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TUTORIAL: PHYLOGENETIC ANALYSIS USING DISTANCE METHODS

June 24, 2021

Example: Plant Type III Chitinases

1. The datasets

In this tutorial, we will demonstrate phylogeny using both protein and DNA alignments as input.

Start by creating a directory called distance, and save chitIII.pro.mafft.fsn  and chitIII.CDS.pal2nal.fsn to this directory. Open these files in blpalign and blnalign, respectively.

blpalign - protein alignment	blnalign - DNA alignment

For comparison only 10 of the 40 sequences in the alignment are shown for both proteins and DNA. The alignments have been scrolled to show a gap of two amino acids (eg. AS--LLL) in the protein, corresponding to three codons (six gap positions) in the DNA (eg. gcctca------ttactctta). The N-terminal regions of the proteins vary in length, which is why gaps are also seen in the N-terminal region of the alignment.

The other item of note is that ":CDSx" is appended to the Accession numbers of the proteins (which are actually the Accession numbers of the GenBank DNA entries from which they were translated. These CDS labels indicate which CDS from each entry was used (remember, long DNA sequences may contain many CDS features). When Ribosome translates proteins, it automatically shortens the CDSx label to _x, in part because some programs have limits to the length of sequence names. As we proceed, the _x naming convention will be used.

2. The problem with gappy regions

The whole point of using multiple sequence alignment as input for phylogeny programs is that over the course of evolution, insertions and deletions (indels) occur in genes. Multiple alignments insert gaps into sequences to make amino acids at homologous positions to line up.

At right we see the alignment scrolled to the C-terminal region. It is obvious that an insertion of 29 amino acids occurred in XM_024786391, which we can assume was a recent event since this insertion was not seen in any of the other sequences. A problem arises because phylogeny programs treat each position (column) independently. That is, the gap would be treated as if it was 29 independent indels, resulting in an artificially long branch on the tree, separating XM_024786391 from all other sequences.

The other problem with gappy regions is that they are usually regions in which any multiple alignment is less reliable, because it is not always obvious where gaps should be inserted.

One common solution to this problem is to edit out ALL positions containing gaps. The downside of this approach is that indels are highly informative from an evolutionary standpoint, because a single indel, regardless of its size, can point to ancient events which instruct the deeper branches of phylogenetic trees. In other words, completely eliminating all gap positions would be throwing away useful data.

Gblocks from the Castresana lab gives us a more sophisticated way to "trim" gaps, while retaining some gap positions.
To create a de-gapped alignment, select all sequences in the protein alignment and choose Alignment --> Gblocks. 

Set "Allowed Gap positions" to "Only positions with <= 50% gaps. This will retain some gap positions, as we'll see below. See the Gblocks documentation for more detailed information on fine-tuning gappy regions in multiple alignments.

Output pops up in a new blpalign window. This output was saved in chitIII.pro.maaft.gblocks.fsa. One of the strong points of Gblocks is the HTML output, excerpted below. The first 100 positions of the alignment, out of 352 positions are shown. Conserved amino acids are highlighted. Based on setting for conserved blocks and flanking posiitons, a subset of 283 positions (underlined in blue) were extracted from the alignment into the blpalign window at right.

Next, let's run Gblocks on the DNA alignment in blnalign. Save this output in chitIII.CDS.pal2nal.gblocks.fsn. (The HTML output for the DNA alignment is not shown.)

3. A quick DNA phylogeny using FITCH

For routine distance tree construction, the method of Fitch and Margoliash is the method of choice. FITCH allows for variable rates of evolution in different lineages, and iterates the tree to minimize the least squares distance across the entire tree. Although Neighbor-Joining is faster, it is also much less thorough, only considering one tree. It is probably the least rigorous method for constructing a phylogeny . Go to the DNA alignment in blnalign and choose Edit --> Select All. Next choose Phylogeny --> DNA Distance methods.  Choose the Fitch-Margoliash method.

DNADIST will calculate a distance matrix, and then FITCH will run, and by default, 3 windows will appear

OUTFILE- the report on the phylogeny
 

TREEFILE - the machine -readable treefile. Readable by programs such as DRAWTREE, DRAWGRAM, and Archaeopteryx.

 
The treefile also pops up on a bltree window, allowing further tasks to be performed using the tree as input.

TREEFILE - the treefile in the Archaeopteryx tree editor.

Note: Do NOT save the contents of the Archaeopteryx window using the .treefile extension. You will overwrite the original treefile. Archaeopteryx saves files in phyloXML format with the extension ".phyloxml".

4. Phylogeny using amino acid sequences.

Some of the parameters for construction of the distance matrix using PROTDIST are different from those for DNADIST.  These include several different methods for constructing distance matrices, as well as a choice of alternative genetic codes, where appropriate. Once the distance matrix is constructed, there is no difference in computation of the phylogenetic tree, so all parameters are the same as previously.

5. Analysis of Results in Archaeopteryx

protein	DNA