Download NCBI databases - Nematode bioinformatics. Analysis tools and data

Biological Databases
What types of data are available?
What is a database?
What are Genbank and Entrez?
What does a typical entry look
like?
How does one use the database?
BIO520 Bioinformatics
Jim Lund
NCBI Biological Databases
Central Dogma-o-centric
•
•
•
•
•
Genomic DNA sequence
mRNA/cDNA sequence
Protein sequence
Protein 3D structure
Literature (Function)
Biological Data
• Genomic DNA sequence (complete)
• mRNA/cDNA sequence
• Gene expression data (NEW)
– Microarrays, SAGE
– Expression catalogs
• Protein sequence
– Protein interaction/complex data (NEW)
• Protein 3D structure
• Literature (Function)
– Organism databases (NEW)
– Annotation and classification projects (NEW)
What is a Biological Database?
An organized body of persistent data and
associated computer software for updating,
querying, and retrieving data records.
• Collection of records and files
• Organized for a particular purpose
• The database is separate from the interface
and can have several interfaces.
– NCBI Protein can be searched by protein name or
using BLAST (Basic Local Alignment Search Tool).
Common database features
• Relational Databases
– Tables
– Relationships between tables
• Version Control
• Consistency enforcement
• Multiauthor/multiuser with security
BIO 520 Student Database
Column
Table
BIO520
.Name
ID Grade
Record
Amy
Joe
Sue
123
456
789
A
B
C
Value
Genbank Entry
LOCUS
BC005255
495 bp mRNA linear PRI 23-JUN-2006
DEFINITION Homo sapiens insulin, mRNA (cDNA clone IMAGE:3950204), complete cds.
ACCESSION BC005255
VERSION BC005255.1 GI:13528923
KEYWORDS MGC.
SOURCE Homo sapiens (human)
ORGANISM Homo sapiens
Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi;
Mammalia; Eutheria; Euarchontoglires; Primates; Haplorrhini;
Catarrhini; Hominidae; Homo.
FEATURES
Location/Qualifiers
source
1..495
/organism="Homo sapiens"
gene
1..495
/gene="INS"
/db_xref="GeneID:3630"
CDS
60..392
/gene="INS"
/translation="MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVCG
ERGFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSL
YQLENYCN"
ORIGIN
1 agccctccag gacaggctgc atcagaagag gccatcaagc agatcactgt ccttctgcca
…
421 ccgcctcctg caccgagaga gatggaataa agcccttgaa ccaacaaaaa aaaaaaaaaa
481 aaaaaaaaaa aaaaa
//
The CORE: DDBJ, EMBL, and
Genbank
Genbank DNA Sequence
Database
• Genbank/EMBL/DDBJ mirror &
exchange sequence records.
• Primary vs. Secondary Databases
– nr (non-redundant database)
• Primary vs. secondary records
– Sequence vs. inferred property (coding
region)
Primary vs. Derivative Databases
• Primary Databases
– Original submissions by experimentalists
– Content controlled by the submitter
• Examples: GenBank, SNP, GEO
• Derivative Databases
– Built from primary data
– Content controlled by third party (NCBI)
• Examples: Refseq, TPA, RefSNP, UniGene, NCBI
Protein, Structure, Conserved Domain
LOCUS
DEFINITION
AY182241
1931 bp
mRNA
linear
PLN 04-MAY-2004
Malus x domestica (E,E)-alpha-farnesene synthase (AFS1) mRNA,
complete cds.
ACCESSION
AY182241
VERSION
AY182241.2 GI:32265057
KEYWORDS
.
SOURCE
Malus x domestica (cultivated apple)
ORGANISM Malus x domestica
Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
Spermatophyta; Magnoliophyta; eudicotyledons; core eudicots;
rosids; eurosids I; Rosales; Rosaceae; Maloideae; Malus.
REFERENCE
1 (bases 1 to 1931)
AUTHORS
Pechous,S.W. and Whitaker,B.D.
TITLE
Cloning and functional expression of an (E,E)-alpha-farnesene
synthase cDNA from peel tissue of apple fruit
JOURNAL
Planta 219, 84-94 (2004)
REFERENCE
2 (bases 1 to 1931)
AUTHORS
Pechous,S.W. and Whitaker,B.D.
TITLE
Direct Submission
JOURNAL
Submitted (18-NOV-2002) PSI-Produce Quality and Safety Lab,
USDA-ARS, 10300 Baltimore Ave. Bldg. 002, Rm. 205, Beltsville, MD
20705, USA
REFERENCE
3 (bases 1 to 1931)
AUTHORS
Pechous,S.W. and Whitaker,B.D.
TITLE
Direct Submission
JOURNAL
Submitted (25-JUN-2003) PSI-Produce Quality and Safety Lab,
USDA-ARS, 10300 Baltimore Ave. Bldg. 002, Rm. 205, Beltsville, MD
20705, USA
REMARK
Sequence update by submitter
COMMENT
On Jun 26, 2003 this sequence version replaced gi:27804758.
FEATURES
Location/Qualifiers
source
1..1931
/organism="Malus x domestica"
/mol_type="mRNA"
/cultivar="'Law Rome'"
/db_xref="taxon:3750"
/tissue_type="peel"
gene
1..1931
/gene="AFS1"
CDS
54..1784
/gene="AFS1"
/note="terpene synthase"
/codon_start=1
/product="(E,E)-alpha-farnesene synthase"
/protein_id="AAO22848.2"
/db_xref="GI:32265058"
/translation="MEFRVHLQADNEQKIFQNQMKPEPEASYLINQRRSANYKPNIWK
NDFLDQSLISKYDGDEYRKLSEKLIEEVKIYISAETMDLVAKLELIDSVRKLGLANLF
EKEIKEALDSIAAIESDNLGTRDDLYGTALHFKILRQHGYKVSQDIFGRFMDEKGTLE
DFLHKNEDLLYNISLIVRLNNDLGTSAAEQERGDSPSSIVCYMREVNASEETARKNIK
GMIDNAWKKVNGKCFTTNQVPFLSSFMNNATNMARVAHSLYKDGDGFGDQEKGPRTHI
LSLLFQPLVN"
ORIGIN
1 ttcttgtatc ccaaacatct cgagcttctt gtacaccaaa ttaggtattc actatggaat
61 tcagagttca cttgcaagct gataatgagc agaaaatttt tcaaaaccag atgaaacccg
121 aacctgaagc ctcttacttg attaatcaaa gacggtctgc aaattacaag ccaaatattt
181 ggaagaacga tttcctagat caatctctta tcagcaaata cgatggagat gagtatcgga
241 agctgtctga gaagttaata gaagaagtta agatttatat atctgctgaa acaatggatt
//
A Traditional
GenBank Record
Header
The Flatfile Format
(formatted text)
Feature Table
Sequence
Genbank Entry
LOCUS
PCU30791
31-MAY-1996
1234 bp
mRNA
PLN
DEFINITION Pneumocystis carinii carinii form 6 guanine
nucleotide binding protein alpha subunit (pcg1) mRNA,
complete cds.
ACCESSION U30791
NID
g1345098
VERSION
U30791.1 GI:1345098
Unique ID
Version Control
Content-Taxonomy
SOURCE
Pneumocystis carinii f. sp. carinii.
ORGANISM Pneumocystis carinii f. sp. carinii
Eukaryota; Fungi; Ascomycota; Archiascomycetes;
Pneumocystidaceae; Pneumocystis.
Reference
REFERENCE 1 (bases 1 to 1234)
AUTHORS Smulian,A.G., Ryan,M., Staben,C. and Cushion,M.
TITLE Signal transduction in Pneumocystis carinii:
characterization of the genes (pcg1)
encoding the alpha subunit of the G protein (PCG1)
of Pneumocystis carinii carinii
and Pneumocystis carinii ratti
JOURNAL Infect. Immun. 64 (3), 691-701 (1996)
PUBMED 96186460
•Unique cross reference
•Can be >1 reference
Features
FEATURES
Location/Qualifiers
source 1..1234
/organism="Pneumocystis carinii f. sp. carinii“
/strain="Form 6“
/note="450 kb chromosome"
/db_xref="taxon:38081“
5'UTR 1..90
gene 91..1155
/gene="pcg1"
CDS
CDS 91..1155
/gene="pcg1”
Related info
in another
database
/note="G-protein alpha subunit"
/codon_start=1
/product= "guanosine nucleotide binding
protein alpha subunit"
/protein_id="AAC49295.1"
/db_xref="PID:g1345099" .
INFERRED
/db_xref="GI:1345099"
/translation="MGCCFSATYNQDTLRSKEIE
SYLRQEQEHACHEAKILLLGAGES…
DNA
BASE COUNT 421 a 171 c 195 g 447 t
ORIGIN
1
tgaattctaa attttatatt …
1201 … tattttttta tgctccagat aaaa
//
Genbank entries
• Combination of required (LOCUS,
SOURCE) and optional fields.
– The entry is hierarchical, some fields
contain subfields.
• REFERENCE->AUTHORS
• Some fields can appear multiple times
(REFERENCE, /gene)
• Some fields are numerical, other are
text. Some fields contain free text,
others use a controlled vocabulary or an
database ID.
Other Genbank output formats
• FASTA
– Simple, little annotation information
– Easy to use
– Common denominator format
• ASN1
– Computer friendly, human unfriendly
• XML, INSDSeqXML, TinySeqXML
• Graph (graphical map of seq features)
…and more
DNA Sequence Files
Common formats
• Genbank (used by VectorNTI)
• FASTA
• GCG
– Accelrys GCG (Genetics Computer
Group) package
– formerly GCG Wisconsin Package
Many others!
FASTA
One annotation line only!
>gi|1345098|gb|U30791.1|PCU30791
TGAATTCTAAATTTTATATTTCTAATTGCATTTTATATT
TTTGATAATACTAGATTTATTCCTGGAAACT
TAAATTAGTTATTTTAAGTTATGGGATGTTGTTTTTCT
GCTACATATAACCAAGATACACTTCGTTCCAA
Submitting sequences to
Genbank
•Sequin
–Stand-alone sequence submission tool.
•BankIt
–Web based sequence submission.
Genbank is an ARCHIVE
•The literature and secondary
databases are the knowledge
sources.
•There are many additional NCBI
annotation databases
NCBI annotation databases!
•Genbank -> RefSeq (Single sequence for each gene)
•Entrez Gene (Gene-based links to annotation sources).
•HomoloGene (Homologs)
•OMIM
•Conserved domains, 3D domains
•GEO (Gene expression datasets)
•DNA, protein, 3D structures
•Interaction data
•Links to other databases!
•NCBI Genomes
•NCBI Map viewer
Finding and editing DNA
files
•
•
•
•
Find DNA: Entrez
Downloading files
Format Conversion
Sequence viewing/editing
Entrez
• Database
searching/browsing
• Example: Pneumocystis Gproteins
– PCR a cDNA to express
in E. coli
– Read about it and
related genes
– Check similarity to
•http://www.ncbi.nlm.nih.gov/
Entrez/
related G-proteins
– View the 3D structure??
Entrez Neighbors-Protein
3D Structure
citation
Protein
BLASTP
Protein
citation
Literature
encoding
DNA
Mapping the menagerie of biological
databases
Nucleic Acid
Manipulations
• On the web:
– Baylor Human Genome Center (BCM)
http://searchlauncher.bcm.tmc.edu/seq
-util/seq-util.html
– European Bioinformatics Institute (EBI)
http://www.ebi.ac.uk/Tools/misc.html
DNA/Protein sequence
format conversion
• Readseq
– Download program:
– http://iubio.bio.indiana.edu/soft/molbio/rea
dseq
– Use online:
– http://www.ebi.ac.uk/cgi-bin/readseq.cgi
– http://searchlauncher.bcm.tmc.edu/sequtil/readseq.html
Beware Information Loss!
Reverse Complementing
5’-GAATCA-3’
5’-TGATTC-3’
NOT
5’-ACTAAAG-3’
Sequence Statistics
•
•
•
•
Nucleotide frequencies (di, tri…)
UV Absorbance
MW
Tm
Restriction Map
• Linear vs Circular
• Enzyme sets
– Which enzymes, where they cut.
• Gel simulation
– Gel-to-map MUCH harder!!
• Useful for:
– Cloning
– Southern blots
– Specialized mol bio techniques
Translation/ORFs
• Translation table
– Standard vs non-standard
• Frame (1,2,3,4,5,6)
• Segmental translation (exon-intron)
• Primary translation vs mature
polypeptide
Sequence Annotation and Editing
•Artemis
•Sequin
•NCBI’s Genbank entry
creation/viewing tool
• Text editor
– Notepad
– Word processor
– vi
MWGTCC
Nonproportional fonts
(courier, monospaced…)
IIIIII
MWGTCC
IIIIII
Primer design program: Primer3
http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi
Primary vs. Derivative Databases
• Primary Databases
– Original submissions by experimentalists
– Content controlled by the submitter
• Examples: GenBank, SNP, GEO
• Derivative Databases
– Built from primary data
– Content controlled by third party (NCBI)
• Examples: Refseq, TPA, RefSNP, UniGene, NCBI
Protein, Structure, Conserved Domain
Other NCBI Databases
•Structure:
imported structures (PDB)
Cn3D viewer, NCBI curation
•CDD:
conserved domain database
Protein families (COGs and KOGs)
Single domains (PFAM, SMART, CD)
•dbSNP:
•Gene:
nucleotide polymorphism
gene records
Unifies LocusLink and Microbial Genomes
Homologene Cluster
Entrez Protein: Derivative Database
Data Source
GenPept
RefSeq
Third Party Annotation
Swiss Prot
Sequences
6,937,176
3,359,561
5,136
255,159
PIR
29,996
PRF
12,079
PDB
91,116
PAT Division
Total
BLAST nr total
(no patents or env)
669,035
10,690,223
4,545,310
Redundant Proteins
>gi|463989|gb|AAC50285.1| DNA mismatch repair prote...
MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIV...
EDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTAD...
>gi|13905126|gb|AAH06850.1| MutL protein homolog 1 ...
MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIV...
EDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTAD...
GenPept
>gi|1079787|gb|AAA82079.1| DNA mismatch repair prot...
MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIV...
EDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTAD...
>gi|4557757|ref|NP_000240.1| MutL protein homolog 1...
MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIV...
EDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTAD...
NCBI RefSeq
>gi|730028|sp|P40692|MLH1_HUMAN DNA mismatch repair...
MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIV...
EDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTAD...
Swiss-Prot
>gi|741682|prf||2007430A DNA mismatch repair protei...
MSFVAGVIRRLDETVVNRIAAGEVIQRPANAIKEMIENCLDAKSTSIQVIV...
EDLDIVCERFTTSKLQSFEDLASISTYGFRGEALASISHVAHVTITTKTAD...
PRF
RefSeq: NCBI’s Derivative Sequence
Database
• Curated transcripts and proteins
– reviewed
– human, mouse, rat, fruit fly, zebrafish, arabidopsis
microbial genomes (proteins), and more
• Model transcripts and proteins
• Assembled Genomic Regions (contigs)
– human
– mouse
– rat
– chicken
– honeybee
– sea urchin
– zebrafish
– cow
– dog
– black poplar
• Chromosome records
– Human genome
– microbial
srcdb_refseq[Properties]
– organelle
ftp://ftp.ncbi.nih.gov/refseq/release/
RefSeq Accession Numbers
mRNAs and Proteins
NM_123456
NP_123456
NR_123456
XM_123456
XP_123456
XR_123456
Gene Records
NG_123456
Chromosome
NC_123455
Assemblies
NT_123456
NW_123456
Curated mRNA
Curated Protein
Curated non-coding RNA
Predicted mRNA
Predicted Protein
Predicted non-coding RNA
Reference Genomic Sequence
also Microbial replicons, organelles
Contig
WGS Supercontig
http://www.ncbi.nlm.nih.gov/RefSeq/key.html