NCBI Entrez queries

Searching NCBI has much more options than Google

(do you know Google options?)

By default the query text is searched in any part of any database

But you can specify the fields where you are looking for

• Title of a paper
• author
• date
• taxonomic id

Entrez Examples

protease NOT hiv1[organism]

This will limit the search to all proteases, except those in HIV 1.

1000:2000[slen]

This limits the search to entries with lengths between 1000 to 2000 bases for nucleotide entries, or 1000 to 2000 residues for protein entries.

Entrez Examples

10000:100000[mlwt]

This limits the search to protein sequences with calculated molecular weight between 10 kD to 100 kD.

src specimen voucher[properties]

This limits the search to entries that are annotated with a /specimen_voucher qualifier on the source feature.

Creating advanced queries

Quotes " are important

The fields are written inside brackets []

Each database page includes an Advanced Search option

Combining queries

Entrez queries can be single words, short phrases, sentences, database identifiers, gene symbols, or names

AND: Finds documents that contain terms on both sides of the operator terms. The intersection of both searches.

OR: Finds documents that contain either term. The union of both searches.

NOT: Finds documents that contain the term on the left but not the term on the right of the operator. The subtraction of the right side from the left side

Example

AND must be in uppercase. It is recommended to also use uppercase for OR and NOT

• Operators are processed left-to-right

    promoters OR response elements NOT human AND mammals

• Parenthesis can be used to control the evaluation order

    g1p3 AND (response element OR promoter)

Dates and Other Ranges

• Certain fields can accept ranges of values

  • Publication Date, Modification Date, Accession, Molecular Weight, and Sequence Length

• Low and high numbers are entered with a colon “:” between them followed by the field

    110:500[Sequence Length]
    2015/3/1:2016/4/30[Publication Date]

NCBI online documentation

We can get a different explanation in the public documentation made by NCBI

https://ftp.ncbi.nlm.nih.gov/pub/education/ Mod_Workshops/2016/June_UWashington/ Workshop1_Navigating_NCBI/

All documents made by NCBI are public domain

Pipelines: putting all together

When we design molecular biology experiments, or when we analyze their results, we need to use several tools in chain

Today we are going to see an example using the NCBI website

Protein domains according to http://pfam.xfam.org/

We need an official definition of each domain

F-box

• motif PF00646.
• alternative motifs (Gupta et al. 2015)
  • PF12937, PF13013, PF04300, PF07734, PF07735, PF08268 and PF08387

WD-40

• motif PF00400

Finding proteins with those domains

Using NCBI Conserved Domain Architecture Retrieval Tool (https://www.ncbi.nlm.nih.gov/Structure/lexington/lexington.cgi)

Use the query:

[pfam00646,pfam00400]

to look for proteins that contain both domains in the specified order

Results

• Several architectures contain both motifs
• Selected the first, with only the relevant domains and nothing else
• Click the button “Lookup sequences in Entrez” to find the list of proteins

Filter results: only Legumes

Most of times is a good idea to check the Taxonomy database

Each sequence on GenBank is tagged with a taxon id

Using taxid is more precise than using common names

For example, a protein from human can be labeled “95% similar to mouse”

Is that a human or a mouse protein?

Downloading

For your convenience you can download the sequences

• Decide Format
• Decide Content

In this case we only need accession ids

Finding more proteins

Now we use BLAST to find other proteins in legumes similar to the ones we have

Notice that CDART only has some proteins pre-processed. New sequences take time to be processed

How would you do that?

Save your search strategy

It is essential that your protocol can be replicated

It is a very good idea to save the search strategy in a file

It is also wise to save the output in a text file

Separate by tab or by comma

Process the output|check domains

How many new proteins we find?

Do all of them have the good domains?

Let’s use CDD again, this time looking for motifs on the new proteins \[protein \to\{domains\}\] (the first time was \(domain\to\{proteins\}\))

Next steps

• Keep only proteins with the good domains
• Download the sequences of all proteins
• Download the sequences of the messengers
• Design primers to measure gene expression
• Find literature about gene expression

It is boring to do it one by one

And takes a lot of time

It is easy to make mistakes

It is hard to replicate

Can we do it automatically?

Map of E-tools

Use your favorite language

There are Entrez libraries for most languages

For example in R it is called rentrez

There is a command line version, and versions for all major computer languages