Download virilis_annot

Annotation of Drosophila virilis
Chris Shaffer
GEP workshop, 2006
Annotation of D. virilis
Outline of general technique and then
one practical example
 This technique may not be the best
with other projects (e.g. corn, bacteria)
 The technique optimized for projects:

–
Moderately close, well annotated neighbor
species
– No EST, mRNA or expression data
available
Helpful Hints

Evolutionary distance between D. virilis
and D. melanogaster is much larger
than chimp to human
–
Conservation will be at the protein domain
level
– Synteny is detectable in some fosmids
– Most genes stay on the same
chromosome (3 exceptions seen in ~40
genes)
D. virilis
Average gene size will be smaller than
mammals
 Very low density of pseudogenes
 Almost all genes in virilis will have the
same basic structure as melanogaster
orthologs; mapping exon by exon
works well for most genes

How to proceed
First, identify features of interest:
1. Genscan results
•
Regions of high similarity with D.
melanogaster protein, identified by BLAST
2.
•
•
3.
Watch out for ends - fused or split genes
Overlapping genes usually on opposite strand
Be vigilant for partial genes at fosmid ends
Regions with high similarity to known
genes (i.e. BLAST to nr) not covered
above
Basic Procedure
For each feature of interest:
1. Identify the likely ortholog in D. m.
2. Use D. m. database to find gene model of
ortholog and identify all exons
3. Use BLASTX to identify locations and
frames of each exon, one by one
4. Based on locations, frames, and gene
predictions, find donor and acceptor splice
sites that link frames together; identify the
exact base location (start and stop) of each
coding exon
5. double check your results by translation
Basic procedure (graphically)
fosmid
feature
BLASTX of predicted gene to melanogaster
proteins suggests this region orthologous to
Dm gene with 5 exons:
BLASTX of each exon to locate region of
similarity:
1
3
3
2
1
Basic procedure (graphically)
1
3
3
2
1
Zoom in on ends of exons and find first met, matching intron Doner (GT)
and Acceptor (AG) sites and final stop codon
1
3
Met
GT
AG
GT
Once these have been identified, write down the exact location of the first base
and last base of each exon. Use these numbers to check your gene model
1121
1187
1402
1591
1754
1939
2122
2434
2601
2789
Example Annotation


Open Safari and go to goose.wustl.edu
Click on Genome Browser
Example Annotation

Settings are:
Insect; D. virilis; Mar. 2005; chr10
(chr10 is a fosmid from 2005)
 Click submit
Example Annotation
Seven predicted Genscan genes
 Each one would be investigated

Investigate 10.4
All putative genes will need to be
analyzed; we will focus on 10.4 in this
example
 To zoom in on this gene enter:
chr10:15000-21000 in position box
Then click jump button

Step 1: Find Ortholog
If this is a real gene it will probably
have at least some homology to a D.
melanogaster protein
 Step one: do a BLAST search with the
predicted protein sequence of 10.4 to
all proteins in D. melanogaster

Step 1: Find Ortholog
Click on one of the exons in gene 10.4
 On the Genscan report page click on
Predicted Protein
 Select and copy the sequence
 Do a blastp search of the predicted
sequence to the D. melanogaster
“Annotated Proteins” database at
http://flybase.net/blast

Step 1: Find Ortholog

The results show a significant hit to the
“A” and “B” isoforms of the gene “mav”
Step 1: Results of Ortholog search
The alignment looks right for virilis vs.
melanoaster- regions of high similarity
interspersed with regions of little or no
similarity
 We have a probable ortholog: maverick

Step 2: Gene model
What does mav look like?
 Go to ENSEMBL to get exons and map
them to regions:

–
Web brower- go to www.ensemble.org
Step 2: Gene model
Click on Drosophila
 Search for mav (top right search box)
 Click on “Ensembl Gene: CG1901”
 Scroll down to map and notice two
isoforms:

Step 2: Gene model




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We now have a gene model (two exon gene,
two isoforms).
We will annotate isoform A since it is the
largest. Due to time constraints, our policy
so far is to have students pick and annotate
only one isoform for each feature.
If more than one isoform exists, pick the
largest or the one with the most exons
Here student should choose to annotate
isoform A (largest)
All isoforms should be annotated eventually
Step 3: Investigate Exons

Given we need to annotate isoform A, we
need exon sequence for exon 1 and 2, so
we do BLASTX search
 Click on [Peptide info] for isoform A on right
just above map
 Scroll down to find peptide sequence with
exons in different colors:
YNASSNKYSLINVSQSKNFPQLFNKKLSVQWINTVPIQSRQTRETRD
IGLETKRHSKPSKRVDETRLKHLVLKGLGIKKLPDMRKVNISQAEYSSKYI
EYLSRLRSNQEKGNSYFNNFMGASFTRDLHFLSITTNGFNDISNKRLRHRR
SLKKINRLNQNPKKHQNYGDLLRGEQDTMNILLHFPLTNAQDANFHHDK
Step 3: Investigate Exons
Start with exon 1
 We will use a varient of the BLAST
program, called blast2seq. This version
compares two sequences instead of
comparing a sequence to a database
 Best to search entire fosmid DNA
sequence (easier to keep track of
positions) with the amino acid
sequence of exon 1

Step 3: Investigate Exons


Create 3 tabs in Safari
In the first tab, go to the goose browser chr10
of virilis; click the DNA button, then click “get
DNA”
 In the second tab, go to www.ensembl.org
and get the peptide sequence for the
melanogaster mav gene
 These first two tabs now have the two
sequences you are going to compare
 In the third tab go to NCBI blast page and
click on “Align two sequences (bl2seq)”
Step 3: Investigate Exons






Copy and paste the genomic sequence from
tab 1 into sequence box 1 of tab 3
Copy and paste the peptide sequence of
exon 1 from tab 2 into sequence box 2
Since we are comparing a DNA sequence to
a protein we need to run BLASTX
Turn off the filter
Leave other values at default for now
Click “align” button to run the comparison
Step 3: Investigate Exons


No significant homology found
Either the mav ortholog is not in this fosmid
(unlikely given the original blastp hit) or this
exon is not well conserved
 Lets look for similarities of lower quality
 Click the back button to go back to the
bl2seq page
 Change the expect value to 1000 and click
align
Step 3: Investigate Exons

We have a weak alignment (50 identities and
94 similarities), but we have seen worse
when comparing single exons from these
two species
 Notice the location of the hit (bases 16866 to
17504) and frame +3
Step 3: Investigate Exons
A similar search with exon 2
sequences gives a location of
chr10:18476-19744 and frame +2
 For larger genes continue with each
exon, searching with bl2seq (adjusting
e cutoff if necessary) and noting
location and frame of region of
similarity

Step 4: Create Gene Model

Pick ATG (met) at start of gene, first met in
frame with coding region of similarity (+3)
 For each putative intron/exon boundary
compare location of BLASTX result with
gene finder results to locate exact first and
last base of the exon and check that the
intron starts with “GT” and ends with “AG”


Exons: 16515-17504; 18473-19744
Intron GT and AG present
Step 4: Confirm Gene Model
As a final check we need to create the putative
mRNA, translate it and make sure the
protein we get out is similar to expected:
1. Enter coordinates for each exon in browser
2. Click “DNA” button at top then “get DNA”
3. Copy the sequence into a text file
4. Repeat for each exon, adding DNA to file
5. Go to http://us.expasy.org/tools/dna.html
6. Enter your entire sequence, hit “Translate
Sequence”; should get one long protein
7. Compare the protein sequence to ortholog
using bl2seq
Step 4: confirm model (Future)
We have a web page under
construction which will simplify
confirmation
 This web site will double check intronexon boundaries, translate the putative
message and create a data file suitable
for uploading

Considerations

Some exons are very hard to find (small or
non-conserved; keep increasing E value to
find any hits (10,000,000 not unheard of)
 Donor “GC” seen on rare occasions
 We have seen one example where the only
reasonable interpretation was that an intron
had moved (out of about 70 genes)
 Without est and expression data you may
get stuck; use your best judgment
Gene Function
In addition to annotation of the genes
we ask the students to look into the
function of each gene and discuss
what they found in their final paper on
annotation
 For genes in Drosophila the best
source to begin your investigation into
gene function is the drosophila online
database called Flybase.

Flybase
www.flybase.org
flybase.bio.indiana.edu
Flybase gene info
Search for gene name
 Will find links to info pages with many
helpful references
 Remember many genes have functions
assigned based only on similarity data
 This is especially true for anonymous
genes “CG#####”. Take any functional
assignment with large amounts of
skepticism, consider it a guess at best

Gene function for Mav