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Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006
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Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

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Page 1: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

Annotation of Drosophila virilis

Chris Shaffer GEP workshop, 2006

Page 2: 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

Page 3: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

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)

Page 4: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

D. virilis

Average gene size will be smaller than mammals

Very low density of pseudogenesAlmost all genes in virilis will have the

same basic structure as melanogaster orthologs; mapping exon by exon works well for most genes

Page 5: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

How to proceed

First, identify features of interest:1. Genscan results

• Watch out for ends - fused or split genes2. Regions of high similarity with D.

melanogaster protein, identified by BLAST• Overlapping genes usually on opposite strand• Be vigilant for partial genes at fosmid ends

3. Regions with high similarity to known genes (i.e. BLAST to nr) not covered above

Page 6: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

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 exons3. 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

Page 7: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

Basic procedure (graphically)fosmid

BLASTX of predicted gene to melanogaster proteins suggests this region orthologous to Dm gene with 5 exons:

feature

BLASTX of each exon to locate region of similarity:

1 3 3 2 1

Page 8: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

Basic procedure (graphically)

Zoom in on ends of exons and find first met, matching intron Doner (GT) and Acceptor (AG) sites and final stop codon

GT AG

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

1 3 3 2 1

1 3

Met GT

1121 1402 1754 2122 26011187 1591 1939 2434 2789

Page 9: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

Example Annotation Open Safari and go to goose.wustl.edu Click on Genome Browser

Page 10: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

Example Annotation Settings are: Insect; D. virilis; Mar. 2005; chr10 (chr10 is a fosmid from 2005) Click submit

Page 11: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

Example Annotation

Seven predicted Genscan genesEach one would be investigated

Page 12: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

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

Page 13: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

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

Page 14: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

Step 1: Find Ortholog

Click on one of the exons in gene 10.4On the Genscan report page click on

Predicted Protein Select and copy the sequenceDo a blastp search of the predicted

sequence to the D. melanogaster “Annotated Proteins” database at

http://flybase.net/blast

Page 15: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

Step 1: Find OrthologThe results show a significant hit to the

“A” and “B” isoforms of the gene “mav”

Page 16: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

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

Page 17: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

Step 2: Gene modelWhat does mav look like?Go to ENSEMBL to get exons and map

them to regions:– Web brower- go to www.ensemble.org

Page 18: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

Click on DrosophilaSearch for mav (top right search box)Click on “Ensembl Gene: CG1901”Scroll down to map and notice two

isoforms:

Step 2: Gene model

Page 19: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

Step 2: Gene model 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

Page 20: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

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:

YNASSNKYSLINVSQSKNFPQLFNKKLSVQWINTVPIQSRQTRETRDIGLETKRHSKPSKRVDETRLKHLVLKGLGIKKLPDMRKVNISQAEYSSKYIEYLSRLRSNQEKGNSYFNNFMGASFTRDLHFLSITTNGFNDISNKRLRHRRSLKKINRLNQNPKKHQNYGDLLRGEQDTMNILLHFPLTNAQDANFHHDK

Page 21: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

Step 3: Investigate ExonsStart with exon 1We 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

Page 22: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

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)”

Page 23: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

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

Page 24: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

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

Page 25: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

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

Page 26: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

Step 3: Investigate ExonsA 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

Page 27: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

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

Page 28: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

Step 4: Confirm Gene ModelAs 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 browser2. Click “DNA” button at top then “get DNA”3. Copy the sequence into a text file4. Repeat for each exon, adding DNA to file5. Go to http://us.expasy.org/tools/dna.html6. Enter your entire sequence, hit “Translate

Sequence”; should get one long protein7. Compare the protein sequence to ortholog

using bl2seq

Page 29: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

Step 4: confirm model (Future)

We have a web page under construction which will simplify confirmation

This web site will double check intron- exon boundaries, translate the putative message and create a data file suitable for uploading

Page 30: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

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

Page 31: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

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.

Page 32: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

Flybase

www.flybase.org flybase.bio.indiana.edu

Page 33: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

Flybase gene info

Search for gene name Will find links to info pages with many

helpful referencesRemember many genes have functions

assigned based only on similarity dataThis is especially true for anonymous

genes “CG#####”. Take any functional assignment with large amounts of skepticism, consider it a guess at best

Page 34: Annotation of Drosophila virilis Chris Shaffer GEP workshop, 2006.

Gene function for Mav