NCBI’s Bioinformatics NCBI’s Bioinformatics ResourcesResources

Michele R. Tennant, Ph.D., M.L.I.S.

Health Science Center Libraries

U.F. Genetics Institute

January 2015

Entrez Nucleotides

Entrez Nucleotides (GenBank)

• Database of nucleotide sequences (ATGC)

• Actually contains data from several databases - GenBank, EMBL, DDBJ, RefSeq

• Hard to search because many submitting scientists send in redundant information and poorly annotated information

Nucleotide Data DomainNucleotide Data Domain

• As of December 15, 2014

• Over 184,938,063,614 bases

• Over 179,295,769 sequence records

• Some complete genomes and chromosomes

So Why So Hard to Search?So Why So Hard to Search?

• No controlled vocabulary - lose power of MeSH - must OR synonyms. Often miss the records you want.

• Archival - quality of annotations depends on the submitter (especially features field); little to no quality control; spelling errors! Often miss the records you want.

• Redundant - lots of records for the same gene; partial records, etc. Often pull up records you don’t want.

GenBank Sample Record

• Before searching, we will look at a GenBank sample record

• Note that the “Features” field provides useful biological information, and may be searched

Click any link in sample record

to access definition of

field and search tips

“Definition” field acts as record title – search

[titl]

Unique identifier; assigned by NCBI;

required by journals/grants

Link to PubMed citation/abstract

The “Features” field provides the most

biological information; search as [fkey]

Numbers indicate

location on the nucleotide sequence

…3158

GenBank IdentifiersGenBank Identifiers

• Accession Number - U49845 [accn]• Unique identifier; does not change• Letter prefix no longer has significance

• Version - U49845.1 • If any change to sequence, version

U49845.2 created• GenInfo Identifier (GI number) [uid]

• Run parallel to accession.version system; change in sequence changes number

Searching “Nucleotides”• Database is difficult to search:

• Redundant records• Archival - poor or missing annotation

• Best searches are done using commands; need a class to learn all

• Practice search – search for sequences for human presenilin 1• Is there anything odd about the some of the

retrieved results?

Search for HUMAN presenilin 1

But end up with rat, mouse, etc.

Choose “nucleotide” from dropdown, then

click “search”

Searching “Nucleotides”• We retrieved the non-human and PSEN2

(rather than PSEN1) records because the computer looked for the terms “human” and “presenilin 1” ANYWHERE in the record (click on details tab to see how the computer parsed your search)

• Use complex boolean searching to clean this up: term [field] AND term [field]

Searching “Nucleotides”• How to get rid of non-human sequences?

• Search human [orgn] (this works for any taxon)

• How to get rid of non-presenilin 1 sequences?• Another trick – search PSEN1 [gene]• Note – you may miss relevant sequences, but should not pick

up irrelevant sequences• The sequences that you miss are the ones that have not been

annotated with the current official gene symbol in the “gene” field

• DO NOT use this method if you need to find every sequence for a particular gene

• Human [orgn] AND PSEN1 [gene]

Use these filters to choose molecule type, confine to

RefSeq records

This is the search that was completed using fields (orgn, gene) and filters

How Can I Find “Best” Sequences

• Non-redundant, curated subset of the sequence data domains

• Contains one record for each gene or splice variant from each organism represented

• Records can be thought of as “review articles” for sequences

• “Best” (usually longest) sequence used as seed• Value-added annotations provided by experts• Easy – a tab now exists to limit retrieval to just RefSeq

Click on the RefSeq link to retrieve only the “best” sequences (highly

annotated, complete, nonredundant)

The typical RefSeq accession number

format: 2 letters, an underscore, and

then numbers

Viewing Formats

• The “Default” view is the standard GenBank record

• Researchers often use the “FASTA” format for analysis

• Change the record format at the “Display” pull-down menu

Entrez Proteins

Entrez ProteinsEntrez Proteins

• Contains data from several databases:

• SwissProt, PIR, PRF, PDB

• Translations from annotated coding regions in GenBank and RefSeq

• Redundant archival data domain of publicly available protein sequences

Searching Entrez ProteinsSearching Entrez Proteins

• Searched like Entrez Nucleotides• “Filters” choices differ; includes

molecular weight and sequence length filters

Entrez Gene

Entrez Gene

• Pulls together information (sequences, structures, literature, gene models, pathways, etc.) for genes

• Best place to start for “gene-centered” info• One record per gene per organism• Search by names, symbols, accessions,

publications, GO terms, chromosome numbers, E.C. numbers, etc.

Search using gene symbol

Could have searched under any of these

aliases (unlike GenBank where you

would have to try them all)

Official gene symbol as

determined by the Human

Genome Nomenclature Commission

Summary of protein, function

and disease-causing mutations; from RefSeq record

Links to PubMed records

that provide evidence of

function – any researcher can

add these

Links to OMIM records of phenotype/

disease

Gene Ontology terms form a controlled vocabulary with three components – biological process, molecular function, and cellular

component

Links to homology

maps

Links to protein interactions

Pathway info may be available from

the Kyoto Encyclopedia of

Genes and Genomes

Sequence and domain links

Links to GeneReviews – clinical resource

Taxonomy Browser

Search Taxonomy BrowserSearch Taxonomy Browser

• How many genera from the family Iguanidae are represented by sequence data?

• How many nucleotide and protein sequences are available for the family?

Entrez Searching Summary

To Find Everything(?) To Find Everything(?) Broaden SearchBroaden Search

• OR together synonyms • OR together related terms (gene name, gene symbol,

protein name, alternate spellings, disorder)• Don’t specify a field- search entire record• Truncation - use * at end of word root• Click “Related Records”• Try using Taxonomy Browser to pick up all taxa in a

particular group

Fewer/Best Records Fewer/Best Records Narrow SearchNarrow Search

• Search particular fields:

• PubMed - MeSH Browser, subheadings, major MeSH

• Nucleotide - features, title, gene, properties, organism

• Use “Filters”• Search only the RefSeq database

Will Entrez Find Will Entrez Find Every Sequence Record?Every Sequence Record?

• No!!! • Entrez relies on annotation of records, so you are

searching solely on “terminology”• Some records are not annotated, some records are

poorly or incorrectly annotated

• To find all useful sequences – need to search on sequence itself • Related sequence link• BLAST

Entrez “Related Records”Entrez “Related Records”

• Will vary depending on data domain• PubMed related articles

• Based on a “word weight” algorithm – MeSH, title, abstract words

• In order by weight (highest weight first)

• Nucleotide and protein related sequences• Based on basic BLAST search• In order by best BLAST score