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

T-COFFEE complete

On to WebLogo

“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