NCBI Boot Camp for Beginners Slides

NCBI
Boot
Camp

NCBI
“
”
...advances science and health by providing access to biomedical and genomic information

NCBI
Sequences
Expression
Genome maps
Structures
Protein Domains
Homology (gene, protein, structure)
Pathways
Genetic Variation

NCBI
tools
databases

databases*
* a brief survey of selected dbs

1
literature

PubMed
Bookshelf
OMIM

PubMed
20,672,941
citations
2,157,529
PubMed Central
5,519
indexed journals

Bookshelf
767

Dr. McKusick
OMIM

Lesch-Nyhan
If you query for Lesch-Nyhan, you
get a very long OMIM record 
OMIM

Clinical Features
Biochemical Features
Inheritance
Pathogenesis
Diagnosis
History
Description
Cloning
Gene Structure
Mapping
Molecular Genetics
Pathogenesis
Evolution
Animal Model
Allelic Variants
See Also
References
Contributors
Creation Date
Edit History
OMIM
Note: there are separate entries for Lesch-Nyhan syndrome and the protein that causes the defect

OMIM
Every OMIM Record has an extensive list of internal and external links

2
sequences

Nucleotide
GenBank
RefSeq

DNA
RNA
Protein
EST: expressed sequence tag
SNP: single nucleotide polymorphism
WGS: whole genome sequencing
CDS: coding sequence
STS: sequence tagged site

NCBI
SNP
Primary
Databases
GEO
GenBank
Protein

GenBank Format
GenBank

LOCUS
Locus name, size, type, division, modification date
Search tips:
Locus names can change!
Division names are historical,
not taxonomical!

DEFINITION
As the author sees fit…
Search tip: No Controlled Vocabulary in Definitions!

ACCESSION/Version
Accession numbers do not change, even if information in the record is changed at the author's request.
Version and GI numbers change

Keywords, Source, Organism
Organism: Tied into Taxonomy Browser
Search tip: Keywords are often blank
When performing a “keyword” style search, use [all] , [word] or [title]

Selected References
Newest First
Last “reference” covers submission information

Features I
Source, gene, misc features

Features II
CDS: links, translation

Sequence

GenBank Format
GenBank
(also for protein)

132,015,054
Sequences in GenBank 3/20/11
+
HARD WORK
-
redundancy
RefSeq

RefSeqs
provides a single record for each natural biological molecule for major organisms ranging from viruses to bacteria to eukaryotes
RefSeq

bio mol
DNA
RNA
Protein
RefSeqs
RefSeq

bio mol
DNA
RNA
Protein
RefSeqs
HELLO
my name is
XX_123456
RefSeq

bio molecules
Genomic DNA
(NC)
Incomplete
(NG)
mRNA
(NM)
Model mRNA
(XM)
Curated Protein
(NP)
Model protein
(XP)
RefSeq

NG_012250.1 NM_000690.2 AY621070.1 EU414258.1 EU414257.1 EU414256.1 EU414255.1 EU414254.1 EU414253.1 EU414252.1 EU414251.1 EU414250.1 EU414249.1 AF164120.1 EU373813.1 EU373812.1 EU373811.1 EU373810.1 EU373809.1 EU373808.1 EU373807.1 EU373806.1 EU373805.1 EU373804.1 AH002599.1 M20456.1 M20455.1 M20454.1 M20453.1 M20452.1 M20451.1 M20450.1 M20449.1 M20448.1 M20447.1 M20446.1 M20445.1 M20444.1 CR456991.1 AB385105.1 CU678321.1 CU678320.1 AF073514.1 AF073513.1 AF073512.1 AF073511.1
NG_012250.1
NM_000690.2
RefSeq

Note: the NP sequence would not normally be found using a nucleotide search – I have included it only to show the complete suite of RefSeq for ALDH2
NG_012250.1
NM_000690.2
NP_000681.2
RefSeq

3
genes/genome

Genome
Gene
HomoloGene

Genome
1090
eukaryota
1483
prokaryota
2507
viruses

Note: genome records are either mitochondrial or chromosome
Note: no common names are listed as genome query results

The genome record shows a variety of stats for different databases, as well as a map of the genome that is scrollable

Searching in BioProject yields common names

BioProject results contain background information

Instead of searching Genome, you can also browse via the Genome Resource Guide

Genome Resources
G
Genome BLAST
B
Map Viewer
M
Genome Project
(BioProject)
P

G
Genes and Human Health
Epigenomics
The Genomic Sequence
Maps and Markers
Transcribed Sequences
Cytogenetics
Comparative Genomics
A standard record in Genome Resources contains many links out along with brief database summaries

M
Map Viewer starts by letting you select a chromosome (or section of a circular genome)

To the left of each gene, there are a variety of links out. Note: these change based on the level of information known about a given gene.
M
HUGO Gene Nomenclature
Sequence Viewer
Protein
Download
Evidence Viewer
Molecular Model
STS, OMIM, CCDS, SNP

Regulatory
Gene
Intron
Exon
Intron

Each gene record provides extensive details. We will go through an example Gene record in the following slides.

Sequence Viewer and MapViewer
Genomic Info

Bibliography
PubMed
NOTE: Gene Reference into Function is an excellent resource for literature related to function. These articles have been submitted for inclusion into GeneRIF and are not the product of an automated text search.
GeneRIF

There’s
Even More!
Interactions
Gene Ontology
Genotypes
Homologues
Protein Information
Interactions will list all known interacting molecules, providing links to

RefSeq
These reference sequences are stable and are independent of genome builds

The NCBI Assembly
~100 individuals
The Celera assembly
~5 individuals
These reference sequences refer to specific builds
HuRef
Just Craig Ventner

LINKS
LINKS: internal, external and commercial

HomoloGene

Homologs
paralogs
orthologs
orthologs
frog α
chick α
mouseα
mouseβ
chick β
frogβ
α-chain gene
β-chain gene
GENE DUPLICATION
Early Gene of Interest

Protein of Interest
(P3H1)
Cross-species identity is automatically calculated
Automatic sequence alignments are easily accessible

Protein of Interest
(P3H1)
Note: UniGene may come up with different results, since it is based on EST clusters and not protein sequence

4
expression
& structure

UniGene
EST, GEO
Structures
CDD, MMDB, PubChem…

UniGene
…an organized view of the transcriptome

SELECTED PROTEIN SIMILARITIES
GENE EXPRESSION
EST
GEO
MAPPING POSITION
SEQUENCES
mRNA
EST

GENE EXPRESSION
EST
This is a “virtual northern” whereESTs are counted to get a rough sense of overall expression levels

GENE EXPRESSION
GEO
Note: the GEO results contain all arrays that assay for this gene; most of these results are for specific disease or altered states and do not necessarily reflect wild type, normal levels of expression

Structures
CDD, MMDB, PubChem…

Cn3D colored by secondary structure
Note: Cn3D has aligned the individual chains for you

Cn3D colored by chain (there are 7)

“structure function”: the hemolysin protein bores a hole into red blood cells and sucks their insides out. The structure kind of looks like a hollow tack.

Note: the structure listing shows each individual chain (along with 3D domains and superfamilies) AND the chemical that was found in the crustal structure (see arrow)

Another example… this time a single chain with distinct domains

Now we are coloring by domain. Also note the funky space-filling model. It makes proteins look fat.

Note that Super Families are defined: clicking on them will take you to the conserved domain database

The Conserved Domains Database provides alignments across species of conserved domains, along with a general description of the domain

3D domains are color coded. Note: 3D domains do not always correlate to Super Families! Clicking on the 3D domain will take you to related structures

You can select structures and then view the 3D alignment in Cn3D

Volia! Structural alignment. Note: the sequences are aligned in the Sequence View box.

PubChem has three primary areas:
BioAssay – registry of assays that can be searched by small molecule
Substance – a redundant registry of compounds
Compound – a non-redundant, curated chemical database

You can search PubChem by chemical name, CAS number, or even by similar structures.
Records contain lots of additional information. Highlights: synonyms (which can be quite extensive in chemical nomenclature). Of particular note: if the compound shows up in Structure, you can link to a view in Cn3D that shows it complexed with protein/DNA/RNA!

BioSystemswill display a short verbal description, a schematic of the system in question and a link to all of the genes, proteins, small molecules found in the system along with links to related systems .

NCBI
discovery
initiative

NCBI
high quality DB
discovery tools

high quality DB
discovery tools
RefSeq
GenBank
Database Ads
check out these resources!
Sensors
are you looking for…
Analysis tools
pre-computed & on the fly

where
do I
start?

anywhere*
*but gene acts as a good hub

Apolipoprotein E
APOE
Cys130Arg

We
Can
Do
It!
Gene and RefSeq
Genome Maps
Allelic Var/Disease
Expression
Homologus G/P
Structure

Search for APOE in Entrez:
Note that there are many different records in several different databases that have hits for APOE.
Select PubMed.

Select APOE in homo sapiens
We have used PubMed for it’s gene sensor , which is fantastically useful. However, you can also search directly in the Gene database.

LOTS of information in this report, including links IN the report, links to other NCBI databases and links to outside resources.

Let’s check out the reference sequences….

Note the genomic, mRNA and protein RefSeq that are independently maintained.

Separate records for ref sequences associated with specific genomic builds…

(many databases here)
Let’s check out the SNP:Variation Viewer

Note, the Cys130Arg variant has been frequently observed and well documented

Let’s observe the Sequence Viewer and MapViewer for this gene.

Note, you can change which sequence you want to observe (Stable reference, reference, celera and HuRef)

The full view shows genes in the area, along with info on SNPs and other variation classifications.

Full screen of MapViewer

ab initio modeling
Ensemble
Genes
UniGene
RefSeq
These are default maps

You can change the maps you wish to view, both in terms of how you are annotating the genome but also in which organisms.

Here we are looking at Chimp, Mouse and Human Gene maps.
You can zoom out to get a larger picture of the area.

APOE is part of the APO gene cluster. Note: lines between maps are mapped homologues.

Each gene has a series of links following its name. We’ll jump to the APOE OMIM record.

Another extensive record! Let’s jump to allelic variants.

Let’s go to the SNP record
Cys130Arg is .0016
.0016
Extensive documentation…

OMIM
Prot 3D
SeqView
GeneView
MapView
VarView
PubMed

NOTE: the default setting doesn’t show much, because it doesn’t include clinically associated variants – click this box and refresh.

This is the one we want! Let’s jump to this reference SNP

Hummmm… the two reference assemblies have a wild type allele, whereas Celera and HuRef carry the mutant allele.
Let’s check out this area in HuRef using the sequence viewer – click on the chromosome position link.

Clicking on sequence will bring up the sequence and the CDS. You will note that HuRef (which means Craig Ventner) carries the mutant allele.

Let’s check out expression in UniGene

Click on EST profile to go to the virtual northern.

Disease
State
BODY SITES
Development

Click on GEO Profiles to see actually gene expression array data.

Note, there are thousands of hits, meaning many gene arrays have assayed for this gene. However, most of these are in reference to a disease or altered state.

Use GDS596 – it is the results for “normal” gene expression.
Click on a chart to see detailed results.

Highest expression in the liver, with lower level throughout the brain.
liver
brain

Let’s check out homologs

You can show a pairwise alignment using BLAST…

E
value
Note the very low E value
1e-158

The alignment shows that the Chimp genome carries an R at the allele in question!

You can also check out homologs found in UniGene – a different a way to search.

Bunnies show up using the UniGene homolog search, but not the HomoloGene search.

Let’s go check out the protein record…

Click here to link to the RefSeq protein record.

Let’s run a BLAST to see if we can identify the giant panda homolog.

I’ve changed the search to focus on the RefSeq protein database and limit it to the giant panda.

Note: BLAST automatically detects domains
The highest hit is a hypothetical protein. Let’s take a look at the alignment.

Note, the panda has the mutant arginine...
… does this mean pandas and chimps both have early onset Alzheimer's disease? Nobody knows!

Let’s check out some related structures.

This is the default setting. Change to all similar MMDB.

Click here to go to an alignent between your query and the structure’s sequence.

Click here to view in Cn3D
Note: the structure sequence contains the mutant arginine

Showing side chains, colored by hydrophobicity.
The arginine is shown in yellow.
Click here to go to the structure summary for 1B68

Click here to find similar 3D domains

Select another structure and then view 3D alignment.

Overall alignment, showing side chains colored by hydrophobicity.
Note, the Cys vs. Arg doesn’t make a huge change structurally.

asdf
science
can be
complex...

…we can
help you
with that.

thank
you

Jackie Wirz, PhD
wirzj@ohsu.edu

NCBI Boot Camp for Beginners Slides

by Jackie Wirz

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