1. The Life Sciences Search Engine
Using Entrez
The Life Sciences Search Engine

2. Searching NCBI Databases Efficiently
Knowing how to retrieve the exact information you need in an efficient way is the fundamental and most important skill in Bioinformatics.
Every NCBI database is designed and created for some specific purposes.
A common mistake Bioinformatics novices make is searching for information in an inappropriate database.
Entrez links among and within databases, making it easier to search for information.

3. What is Entrez?
Entrez is an NCBI retrieval system designed for searching several linked databases.
Entrez is a search tool for integrated access to the biological literature and sequence data.
Entrez is extremely powerful, enabling the user to quickly move between the different specialized databases.

4. Entrez
Entrez is divided into sites for nucleotide, protein, structure, genomes, OMIM, and more. You can use limits (such as RefSeq) to focus your Entrez search.
When you conduct a search via Entrez, your query generates this screen, telling you the number of hits to your query.

5. The Entrez System

6. The Big Picture Books UCSC PopSet PubMed e! GDB ProbeSet Nucleotide
MGC
Genome
Protein
Entrez
LocusLink
HGMD
Taxonomy
OMIM
Structure
Homologene
SNP
CDD
3D Domains
UniSTS
MapViewer
UniGene

7. Entrez and LocusLink
Entrez doesn’t link to all the databases that contain sequences, however!
LocusLink has its own groups of links to specialty databases, since it doesn’t cover all the genomes yet.

8. Entrez: Database Integration
Word weight
PubMed abstracts
Phylogeny
Taxonomy
3 -D Structure
3-D Structure
VAST
Genomes
Nucleotide sequences
Protein sequences
BLAST
BLAST

9. The (ever) Expanding Entrez System
PubMed
Nucleotide
Entrez
UniGene
Protein
Journals
Structure
CDD
Genome
PopSet
SNP
OMIM
3D Domains
Taxonomy
UniSTS
ProbeSet
Books

10. Entrez Databases PubMed Biomedical literature Books Online textbooks
Nucleotide GenBank, EMBL, DDBJ, RefSeq, PDB
Protein [GenBank, EMBL, DDBJ], RefSeq,
SWISS-PROT, PIR, PRF, PDB
Genome Complete genomes
Taxonomy Organisms in NCBI sequence databases
Structure MMDB: experimental 3D structures
Domains CDD: conserved protein domains
3D Domains Compact 3D protein domains in MMDB
OMIM Online Mendelian Inheritance in Man
SNP Single nucleotide polymorphisms
UniSTS Sequence Tagged Site markers
ProbeSet Gene expression and microarray datasets
PopSet Population study datasets
UniGene Gene-based expressed sequence clusters

11. Nucleotide Database
The Nucleotide database contains sequence data from GenBank, EMBL, and DDBJ, the members of the tripartite, international collaboration of sequence databases.
EMBL is the European Molecular Biology Laboratory at Hinxton Hall, UK;
DDBJ is the DNA Database of Japan in Mishima, Japan.
Sequence data are also incorporated from the Genome Sequence Data Base (GSDB), Santa Fe, NM.
Patent sequences are incorporated through arrangements with the U.S. Patent and Trademark Office (USPTO) and via the collaborating international databases from other international patent offices.

12. Entrez Nucleotides Primary GenBank / EMBL / DDBJ 35,116,960 Derivative
RefSeq ,219
Third Party Annotation ,182
PDB ,703
Total ,384,248
These slides are obtained and/or modified from the ncbi website “field guide to ncbi” clides and from

13. Database Searching with Entrez
Using limits and field restriction to find plant g6pdh
Linking and neighboring with g6pdh

14. glucose 6 phosphate dehydrogenase
Entrez Nucleotides
glucose 6 phosphate dehydrogenase
The G6PD enzyme catalyzes the oxidation of glucose-6-phosphate to 6-phosphogluconate, while reducing nicotinamide adenine dinucleotide phosphate (NADP+ to NADPH). In terms of electron transfer, glucose-6-phosphate loses two electrons to become 6-phosphogluconate and NADP+ gains two electrons to become NADPH. This is the first step in the pentose phosphate pathway. This pathway, or shunt, as it is sometimes called, produces the 5- carbon sugar, ribose, which is an essential component of both DNA and RNA.

15. You will find MANY hits for this and can’t easily tell which ones are plants.

16. Limits Are Helpful
Limits allow restriction of a search to a defined subset of the database.
Limits can be set to restrict a search to a particular database field (e.g., the Author field).
Limits can be set to search everything but a particular type of data (e.g., “exclude patent records”).
Alternatively, limits can be set to search only a particular type of data (e.g., Genomic RNA/DNA) or to search only data from a particular source database (e.g., EMBL). Date limits and sequence length limits are also possible.
The contents of each Entrez database differ, and therefore the Limits available for each database differ.

17. Entrez Nucleotides: Limits & Preview/Index
glucose 6 phosphate dehydrogenase
Try using the Limits and Preview function to hone your search
To find the Plant G6PD genes.

18. Entrez Nucleotides: Limits
Accession
All Fields
Author Name
EC/RN Number
Feature key
Filter
Gene Name
Issue
Journal Name
Keyword
Modification Date
Organism
Page Number
Primary Accession
Properties
Protein Name
Publication Date
SeqID String
Sequence Length
Substance Name
Text Word
Title Word
Uid
Volume
Field Restriction
glucose 6 phosphate dehydrogenase
Exclude bulk sequences
You can select from a long list of field limits to search through the databases.

19. Entrez Nucleotides: Limits
glucose 6 phosphate dehydrogenase
Title == Definition
Exclude Bulk Sequences
mRNA molecule type
Nuclear gene
You can limit the search by making some selections from among the choices in the flat fields of the files.

20. Document Summaries: Limits
Now you have many less files than you started out with, because of the limits choices. However, you still have a lot of choices.

21. Adding Terms: Preview/Index
Accession
All Fields
Author Name
EC/RN Number
Feature key
Filter
Gene Name
Issue
Journal Name
Keyword
Modification Date
Organism
Page Number
Primary Accession
Properties
Protein Name
Publication Date
SeqID String
Sequence Length
Substance Name
Text Word
Title Word
Uid
Volume
green plants
green plants
You may not know all the terms that are in the database to select from. You can use the Preview/index to give you some lists of searchable keywords. This could help you set more meaningful limits.

22. Plant cytosolic g6pdh mRNAs
Using the green plants limits, you are now down to only one page of G6PD genes and they are all from green plants.

23. Database Neighbors and Interlinking
What makes Entrez more powerful than many services is that most of its records are linked to other records, both within a given database (such as Nucleotide) and between databases.
Links within a database are called “neighbors” (e.g., Nucleotide neighbors).

24. Links Between Databases
Protein and Nucleotide neighbors are determined by performing similarity searches using the BLAST algorithm to compare the entry amino acid or DNA sequence to all other amino acid or DNA sequences in the database. We will discuss more about BLAST later.
Nucleotide sequence records in the Nucleotide database are linked to the PubMed citation of the article in which the sequences were published.
Protein sequence records are linked to the nucleotide sequence from which the protein was translated.

25. Plant cytosolic g6pdh mRNAs
Summary
Brief
GenBank
ASN.1
FASTA
GI list
LinkOut
PubMed Links
Protein Links
Nucleotide Neighbors
PopSet Links
Structure Links
Genome Links
Taxonomy Links
OMIM Links
Formats
Links and neighbors
(related records)
Now you can look for other information that might be close to what you are studying using links and neighbors, which are related recods. This helps you broaden your search a little, in case the files didn’t add your search terms as keywords.

26. LinkOut
LinkOut is a feature of Entrez that is designed to provide users with links from PubMed and other Entrez databases to a wide variety of relevant web-accessible online resources:
Full-text publications
Other biological databases
Consumer health information
Research tools
The goal is to facilitate access to relevant online resources beyond the Entrez system to extend, clarify, or supplement information found in the Entrez databases.

27. Protein Database
The protein database includes proteins from translate regions of DNA in GenBank as well as sequence from PIR
The entry includes:
The name of the protein
How the protein sequence was derived
An accession and a PID number
The number of amino acids

28. Protein Entry The Entry also includes:
Structural information for the protein (if known)
Helices and -Sheets
Domains
Etc
The sequence of amino acids comprising the protein

29. Setting Protein Database search limits
Choose Protein from the drop-down menu
Can do a Boolean search
Or can set LIMITS
Fields (eg Author, Journal, etc.)
Gene Location (genomic, mitochondrial etc)
Segmented Sequence
Only from (Database to check)
Modification date

30. Linking Between Databases
Sometimes you will pull up a record and you have no idea what organism the gene you are looking at is from.
For Example, the following record- what is Medicago sativa ?

31. Entrez GenBank / GenPept

32. Taxonomy to the Rescue
Entrez lets you click a live link from the record and determine what organism Medicago sativa is.
It is alfalfa.
You can also tell what it is related to taxonomically, because sometimes the common name isn’t very useful either!

33. Taxonomy Link

34. Advanced Neighbors: BLink

35. What is BLink BLink - BLAST Link
Someone has done a BLAST search already, and you can just retrieve it!
BLink displays the graphical output of pre-computed blastp results against the protein non-redundant (nr) database.

36. 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

37. PopSet Links
The PopSet database contains aligned sequences submitted as a set resulting from a population, phylogenetic, or mutation study.
These alignments describe such events as evolution and population variation.
The PopSet database contains both nucleotide and protein sequence data.

38. Protein Neighbors->PopSet Links

39. Protein Neighbors->Genome Links

40. PopSet search results The results or a PopSet search
The PopSet database includes alignments of genes from multiple organisms OR different gene families OR mutational analyses

41. PopSet Entry The PopSet entry includes: The title of the paper/study
The length of the sequence(s) aligned
The number of aligned sequences

42. PopSet Entry without alignment
The PopSet Entry without an alignment
Title of the study
The number of sequences included
Links to the sequences

43. Entrez Structures

44. Protein Structures can also be in databases
This is an excellent review of this topic
is a useful review
Tutorial.

45. Entrez links to structure databases
The Structure database or Molecular Modeling Database (MMDB) contains experimental data from crystallographic and NMR structure determinations.
The data for MMDB are obtained from the Protein Data Bank (PDB).
The NCBI has cross-linked structural data to bibliographic information, to the sequence databases, and to the NCBI taxonomy.
Use Cn3D, the NCBI 3D structure viewer, for easy interactive visualization of molecular structures from Entrez.

46. Structure Search results
The structure of proteins are also in a database
Search as before
Your search results are similar

47. Structure Entry The structure Entry has links to the other databases
And it will allow you download a file to open with a structure viewer program

48. Proteins with similar structures and functions have been identified in the databases

49. BLink: Advanced Protein Neighbors

50. BLink: Related Structures
Related biological functions usually have related structures.

51. Viewing Structure in Cn3D
You can download Cn3D (a structural viewer program) from NCBI
This will allow you to view the structures from the structure database

52. Cn3D Text Window
The Text window of Cn3D will align two or more proteins so you can compare the structure of multiple proteins

53. BLink: Human Homologue
Often in biomedical sciences, we want to find the human gene rather than just the mouse, dog, honeybee or rabbit gene. Selecting the human homologue can do this.

54. Human RefSeqs: Genome Reagents

55. MMDB: Molecular Modeling Data Base
Derived from experimentally determined PDB records
Value added to PDB records including:
Addition of explicit chemical graph information
Validation
Inclusion of Taxonomy, Citation,
and other information
Conversion to ASN.1 data description language
Structure neighbors determined by
Vector Alignment Search Tool (VAST)

56. Structure Summary Cn3D viewer Structure Neighbors 3D Domain Neighbors
Conserved Domains

57. Cn3D 4.1
You can view the structure in the database in several different formats.

58. Cn3D 4.1: Structural Alignment
Conserved ATP binding site
Two genes can be compared with both their sequences and their structures to see if they aligh into the same shape of molecule. Often the structure and function of a gene is more conserved that its exact primary amino acid sequence.
Src Kinase H. sapiens
Casein kinase S. pombe

59. Cn3D: Simple Homology Modeling
Here you can see the 3-D structure of two related genes- one from human and one from swordtail, a type of fish. These genes are red where the sequence matches and blue where it doesn’t. The 3-D sequence can be highlighted to show the regions where the sequence isn’t the same.
human
swordtail

60. Using Cn3D to model domains
Domains are functional parts of a protein. Here the same small functional part of a series of proteins has been lined up and the sequence is compared. Some regions match very closely and are in pink. Others are similar. The important serine is highlighted in yellow.

61. Other services and databases from the NCBI
LocusLink to all possible information from NCBI and beyond for a few well characterized model organisms.
LocusLink is a great starting point: it collects key information on each gene/protein from major databases. It now covers 8 organisms.
RefSeq provides a curated, optimal accession number for each DNA (NM_006744) or protein (NP_007635)

62. Locus Links Results of a Locus links search, includes: Locus ID
Species
Locus symbol
Locus name
Locus location
Links
Protein Database
OMIM
Reference Sequence
Related GenBank Sequences
Homologene Data
UniGene
Variation Data
When you find a gene is in locus link, you can easily compare it with other useful information. Not all genes have the same amount of information known.

63. LocusLink: Selected Higher Genomes
OMIM
RefSeq
GenBank
dbSNP
UniGene
Full report
PubMed
HomoloGene
Map Viewer
Protein

64. Protein Database
The Protein database contains sequence data from the translated coding regions from DNA sequences in GenBank, EMBL, and DDBJ as well as protein sequences submitted to:
Protein Information Resource (PIR)
SWISS-PROT
Protein Research Foundation (PRF)
Protein Data Bank (PDB) (sequences from solved structures)

65. NCBI Protein Databases
GenPept GenBank, EMBL, DDBJ CDS translations
RefSeq mRNA based (NP_) and genome based (XP_)
Swiss-Prot curated high quality protein reviews
PIR protein information resource Georgetown University
PRF protein resource foundation
PDB Protein Databank sequences from structures

66. Entrez Protein GenPept (GB,EMBL, DDBJ) 3,442,298 RefSeq 856,191
Third Party Annotation ,834
Swiss Prot ,508
PIR ,821
PRF ,079
Total ,442,298
BLAST nr ,642,191

67. Protein Link
BLAST Link
Conserved Domains

68. Related Proteins: Redundancy
Redundant Sequences
Since the protein database pulls in sequences from all these other databases, some of the entries can be considered redundant or duplicates. You can do some searches with the “nonredundant” database. Sometimes, however, you might want more information that is found in some of the specialized databases. SwissProt is an example- lots more information has been added to these entries as they are more heavily curated.

69. Related Proteins: Links
Sequence from MutL structure

70. BLink: non-redundant relatives
Arabidopsis homolog
Conserved Domain

71. MLH1 Domain Structure: CDD
CDD is a database of protein domains. The source databases for CDD are Pfam, Smart, and COG.
This link has more information on domains.
ATPase Domain
Mismatch Repair Domain

72. MLH1: ATPase Domain

73. 1BGQ: ATPase Domain in Cn3D
Yeast HSP90
ATP Binding site helix
You can highligh at region in the primary sequence and then identify it in the 3-D model. This is very handy!

74. Variations Human MLH1

75. Finding structural models
BLink
Finding structural models

76. Mapping Variation Onto Structure
Loads sequence alignment and structure in Cn3D
Bacterial DNA mismatch repair proteins

77. Mapping Variation Onto Structure
Asn
Ile
Ile – Val
Conserved Asn

78. NCBI Genome Databases
The Genome database provides views for a variety of genomes, complete chromosomes, sequence maps with contigs, and integrated genetic and physical maps.

79. Microbial Genomes
ZWF

80. Genome search results Genome Search Results
The Genome database includes full (and some partial) genomes from viruses to complex organisms

81. Genome Entry Genome entries include Maps of the genome
Links to the sequence
The organism for the genome

82. Genes Database: All Genomes
Coming soon!

83. Genes Database: All Genomes

84. Genes Database: All Genomes

85. But wait! There’s more!
There is even more at NCBI that I have covered here.
This site map is also a guide to NCBI resources. Each link leads to a brief description of the resource on this page, then to the resource itself.

86. There are many bioinformatics servers outside NCBI.
Try ExPASy’s sequence retrieval system at
(ExPASy = Expert Protein Analysis System)
Or try ENSEMBL at for a premier human genome web browser.