Download Exercises Biological databases PART

Exercise databases
Bioinformatics (updated 2015 january)
EXERCISES BIOLOGICAL DATABASES PART
Exercises Biological databases PART .......................................................................................................................... 1
Discovering genome projects in NCBI ............................................................................................................................. 1
View the genome sequence initiatives (Go to Bioproject) ......................................................................................... 1
Get a look at the large initiatives: ............................................................................................................................... 6
Using the Entrez search engine to discover distinct databases at NCBI ......................................................................... 7
Pubmed database ....................................................................................................................................................... 7
Redundant sequence database: Nucleotide, Protein, genome, EST… ........................................................................ 8
Performing advances searches using Entrez in other redundant databases ............................................................ 13
Comprehensive database : unigene ......................................................................................................................... 14
comprehensive database Gene ................................................................................................................................ 18
DISCOVERING GENOME PROJECTS IN NCBI
VIEW THE GENOME SEQUENCE INITIATIVES (GO TO BIOPROJECT)

How many prokaryotic genomes have been sequenced? October 2006: 381 How many are in progress
(October 2006: 267)? Note also the nice taxonomic overview of all prokaryotic species that have been
sequenced.
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How many plant species have been fully sequenced
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What is the 1000 genomes project
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What is HMP
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Exercise databases
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
Can you find the mammoth sequencing project

Search for the genomic map of the Chimp link (Pan troglodytes)
Microbial genomes
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(Browse genomes is at the bottom of the page if you wait long enough)
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Plant genomes
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Find a full genome (chimp i.e. pan)
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GET A LOOK AT THE LARGE INITIATIVES:
What is HMP?
Why is this an example of a metagenomics project?
What is the 1000 genomes project
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Why can it be useful
See also http://www.1000genomes.org/about
The 1000 Genomes Project (human)
“The purpose of the project is to support the discovery and understanding of genetic variants that influence human
disease. Specifically defined goals are (a) the discovery of single nucleotide variants at frequencies of 1% or higher in
diverse populations, (b) even more comprehensive discovery (variants down to frequencies of 0.1 - 0.5%) in functional
gene regions, and (c) discovery of structural variants, such as copy number variants, other insertions and deletions, and
inversions, including sequence-level understanding of breakpoints. The volume of data generated by 1000genomes
project is unprecedented. The data is accessible from two mirrored ftp sites at EBI and NCBI.”
USING THE ENTREZ SEARCH ENGINE TO DISCOVER DISTINCT DATABASES AT NCBI
PUBMED DATABASE
Search for articles on pax6.
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REDUNDANT SEQUENCE DATABASE: NUCLEOTIDE, PROTEIN, GENOME, EST…
This database contains all the redundant information that is
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?CMD=search&DB=nucleotide
used
by
ENSEMBL
and
GENE.
Search for sequence entries that contain pax6 and human using a simple versus an advanced query
1.
First just search for Pax 6, what do you retrieve?
1.
Search Pax6 advanced search (organism is human and gene name is pax6)
You retrieve many different sequences, these can be mRNAs, genomic DNA,…
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Use an advanced search to find pax6 (gene name) in human (organism) and limit to genomic sequences only.
There are many entries, most of which only contain part of the sequence (incomplete e.g. only a certain exon and
many sequences that come from genomic surveys).
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Indicate that you only want the ref seq sequences. 8 entries contains the complete genomic sequence, derived from
alternative assemblies. Use the accession number NG_008679.1 to view the genbank file
2.
Open the Genbank file of a gene entry and interpret the output (what is the difference between and exon
and an mRNA, do you find the Gene ID, this is the link to the comprehensive NCBI database?)
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View the pax 6 graphically
Repeat the exercise (Use an advanced search to find pax6 (gene name) in human (organism)) but do now restrict the
search to mRNA only. In this case you will find accession numbers that start with NM_: these are REFSEQ sequences or
XM_ representing respectively the transcripts or predicted transcripts.
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PERFORMING ADVANCES SEARCHES USING ENTREZ IN OTHER REDUNDANT DATABASES
1. This problem practices using the Entrez search program at the national Center for Biotechnology Information (NCBI)
to perform a search for the amino acid sequence of the human heat shock factor HSF1. Normally a large number of
matches are found in such searches. We will use the Entrez Boolean search features, which restrict the reported
matches to a series of required conditions. This feature allows us to narrow the search to the sequence we want.
a)
Go to the Entrez Web Site and choose Protein
b) Enter the terms heat shock factor in the search window and click GO [heat shock protein AND human]. This
search is to find any sequence entry in the protein sequence database that include this phrase.
c)
Now limit the search by clicking the mouse on advanced search, go to add terms, choose organism in the first
box, type human in the second, then click AND to limit the search to just human proteins and then click
preview. The history will show the results of a search for database entrees with the term heat shock protein
AND originating from humans as the organism. How many hits are there now?
d) We can limit the hits to matches to RefSeq, which is Genbank’s annotated sequence database, to give a best
representative sequence entry for each protein. Click the mouse on Limits, and in the Limited to section of
the page, ignore the boxes on the left and choose RefSeq in the right box. Then click GO and history. Now we
have all human heat shock factors in RefSeq. The gene of interest is HSF 1. Add this term to the query using.
How many hits did you receive? [no limit on gene name]
e)
The gene of interest is HSF. Click clear in the text entry box at the top of the page, type HSF 1 and click
preview. You obtain more hits because you performed a keyword search. It is better to search via the limits
option.
f)
There are other ways of arriving at this final sequence. As another example, pull out all human protein
sequences in RefSeq and all HSF 1 sequences in all organisms and then select the human one using another
Boolean search feature of Entrez. First clear history, clear the upper text box, and reselect advanced search.
Enter human and organism in the text box, click Limits, and limit to RefSEq. Click GO and then History. Now
we have a complete list of human sequences in RefSeq.
g)
Now click Advanced search choose gene name in the left box and HSF1. Combine this search with the
previous one using Booleans in the history. The result should be a small number of HSF 1 proteins.
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h) Finally note the RefSeq accession number starting with NP and use the mouse click to display the FASTA
format. NP identifies the protein as curated protein sequence. The sequence may be copied and pasted into
the page of a simple text editor and save as a local computer file.
i)
While on the page with the target sequence click on LINKs and choose Gene option. Now the gene entry
becomes visible. Note that the RefSEq numbers in the GENE database start with NM for annotated mRNA and
NT for annotated genome/ chromosome.
COMPREHENSIVE DATABASE : UNIGENE
Go to Unigene, http://www.ncbi.nlm.nih.gov/unigene/
Go the overview page for human (Unigene statistics).
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How many unigene clusters contain only 1 sequence (i.e. unclustered sequences). What will happen if more EST
sequences become available. How many clusters contain both an mRNA sequence and an EST. How many only an EST.
What will be the most reliable clusters? (HTC = a high throughput cDNA; Sequences in this division may still have 5'
and 3' UTRs at their ends, partial coding regions, and introns.)
Search for the homo sapiens unigene cluster corresponding to the human pax6. Interpret the output (based on which
sequences the cluster was built),
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View the expression of the Pax6 gene based on the analysis of EST counts (expression, EST counts). In which tissues do
you expect the gene to be expressed? Is this the case?
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From the Unigene page

Go to the DDD (digital differential display) Compare the difference in expression between two human tissues
a)
Go to the NCBI Gene. In the first box choose Gene, then Brief in the second and human as search organism in
the third, The enter HSF1 as the query and click GO. A small number of entries match the query and one of
them should be HSF1. The position column shows the relative numbered position on the long arm (q for the
long arm) of chromosome 8. The colored boxes provide sequence of the gene with direct links to RefSeq.
Clicking on the green will give the protein sequence.
b) Click on the empty check box beside the sequence and then click view to produce a page with a great deal of
information about the gene, including gene structure, genome location, RefSEQ protein and nucleotide
identifiers AND much useful information about the evidence on which this gene is based. Click on OMIM
(online inheritance in man) to see a biological summary of the HSF1 gene functions.
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What is the length of the genome, Click on the map to see the genes that are present and then go to the blocks to see
the sequences.
COMPREHENSIVE DATABASE GENE
Check out the Gene database. This is the major curation project at Ncbi. They try to convert the redundant sequence
databases into 1 non redundant, comprehensive sequence database in which each locus in the genome is completely
described by a representative mRNA sequence(s).
Entrez Gene is the American counterpart of ENSEMBL. http://www.ncbi.nih.gov/entrez/query.fcgi?db=gene
Search in the gene database for pax6 human (note the difference in result when searching with AND or using the
preview/index).
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Find the accession numbers of the pax6 transcripts, proteins, genome REFSEQ sequence

Find the Pax6 gene ID
Compare the results with what you found for Pax6 at Ensembl (see later).
The Gene database contains for each locus in the genome all associated features (indicated by the corresponding
Gene ids). A transcript is indicated by NM (XP if the transcript is still under review), a protein by NP, a genomic contig
by NT. All features (mRNA, genomic DNA, EST) associated with the same locus obtain the same Gene ID. The output is
less graphical than Ensembl (see below). In Gene non redundant sequence features are also grouped to generate a
comprehensive view of the gene.
How many transcripts are known?, corresponding to how many different isoforms?
Note you find 2 splice variants (now there are more…sequence databases get continuously updated)


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Find the sequence entries from which Gene was derived.
What is the meaning of 2 alternative assemblies?
Select the genome view
(Can you see the two representative isoforms (red, proteins)?, what is the meaning of the purple squares , variations
with a pathogenic phenotype)
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
Find the GO categories of Pax6 (note there are three ontology classification systems (function, process,
component)
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Find the diseases in which Pax6 is involved
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How is this gene found to be related to these diseases?
(via GWAS study, how many variants have been detected in this gene? GWAS was performed against which trait?)
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