • 160,000 organisms in the nucleotide sequence database (Genbank)
Entrez gene
• each record represents a single gene from a given organism Gene record includes: – a unique identifier or GeneID assigned by NCBI – a preferred symbol – and any one or more of: – sequence information – map information – official nomenclature from an authority list – alternate gene symbols – summary of gene/protein function – published references that provide additional information on function – expression – homology data – and more
Gene / Protein Chromosomal Localization Homologous Sequences Genomic Sequence mRNA Sequence Exon-Intron Structure SNP Expression Profile Amino acid Sequence 3D Structure Interacting Partners Disease
Searching Entrez Gene
Entrez gene
Find: • gene symbols and aliases • sequences: genomic, mRNA, protein • intron-exon architecture • genomic context: neighboring and antisense genes • Interacting partners • associated gene ontology terms: function, cellular component and biological process
Entrez Gene record
Query: BRCA1 Search Tips: Query text box: BRCA1 Limits: •To limit your search to a specific field, select: “Gene name” from drop-down menu •Limit by taxonomy: select “Homo sapiens” Name and aliases Chromosoma l location
Sourse: NCBI
Entrez Gene: sequences and genomic context
mRNA Seq Genomic Seq
Sequences: mRNA, Genomic, Protein
ProteinSeq
Transcription and alternative splicing Alternative splicing: http://www.exonhit.com/UserFiles/Image/epissage.swf?PHPSESSID=d9u8tiu2sioqa8u29bkop3l0l2
Entrez Gene: intron-exon architectures Tips: Change Display to “Gene Table” from “Summary”
mRNA Seq Genomic Seq ProteinSeq
Gene Ontology
– Controlled vocabulary tagging
• Function • Biological Processes • Cellular Component
Entrez Gene : Gene Ontology
Homologous sequences
Entrez Gene: Homologous sequence Tips: change Display settings from" summary” to “Alignment score” to “Multiple Alignment”
Single nucleotide polymorphisms
Single nucleotide polymorphisms (SNP) are DNA sequence variations that occur when a single nucleotide (A,T,C,or G) in the genome sequence is altered. For example a SNP might change the DNA sequence A
A
GGCTAA to A
T
GGCTAA
SNPs
Coding SNPs
Entrez Gene: SNPs
Protein Info: HPRD
Protein Info: HPRD
Entrez Gene: Links
Entrez Gene: Linkout
Seq to Entrez gene: UCSC BLAT Query Seq: SGLTPEEFMLVYKFARKHHITLTNLITEE
CLICK CLICK
BLAT to Entreze Gene
Hands-On Exercise Question Find chromosomal location of your gene of interest. How many exons have been reported for your gene?
What are its neighboring genes ?
Query sequence:
IHYNYMCNSSCMGGMNRRPILTII
Exercise:
Find the protein sequence for rat leptin.
BLAT this sequence vs. the human genome to find the human homolog.
Look for SNPs in the coding region of this gene —are there any?
Sequence alignment • Pair wise alignment • Multiple alignment
Pairwise alignment • Global –
Needleman Wunsc
(1970) • Local –
Smith-Waterman
(1981) – Lipman and Pearson /FASTA (1985) – Basic Local Alignment Search Tool (BLAST:1991)
BLAST
To find homologous sequence for a sequence of interest by searching sequence databases: Nucleotide: TTGGATTATTTGGGGATAATAATGAAGATAGCAA TTATCTCAGGGAAAGGAGGAGTAGGAAAATCTTC TA TTTCAACATCCTTAGCTAAGCTGTTTTCAAAAG AGTTTAATATTGTAGCATTAGATTGTGATGTTGAT Protein: MSVMYKKILYPTDFSETAEIALKHVKAFKTLKAEEVILLHVIDER EIKKRDIFSLLLGVAGLNKSVEEFE NELKNKLTEEAKNKMENIK KELEDVGFKVKDIIVVGIPHEEIVKIAEDEGVDIIIMGSHGKTNLKEILLG
BLAST
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To Find statistically significant matches, based on sequence similarity, to a protein or nucleotide sequence of interest.
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Obtain information on inferred function of the gene or protein.
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Find conserved domains in your sequence of interest that are common to many sequences.
•
Compare two known sequences for similarity.
What you can do with BLAST
•Find homologous sequence in all combinations (DNA/Protein) of query and database.
–DNA Vs DNA –DNA translation Vs Protein –Protein Vs Protein –Protein Vs DNA translation –DNA translation Vs DNA translation
BLAST exercise •
Find homologous sequences for uncharacterized archaebacterial protein, NP_247556, from
Methanococcus jannaschii
Descriptions of hits
BLAST search
Sort by E values Link to Entrez Sequence description 2X10 -65 number of display cut off (100)over rides E value cut off (10)
BLAST search
•Orthologs from
closely related species
will have the highest scores and lowest E values –Often
E = 10 -30 to 10 -100
•
Closely related homologs with highly conserved function and structure
will have high scores –Often
E = 10 -15 to 10 -50
•
Distantly related homologs
may be hard to identify –Less than
E = 10 -4
• Wikipedia
Protein domains
SH2
Src homology 2 domains; Signal transduction, involved in recognition of phosphorylated tyrosine (pTyr). SH2 domains typically bind pTyr-containing ligands via two surface pockets, a pTyr and hydrophobic binding pocket, allowing proteins with SH2 domains to localize to tyrosine phosphorylated sites.
Searching CDD
• CDD SEARCH Query sequence:
Blink
•
BLink displays the graphical output of pre-computed blastp results against the protein non-redundant (nr) database. This graphical output includes:
– Alignment of up to 200 BLAST hits on the query sequence – Best Hits to each organism – List of known protein domains in the query sequence – Filter hits by selecting the BLAST cutoff score – Distribution of hits by taxonomic grouping – Display of similar sequences with known 3D structure – Filter hits by database and/or by taxonomic grouping – Display a taxonomic tree of all organisms with similar sequences Access :
Link out from NCBI protein records
Link toTP53 Blink: http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=NP_000537.2&dopt=gp
Protein structure
Protein data bank (PDB)
• international database of 3-D biological macromolecular structures • accepts direct submissions of structure data • maintained by a nonprofit organization, the Research Collaboratory for Structural Bioinformatics (RCSB), associated with Rutgers University, San Diego Supercomputer Center, and the Biotechnology Division of the National Institute of Standards and Technology • contains molecular structures of proteins and nucleic acids, primarily structures experimentally-derived by X-Ray crystallography and NMR • also includes some theoretical models, though they are not encouraged.
3D structure viewing software
• NCBI Cn3D The Cn3D home page includes a link in the blue sidebar for instructions on
installing Cn3D
, which is available for PC, Mac, and Unix. • First glance in Jmol A simple tool for macromolecular visualization.
Cn3D
• View the 3-dimensional structure for 1TUP and practice using some of the Cn3D features that allow you to: – spin the structure using your mouse – use the control+left mouse button combination to zoom in and out of the structure – use the shift+left mouse button combination to move the structure across the viewing window – use the Style menu to render the structure in different ways (e.g., worms, space fill, ball and stick, ...) – use the Style menu to color the structure in different ways (e.g., secondary structure, domain, ...) – use the Style/Edit Global Style to label every 20th amino acids
What is it?
Genome Browser is a computer program which helps to display gene maps, browse the chromosomes, align genes or gene models with ESTs or contigs etc.
Genome Sequence Project Time Line
1976 : RNA Bacteriophage MS2 1995: Haemophilus influenzae 2003: Human genome reference sequence 2005: 265 genomes; 21 archaeal, 211 bacterial, 33 eukaryotic
http://www.genomesonline.org/
Genome Browsers