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README.md

Rooting and comparing trees; Dating

  • phylo-5.ipynb - contains this whole pipeline done. Due to a lot of screenshots in this notebook it cannot be displayed in GitHub. Please download it in case you want to look at it.
  • scripts - contains all Python and R scripts used in this pipeline

For this work, we will use a filtered alignment (this is the same one we got in the Trees step)

Part 1 - Rooting trees

1) Rooting a tree by a known external clade in IQ-TREE

Input

iqtree2 -s SUP35_aln_prank.trim.fas -m TIM3+F+G4 -pre SUP35_TIM3_ufb -bb 1000

Input

iqtree2 -s SUP35_aln_prank.trim.fas -m TIM3+F+G4 -pre SUP35_TIM3_ufb_alrt_abayes -bb 1000 -alrt 1000 -abayes

Input

iqtree2 -s SUP35_aln_prank.trim.fas -m TIM3+F+G4 -pre SUP35_TIM3_root_outgroup -bb 1000 -alrt 1000 -abayes  -o SUP35_Kla_AB039749,SUP35_Agos_ATCC_10895_NM_211584

2) Rooting a tree using midpoint rooting

Input

! python3 midpoint_root.py SUP35_TIM3_ufb.treefile >SUP35_TIM3_ufb_midpoint.treefile

Input

! Rscript midpoint_root.R

2.1) Visualisation of the rooted trees from the previous paragraphs

Input

! Rscript draw_tree.R SUP35_TIM3_ufb.treefile SUP35_TIM3_ufb.png
! Rscript draw_tree.R SUP35_TIM3_ufb_midpoint.treefile SUP35_TIM3_ufb_midpoint.png
! Rscript draw_tree.R SUP35_TIM3_root_outgroup.treefile SUP35_TIM3_root_outgroup.png
! Rscript draw_tree.R SUP35_TIM3_ufb_alrt_abayes_rooted.treefile SUP35_TIM3_ufb_alrt_abayes_rooted.png

Output

SUP35_TIM3_ufb.png
SUP35_TIM3_ufb_midpoint.png
SUP35_TIM3_root_outgroup.png
SUP35_TIM3_ufb_alrt_abayes_rooted.png
2.1.1) Comparison of trees
  • Unrooted and rooted by external group are completely identical. 0 differences.
  • Rooted by midpoint looks neater. Topology looks better.

3) Rooting a tree using an irreversible (non-reversible) model (iq-tree2)

If we have a rather complex tree structure (it is huge, there are long branches, imbalance in sampling by different taxa), rooting the tree by external group will not give us the result we expect.
There are more intelligent models for this.
One of them is the easy-to-apply non-reversible model iq-tree2.
The idea is that they allow you to predict where the root was! This, by analogy with bootstrap is called rootstrap.

Input

iqtree2 -s SUP35_aln_prank.trim.fas -m TIM3+F+G4 -pre SUP35_TIM3_root_auto --model-joint 12.12 -B 1000
# -B 1000 -  it's not `bootstrap`, it's how many times to run `rootstrap`

Input

cat SUP35_TIM3_root_auto.rootstrap.nex
# - contains information about the algorithm's confidence in where the root is located

Output

#NEXUS
[ This file is best viewed in FigTree. ]
begin trees;
  tree tree_1 = ((SUP35_Kla_AB039749:0.2581582648[&id="2",rootstrap="26.8"],SUP35_Agos_ATCC_10895_NM_211584:0.3420323394[&id="3",rootstrap="5.4"]):0.1209998432[&id="1",rootstrap="42.4"],(((((((SUP35_Scer_74-D694_GCA_001578265.1:0.0004800339[&id="11",rootstrap="0"],SUP35_Scer_beer078_CM005938:0.0000010000[&id="12",rootstrap="0"]):0.0000010000[&id="10",rootstrap="0"],SUP35_Sbou_unique28_CM003560:0.0004800702[&id="13",rootstrap="0"]):0.0463459057[&id="9",rootstrap="0"],SUP35_Spar_A12_Liti:0.0325384431[&id="14",rootstrap="0.1"]):0.0354767121[&id="8",rootstrap="0.2"],SUP35_Smik_IFO1815T_30:0.0736998639[&id="15",rootstrap="0.6"]):0.0322607827[&id="7",rootstrap="0.5"],SUP35_Skud_IFO1802T_36:0.0970836557[&id="16",rootstrap="0.7"]):0.0154599513[&id="6",rootstrap="1.8"],SUP35_Sarb_H-6_chrXIII_CM001575:0.0787155739[&id="17",rootstrap="4.8"]):0.0099429593[&id="5",rootstrap="8.6"],SUP35_Seub_CBS12357_chr_II_IV_DF968535:0.0912344001[&id="18",rootstrap="5.1"]):0.1942253516[&id="4",rootstrap="42.4"]):0.0000010000[&id="0",rootstrap="42.4"];
end;

This is basically a Newick file, but strange Newick because it has square brackets in it. Programs that read Newick format will not be able to read this tree. According to the developers of iqtree it is better to read this tree in FigTree.

4) Root-supported tree visualisation (rootstrap)

What can we say about the algorithm's confidence in root selection?

Input

figtree SUP35_TIM3_root_auto.rootstrap.nex

Output

It can't say anything specific about where the tree splits. There's a 42.4% chance the root is either in one place or the other.

Part 2 - Dating

5) Analysing the age of the common ancestor of the two species of smoky leopards from the article https://doi.org/10.1016/j.cub.2006.08.066 based on sequencing data of the atp8 gene region, relying on known data on the frequency of substitutions in mtDNA (approximately 2% per million years) in beauti and beast

  • Check the quality in Tracer
  • Combine trees in treeannotator.
  • Draw the final tree (can be in FigTree, bonus for ggtree)
  • Be sure to show estimates of the age of the common ancestor at the nodes!

Input

efetch -db popset -id 126256179 -format fasta >felidae_atp8.fa

Input

cut -d ' ' -f 1,2,3 felidae_atp8.fa | sed -e 's/ /_/g' > felidae_atp8.renamed.fa

Input

mafft --auto felidae_atp8.renamed.fa >felidae_atp8.aln

Input

trimal -in felidae_atp8.aln -out felidae_atp8.trim.fas -nogaps

Input

iqtree2 -s felidae_atp8.trim.fas -o EF437591.1_Felis_catus -alrt 1000 -abayes

Input

from Bio import Phylo

Input

tree = Phylo.read("felidae_atp8.trim.fas.treefile", "newick")

Input

Phylo.draw_ascii(tree)

Output

                                          , EF437567.1_Neofelis_nebulosa
                                          |
                                          | EF437569.1_Neofelis_nebulosa
                                          |
                                          | EF437570.1_Neofelis_nebulosa
                                          |
                                   _______| EF437568.1_Neofelis_nebulosa
                                  |       |
  ________________________________|       |_ EF437571.1_Neofelis_nebulosa
 |                                |
 |                                |            , EF437572.1_Neofelis_diardi
 |                                |____________|
 |                                             | EF437573.1_Neofelis_diardi
 |
 |                             __ EF437581.1_Panthera_onca
 |                           ,|
_|                      _____||____ EF437587.1_Panthera_tigris
 |                     |     |
 |            _________|     |_______ EF437583.1_Uncia_uncia
 |           |         |
 |___________|         |________ EF437585.1_Panthera_leo
 |           |
 |           |______________ EF437589.1_Panthera_pardus
 |
 |______________ EF437591.1_Felis_catus

The outside group is the house cat. Because everyone else is a big cat.
Fundamentally our tree is similar to that published in articles.
In foreign colleagues the tree was based on several genes, we take only 1 piece of data.

Beauti

Beauti is the GUI application. So I will just provide as many screenshots as possible.

When loading the file, we select that we have nucleotide sequences

Everything is okay.

In Site model select TN93 and empirical frequencies

In Clock model we choose 0.02. Why? Because we rely on the known data on the frequency of substitutions in mtDNA (approximately 2% per million years)

Everything is okay.

Save everything to felidae_2percent.xml.

Input

beast felidae_2percent.xml

Tracer

Tracer is the GUI application. So I will just provide as many screenshots as possible.

All ESS scores are in perfect order.

The so-called "hairy caterpillar".

TreeAnnotator

TreeAnnotator is the GUI application. So I will just provide as many screenshots as possible.

Set parameters, and set input and output. Useful hint - output can be named the same way, but not .trees, just .tree!

FigTree

FigTree is the GUI application. So I will just provide as many screenshots as possible.

Fiddled with the parameters and got these results

Well. The common ancestor of our smoky leopards is about 2.5 million years old.

6) Comparison of the results of my analysis (age of the last common ancestor of Neofelis) with published articles (https://www.science.org/doi/10.1126/sciadv.adh9143, https://www.sciencedirect.com/science/article/pii/S2589004222019198)

What conclusions can be drawn?

In the first article - https://www.science.org/doi/10.1126/sciadv.adh9143 there was a full genome analysis. Their estimate of the age of the common ancestor of smoky leopards is 2.2 million years. And we hit 100 nucleotides pretty good!

But in the second article - https://www.sciencedirect.com/science/article/pii/S2589004222019198 - the age of the ancestor is 5.1 million years old.... Well. Interesting. I can't explain it yet. The only thing I can say is that this article has a cool map of leopard populations. Too bad I didn't see them in Sumatra or Kalimantan when I was there...(