NCBI Boot Camp for Beginners Slides

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

databases* * a brief survey of selected dbs

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

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

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

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

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 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 HELLO my name is provides a single record for each natural biological molecule for major organisms ranging from viruses to bacteria to eukaryotes 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

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

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

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 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

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.

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.

(many databases here) 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

(many databases here) 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...

Jackie Wirz, PhD wirzj@ohsu.edu

NCBI Boot Camp for Beginners Slides

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