Creating a Phylogenetic tree using DNA sequences

I was wondering given DNA samples, how can we create a phylogenetic tree? I mean I don't even want to have actual DNA sequence. Can I generate small samples, like reads of a length 100 or 1000 of nucleotide chains and figure out how they are related.

Suppose we have two reads of DNA samples, for brevity let's have them really small like:AAAGGTCTTGAandATAAAGTGTA. I can run the sequence alignment algorithm on the strings and I will get{"A", {"", "T"}, "AA", {"G", "A"}, "GT", {"C", "G"}, "T", {"TG",""}, "A"}So basically what alighns then where you have indels or substitutions. I can easily now write up a function that calculates how close are these two strings by giving positive weights for matches strong negative weights for indels, etc. Now I want to know if these two guys with really short DNA are related or no. What is the mathematical measure of relatedness? And after I do Multiple Sequence Alignments and get similar results how do I build a tree from it.

There is no single mathematical measure of relatedness.

In the context of phylogeny, you need a model of evolution in order to be able to evaluate some relatedness between your strings of nucleotides. Then you would have various option for how to build a tree so that it reflects this relatedness. See for instance the wikipedia page about computational phylogenetics:

Through what kind of historical process were your sequences generated ? If there is no historical process underlying the generation of your sequences, then it is not meaningful to build a phylogeny with them. You can still define distances between them and use clustering techniques to build a tree, but that tree will not be a phylogeny.

Now I want to know if these two guys with really short DNA are related or no. What is the mathematical measure of relatedness

First of all this line of thought is dangerous, and creates a lot of room for misrepresentation, which have far reaching consequences when dealing with real DNA samples. Failed marriages are just one example.

Never infer phylogeny from short DNA sequences. You can read up on DNA fingerprinting here, which is used to infer ancestry and used in criminal prosecutions. But I think you already know about this.

Coming to inferring phylogeny on DNA samples where you have real DNA samples. For short sequences (upto 10kb, after that it becomes very time intensive), what you want is a Multiple sequence alignment such as Clustal Omega.

But the other procedure is to align entire genomes. Towards this end, you can check out this article from the UCSC team on the creation of their phylogenetics track, with the multiz package, the how to is described here. This procedure is based on an existing evolutionary model as stated by @bli. Finally, these alignments were used to generate conservation scores for the template genome (hg18,hg19 etc. etc.) using phastcons. If you go here, you will find the various multiz alignments between different phylogenetic trees, with the patternmultiz*way. Similarly, the inferred conservation from the respective multiz alignments with the use of phastcons and phylop is provided withphastcons*wayorphylop*way. The help page for phastcons is here and for Phylop is here

Deoxyribonucleic Acid or DNA is a universal attribute of every living organism. The advent of DNA sequencing has allowed the elucidation of evolutionary relationships among organisms. It has unlocked the mystery behind the human ancestry. DNA is made up of four nucleotide bases, namely adenine (A), thymine (T), guanine (G), and cytosine (C). The combination of the three nucleotide bases is called codons or the triplet code. A codon codes for an amino acid which later forms the proteins.

Genomic DNA or gDNA of an organism is now possible to be extracted. Once the gDNA is available, a plethora of analyses can be done. DNA sequencing has also improved ever since it was developed. Now, there are various industries which offers fast and accurate DNA sequencing platforms.

Open source Bioinformatics tools such as Basic Local Alignment Search Tool or BLAST is used to infer sequence homology using the DNA sequences. BLAST search will match the nucleotide bases from reported organisms in the National Center for Biotechnology Information or NCBI Database. Phylogenetic analysis using the DNA sequences and the information gathered from the BLAST search will determine the identity of the organism under study.

What is Phylogenetics?

Phylogenetics is the study of the evolutionary history and relationships among individuals or groups of organisms.

The relationships among organisms are discovered through phylogenetic inference methods where heritable traits, such as DNA sequences or morphologies can be observed under a certain model of evolution. The result of these analyses is a phylogeny (also known as a phylogenetic tree). It is a diagram depicting a hypothesis about the history of the evolutionary relationships of a group of organisms or a family of genes.

Molecular phylogenetics is a branch of phylogenetics that analyses how certain molecules, mainly DNA sequences and proteins have changed over time, to determine evolutionary relationships of a group of organisms or a family of genes.

TreeCluster: Clustering biological sequences using phylogenetic trees

Clustering homologous sequences based on their similarity is a problem that appears in many bioinformatics applications. The fact that sequences cluster is ultimately the result of their phylogenetic relationships. Despite this observation and the natural ways in which a tree can define clusters, most applications of sequence clustering do not use a phylogenetic tree and instead operate on pairwise sequence distances. Due to advances in large-scale phylogenetic inference, we argue that tree-based clustering is under-utilized. We define a family of optimization problems that, given an arbitrary tree, return the minimum number of clusters such that all clusters adhere to constraints on their heterogeneity. We study three specific constraints, limiting (1) the diameter of each cluster, (2) the sum of its branch lengths, or (3) chains of pairwise distances. These three problems can be solved in time that increases linearly with the size of the tree, and for two of the three criteria, the algorithms have been known in the theoretical computer scientist literature. We implement these algorithms in a tool called TreeCluster, which we test on three applications: OTU clustering for microbiome data, HIV transmission clustering, and divide-and-conquer multiple sequence alignment. We show that, by using tree-based distances, TreeCluster generates more internally consistent clusters than alternatives and improves the effectiveness of downstream applications. TreeCluster is available at

Conflict of interest statement

The authors have declared that no competing interests exist.


Fig 1. When the phylogenetic tree is…

Fig 1. When the phylogenetic tree is ultrametric, clustering is trivial.

Fig 2. Comparing Greengenes and TreeCluster.

Fig 2. Comparing Greengenes and TreeCluster.

(A) Cluster diversity (Eq 1) for Greengenes and TreeCluster…

Fig 3. Effectiveness of transmission clustering.

Fig 3. Effectiveness of transmission clustering.

Effectiveness is measured as the average number of individuals…

Fig 4. Alignment error for PASTA using…

Fig 4. Alignment error for PASTA using the centroid and the mincut decompositions.

Fig 5. An example showing that number…

Fig 5. An example showing that number of minimal clusterings under a diameter threshold can…

Fig 6. Execution times of Cluster Picker,…

Fig 6. Execution times of Cluster Picker, HIV-TRACE, and TreeCluster in log-scale.

Constructing A Phylogenetic Tree Worksheet

And genetic data such as mitochondrial dna sequences ribosomal rna genes and any genes of interest. A phylogenetic tree may be built using morphological body shape biochemical behavioral or molecular features of species or other groups.

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Verify the result statistically.

Constructing a phylogenetic tree worksheet. Some of the worksheets for this concept are reading phylogenetic trees ap biology phylogeny review work tree 1 creating phylogenetic trees from dna sequences dna sequence evolution simulation and phylogeny building fill out the following character mark an x if an chapter 26 phylogeny and the tree of. To determine how many base changes your group will make before the next divergence. These diagrams are meant to show how closely related different species are in comparison to teach other.

This marks the beginning of the first. Worksheets are arningevolutionusing dna sequence evolution simulation and phylogeny building creating phylogenetic trees from dna sequences wojciech grajkowski reading phylogenetic trees making cladograms background and procedures phylogeny essential knowledge phylogenetic trees and learning evolution using phylogenetic analysis. He explains how it teaches students about dna sequence alignment and how those sequence differences allow researchers to determine relationships between species.

The base or trunk of the tree begins to divide into smaller and smaller branches. Based on the distance construct a tree. Procedure divide your group into two new groups.

In building a tree we organize species into nested groups based on shared derived traits traits different from those of the group s ancestor. Start with a trait that all organisms share. Calculate all the distance between leaves taxa.

Rotation around node x does not affect meaning of tree. How to construct a phylogenetic tree. Each of these new groups gets a nucleotide sequence strip from the ancestral form.

Choose what method we are going to use and calculate the distance or use the result depending on the method. Ap biology nodes indicate traits that define a new branch of the tree. Which phylogenetic tree is accurate.

Construct an phylogenetic tree one of the best ways to visualize evolutionary relationships is by drawing a evolutionary tree also known as a phylogenetic tree or a tree of life. The tree of life one method commonly used to display evolutionary relationships is by constructing a phylogenetic tree. This activity has students construct their own tree from a set of animals provided using only specific observable physical differences.

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Phylogenetic trees click and learn paul strode describes the biointeractive click learn activity on dna sequencing and phylogenetic trees. Displaying all worksheets related to phylogeny.

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Creating Phylogenetic Trees from DNA Sequences

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What a phylogenetic tree is!

The phylogenetic tree shows the evolutionary relationship among organisms in terms of relatedness and differences. It is the best way to describe an organism and to know from where it was evolved, however, the technique is not 100% accurate to describe the alterations or divergences.

The term phylogenetics or related terminologies like the phylogeny is derived from the Greek word ‘Phulon’ means ‘race’ or ‘lineage’ and ‘genesis’ means ‘origin’ .

Though Charles Darvin had explained the concept of the tree of life, the first evidence of explaining relationships between different species was explained in the Elementary Geology book written by Edward Hitchcock.

Type of evolutionary tree:

Based on the various characteristics of the tree, various phylogenetic trees are classified in different groups such as a rooted tree, a non-rooted tree, a bifurcating tree, a multifurcating tree and an Enumerating tree.

Note that there is so much literature on these topics describing different types of trees but are most confusing, I am trying to explain it in simple language so that you can understand the concept behind it.

The rooted tree is described as a phylogenetic tree sharing the common ancestor on the node. Therefore the classification ends at one point usually on the node which is the common ancestor of all the branches of the tree.

Contrary to the rooted tree, the non-rooted tree doesn’t have a common ancestor. The unrooted phylogenetic tree is always prepared from the rooted tree by excluding the common ancestor or the node of the tree.

The example of the rooted-tree, unrooted tree, bifurcating tree and multifurcating tree.

The phylogenetic tree only has two branches or we can say leaves are known as bifurcating trees. It is also classified in rooted bifurcation trees and unrooted bifurcating trees.

The multifurcating tree is described as having multiple branches on the single node. Again it is classified into a rooted multifurcating tree and an unrooted multifurcating tree.

It was believed that eukaryotic multicellular organisms evolved from the primitive prokaryotes. There are several evolutionary forces that govern the process of evolution. Genetic factors are one of them.

The phylogenetic tree is manufactured based on the phenotypic as well as genotypic variations and similarities between species.

This means when we will construct a phylogenetic tree, visible changes as well as changes in the DNA sequences are taken into account to achieve the accuracy.

How to construct a phylogenetic tree?

First, the taxons are the organisms we are using to prepare a phylogenetic tree, taxons are denoted on the tip of the tree, shown in figure 1 below.

The graphical representation of the phylogenetic tree.

The Taxa is the group of all organisms who are sharing the common clade.

Here the taxon 1 and 2 are considered in the single taxa and sister group because of having the common ancestor.

The Taxon 3 is considered as the outgroup to the entire taxa. Furthermore, node 1 is the common ancestor point for taxon 1 and 2 while node two is a common ancestor for node 1 and taxon 3.

From the above tree, we can say that the organism of taxon 1 and 2 are more similar in terms of phenotype and genetic sequences while the organism of taxon 3 is not so similar because it is not on the same lineage or node, though all three are sharing the common ancestor on node 2.

The node is also known as the branch point at where different branches of organisms share a common ancestor. The 1, 2, 3 and 4 are different organisms or species of interest to study and located on branches.

The end of the phylogenetic tree is known as the “root” where the tree ended for the selected organisms. Notably, each node represents the most common recent ancestor of the group of species.

The phylogenetic tree can’t give us the quantitative data for the divergence of species. For instance, if we add another species to this tree as the Taxon 4, with a common clade 2, we can’t predict which of the two between the 1 & 2 and 3 & 4 are closely related.

Also, we can’t even predict how diverse the two sister groups are (sister group of 1 & 2 and sister groups 3 & 4).

Constructing a Phylogenetic tree by a software:

There are so many different software nowadays available to make a phylogenetic tree, some are complicated and some are simple to use. Here in the present section, I am explaining to you some of the simple methods to make a phylogenetic tree online.

You can start making a phylogenetic tree using the phylogeny tool available in EMBL-EBI.

Select the option services and choose the phylogeny option from the tools. Click on more tools or search it in the search box.

Select the simple phylogeny option.

You may see a three-step phylogeny process, step 1 enters your file of multiple sequence alignment or you can directly paste the data into the box given.

Now in the next step, select the options you want to study.

Tree formation: select any one of the clusters, distance matrix, or Nexus. I recommend making it default.

Distance: you can on or off the option to see the distance between the sequence or the species.

In the next option of “GAPS” select on or off as per your convenience whether or not to use the gap data.

In the cluster method section select UPGMA.

In step three select the ‘be notified’ option and submit the data.

Your tree will be constructed within a few minutes.

How to read a phylogenetic tree?

For a novice reading, a phylogenetic tree is yet another challenging task, it’s always a question from where to start!

The phylogenetic tree is unidirectional starting from a common ancestor to divergence of species, though it is read and constructed from species to a common ancestor.

The species are located on the “tip” of the branches popularly known as the taxons or leaves of plants, whatever you want to call. If we read it, on a tree, every branch has different characteristics or DNA sequences.

Some of them end in the node where they are sharing the common ancestor. Commonly phylogenetic trees are rooted end at the common node.

In phylogeny, the node is also known as a “clade” as well. Though there are so many different variations of the phylogenetic tree, every method of making a tree depicts the same type of information. Take a look at various tree shown in the figure below,

The figure represents different forms of a single type of phylogenetic tree.

Keep in mind that whatever the shape, topology or structure of the tree is, it must have a common node if rooted and branched.

To read a tree, start with the tip of the branches and see where the branch ends (the node), based on that information you can depict or conclude that which organism is nearer or closer and which are distantly related.

Different topological forms of a single phylogenetic tree.

A phylogenetic tree of life:

The tree of life is the phylogeny of almost all organisms on the earth. The tree of life or the phylogenetic tree of life gives us all information on how different organisms are different from one another, the relative distance between two species and with whom they are similar.

Although for us it is very hard or near to impossible to make a tree of life covering all the organisms on the earth.

Here I am explaining a tree of life that I had found whilst doing research online.

Go to the online tool known as ITOL- interactive tree of life- .

Select the options tree of life from the menu bar. You may see the image shown above.

Now what to do with the tree of life?

There are several options on the side of the window using which you can generate or obtain some information.

For example, you can first zoom in or zoom out the three because it is so big.

Using the ‘Basic’ option you can choose which type of tree you want to make!- circular, rooted or unrooted.

There are so many options there, try each one.

After that, you can denote or indicate or label a different group of species like you can choose the option Eukaryota, Bacteria or Archaea given below on the window. Even you can mark them in different colors.

Using the option ‘datasets’ you can give indications of genome size, publications and domains per genome data on the tree.

On the other side, you have the option to manually annotate, you can use it.

You can also share the tree of life on your social media platforms as well.

Applications of a phylogenetic tree:

The phylogenetic tree is constructed to make an evolutionary link between various organisms. By doing so, we can get an idea about how and from whom different organisms are evolved.

Also, it helps to classify organisms and species in different taxa and groups based on their DNA sequence and phenotypic similarities and differences.

In addition to this, it is useful to study the force of evolution and characteristics of different organisms.

It is applicable to study the events occurring during the course of evolution and to classify species based on the divergence of structure and function.

Limitations of a phylogenetic tree:

Firstly, the manual process of constructing a phylogenetic tree is a tedious and time-consuming process. We need special software to create one!

With this to construct a tree based on the DNA or protein sequence variations, a lot of lab work is needed prior to making it.

For instance, if we want to construct a phylogenetic tree of 100 different organisms for a couple of genes. DNA extraction, amplification, and DNA sequencing is performed on all the 100 samples prior to proceeding for phylogeny.

The biggest limitation of the present technique is its accuracy. The Phylogeny methods to differentiate organisms or species are not so accurate.

In addition to this, not all the characteristics can be taken into account for constructing a single tree, it will confuse things and will not give accurate divergence.

Another limitation of the present technique is that we can’t quantify the divergence between two species. For instance, get back to example 1 at the starting of the article. Taxon 1 and 2 share common ancestor node 1 and taxon 3 and 4 share common ancestor node 2 but in which amount taxa 1 and 2 carry the diversity we don’t know.

Furthermore, It is also difficult to explain which organism can be placed first in the sister group.

Direct access to the individual tools available on this server.
Multiple Alignment:Phylogeny:Tree viewers:Utilities:
MUSCLE PhyML TreeDyn Gblocks
T-Coffee / 3D-Coffee TNT Drawgram Jalview
ClustalW BioNJ Drawtree Readseq
ProbCons MrBayes ATV Format converter

This project is funded by the Réseau National des Génopoles (RNG).
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Department of Chemistry and Bioscience, Graduate School of Science and Engineering, Kagoshima University, 1-21-35 Korimoto, Kagoshima, 890-0065, Japan

Dai-ichiro Kato, Atsuhiro Tsuruta, Juri Maeda, Kazunari Arima & Yuji Ito

Japan Fireflies Society, 2-1-24 Shinmei, Hino, Tokyo, 191-0016, Japan

Department of Biology, Keio University, 4-1-1 Hiyoshi, Kohoku-ku, Yokohama, 223-8521, Japan

Analytical Research Center for Experimental Sciences, Saga University, 1 Honjo-machi, Saga, 840-8502, Japan

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D.K. designed, analysed and interpreted all of the experiments and wrote the manuscript. H.S. collected the fireflies, performed the haplotype analysis of the mitochondrial CO II gene, and wrote the manuscript. A.T. and J.M. planned the experiments and analysed the data. Y.H. helped to identify the genetic markers by providing a custom Ruby program. K.A. and Y.I. performed the biochemical experiments and analysed the data. Y.N. performed the RAD-Seq analysis and helped to write the manuscript.

Corresponding author

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