t-coffee multiple alignments of orthologous sequences horizontal gene transfer (phylogenetic trees)...
TRANSCRIPT
T-COFFEE Multiple Alignmentsof Orthologous Sequences
Horizontal Gene Transfer(Phylogenetic Trees)
WebLogo
Overview
• T-COFFEE– Tree-based Consistency Objective Function for
alignment Evaluation• Focuses on orthologous gene sequences• Used to generate multiple sequence alignments
• WebLogo• Constructed from multiple sequence alignment
• Phylogenetic Trees• Used to determine if your gene is derived from horizontal
gene transfer
“Click”
Enter ortholog sequencesinto query box –
Where do I get these?
RECALL: What are orthologs?
• Homologs– Orthologs
• Genes duplicated via appearance of new species
– Identical function in different organisms
– Paralogs• Genes duplicated within a species
– Perform slightly different tasks in cell» Can develop new capabilities» Can become pseudogene if
functionality lost but sequencesimilarity retained
Insert Figure 8-41 fromMicrobiology – An Evolving Science
© 2009 W.W. Norton & Company, Inc.
Under Homolog Selection, choose “Paralogs/Orthologs”from drop-down menu
Where do I find orthologs?
Scrolldown
Scroll down to table containing list of orthologs
Select the genes by clicking these boxes
The genes are rankedby ascending E-values
Add the top 5 orthologs to Gene Cart
Notice orthologous genes are from
different organisms
“Click”
Scroll down to bottom of page
Only 5 genes were selected, why are6 genes shown in the Gene Cart?
One of the genes shown is your ASSIGNED gene
(the one you are annotating)
Generate amino acid sequences for orthologs in FASTA format
Select “FASTA Amino Acid
format”
Scroll down to “Export Genes”
“Click”
Amino Acid sequences in FASTA format for all6 genes will appear
Scroll down
Your assigned gene is located at the bottom
of this list(inspect Gene OID number)
Copy / paste all 5 ortholog sequencesinto your notebook for this module
EXCLUDE your gene, whichshould already be in your notebook
Scroll down
Recording results in your notebook
The amino acid sequences in FASTA format for the top 5 orthologs
Add heading and box
STEP 1: Copy / paste the amino acid sequence in FASTA format for your assigned gene into the query box for T-COFFEE
Return to T-COFFEE database
STEP 2: Copy / paste the amino acid sequences in FASTA format for the top 5 orthologs into the same query box as your gene
T-COFFEE database entries
Separate individual sequences by a
hard return
“Click”
Wait a few moments . . .
Select “Start JalView” to examine the multiple
sequence alignment of the ortholog sequences
T-COFFEE Results
Reminder:Light Blue = Low FrequencyDark Blue = High Frequency
Alignment inspection using JalView
Select “Percentage Identity”under “Colour” menu
Compare to consensus sequence
Copy / paste this alignment into your lab
notebook
Return to T-COFFEE Results
Identify organism in alignment by Gene OID
Recording results in your notebook
“Click”
“Right Click” and open in IE tab (not Firefox)
Copy/paste multiple sequence alignment
Scroll down
1- Select “amino acid” as sequence type
2- Select box for multiline logo
“Click”
WebLogo Results
Zoom in
In IE, save picture as .png file for upload to notebook
Recording results in your notebook
WHAT ARE OUR GOALS?
1. Build a phylogenetic tree
2. Determine if assigned genes are derived from horizontal gene transfer
Phylogenetic Phylogenetic tree of tree of BacteriaBacteria
showing showing established & established &
candidate phylacandidate phyla(organismal (organismal
phylogenyphylogeny))
Domain Phylum Class Order Family Genus Species
Insert Figure 1 from Handelsman (2004) Microbiol. Mol. Biol. Rev. 68: 669-685.
Three bacterial phyla closely related to Three bacterial phyla closely related to Planctomycetes by 23S rRNA analysisPlanctomycetes by 23S rRNA analysis
(organismal phylogeny)(organismal phylogeny)
Insert Figure 4A from Pilhofer et al. (2008)Characterization and Evolution of Cell Division and Cell Wall SynthesisGenes in the Bacterial Phyla Verrucomicrobia, Lentisphaerae, Chlamydiae,and Planctomycetes and Phylogenetic Comparison with rRNA Genes.J Bacteriology 190: 3192-3202.
16S rRNA gene supports the16S rRNA gene supports themonophyletic grouping Planctomycetalesmonophyletic grouping Planctomycetales
(organismal phylogeny by rDNA analysis)(organismal phylogeny by rDNA analysis)
http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=126
Closest phylogenetic relativesof P. limnophilus (same family)
How do we build a phylogenetic tree?
include P. limnophilus gene (first module)
include the top 5 orthologs (second module)
include genes from organisms closely related to P. limnophilus (i.e., same family)
include genes from organisms less closely related to P. limnophilus (i.e., from phyla Verrucomicrobia, Chlamydiae, and Lentisphaerae)
include genes from organisms that are distantly related to P. limnophilus
Recall: We want to include genes from organisms more closely related to P. limnophilus AND genes from organisms that are less closely related to P. limnophilus.
So…depending on the organisms in your top 5 orthologs,there are 2 paths you can take:
Select top 5 orthologs
If top 5 are closely relatedto P. limnophilus. . .
If top 5 are less closely related to P. limnophilus. . .
Choose 5-10less closely related
organisms
Choose 5-10more closely related
organisms
PATH 1 PATH 2
http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?id=126
Building a phylogenetic tree
EXAMPLE: Organisms closely related to P. limnophilus (i.e., same family)
Building a phylogenetic tree EXAMPLE: Organisms less closely related to P. limnophilus (i.e., from phyla Verrucomicrobia, Chlamydiae, and Lentisphaerae)
Insert Figure 4A from Pilhofer et al. (2008)Characterization and Evolution of Cell Division and Cell Wall SynthesisGenes in the Bacterial Phyla Verrucomicrobia, Lentisphaerae, Chlamydiae,and Planctomycetes and Phylogenetic Comparison with rRNA Genes.J Bacteriology 190: 3192-3202.
Inspect your top 5 orthologs:Which path?
Example: PATH #1 – Most are in same family as P. limnophilus, so choose 5-10 sequences from less closely related organisms
Under Homolog Selection, choose “Paralogs/Orthologs”from drop-down menu
Where do I find sequences?
Scrolldown
Scroll through the ortholog list and select some genes fromless closely related as well as some distantly related organisms
Once 5-10 orthologs are selected, add them
to your gene cart
Generate amino acid sequences for orthologs in FASTA format
Select “FASTA Amino Acid
format”
Scroll down to “Export Genes”
“Click”
Amino Acid sequences in FASTA format
Scroll down
Remember: Your assigned gene is at the
bottom of the list
Recording results in your notebook
Create another box in your lab notebook, and copy/paste
ONLY the 5-10 NEW ortholog FASTA sequences
(i.e., exclude those alreadyin first & second module)
Recording results in your notebook
What if your top 5 orthologs are distantly related to P. limnophilus?
Example: PATH #2 – Most arenot in the same phylum as
P. limnophilus, so choose 5-10 sequences from more closely
related organisms
Scroll through the ortholog list and select some genes fromclosely related as well as other distantly related organisms
Once 5-10 orthologs are selected, add them
to your gene cart
Copy / pasteFASTA format
protein sequencesinto notebook
Use Phylogeny.fr site to createa phylogenetic tree
“Click”
Select“A la Carte” from menu
Creating a phylogenetic tree
1- Select “T-Coffee” for multiple alignment
2- Leave other settings as default
Scroll down
“Click”
Scroll down
Copy/paste sequences in query box.
Scroll down & select “submit”
Your P. limnophilus gene
Your top 5 orthologs
5-10 new orthologs
Results of phylogenetic analysis
Download and save as .png for upload to notebook
Recording results in your notebook
How do I interpret the tree results?
Possible scenarios resulting fromPossible scenarios resulting fromconstruction of a phylogenetic treeconstruction of a phylogenetic tree
P. limnophilus
Carboxydothermus
Carboxydothermus
Bacillus
P. limnophilus
Blastopirellula
P. maris
Pirellula
No HGT since P. limnophilus and Blastopirellula are in the same family and are clustered together (i.e., gene phylogeny matches organismal phylogeny).
Possible HGT since P. limnophilus and Carboxydothermus are very distantly related yet clustered together (i.e., gene phylogeny does NOT match organismal phylogeny).
Bacillus
Clostridium
Carboxydothermus
P. limnophilus
P. maris
Blastopirellula
Clustered
Not clustered
Maybe HGT, but unsure because there is also an unresolved or multifurcating branch
Interpreting your phylogenetic tree If your Planctomyces limnophilus gene is clustered with that from an organism in the P. limnophilus family probably not horizontal gene transfer
Planctomyces limnophilusBlastopirellula marinaPlanctomyces marisPedospheara parvula (Ellin 514)
Lentisphaera araneosaVerrucomicrobium spinosumSorangium cellulosumEscherichia coli K12Rhodopirellula balticaThiobacillus denitrificansMoorella thermoaceticaBrucella canisHydrogenivirga sp.Clostridium perfringensGemmata obscuriglobus
If your Planctomyces limnophilus gene is clustered with that from an organism that is NOT in the P. limnophilus family may be horizontal gene transfer
If your Planctomyces limnophilus gene is clustered with more than one organism in the tree (multifurcating branch) unresolved phylogeny
In the example below, is the gene derived by HGT? Why or why not?
In the “Interpretation” box to your lab notebook
Is there evidence of horizontal gene transfer?
What organisms does your gene cluster with? The same family? Or the three more closely related phyla (Verrucomicrobia, Chlamydiae, Lentisphaerae)?
Do the gene and genome GC content differ by more than 5%?
Do the neighborhoods for the top 5 orthologs look similar or different to that of your gene in P. limnophilus?
Recording results in your notebook