First step that we have to do is getting cytochrome b sequence of all species, by go to NCBI homepage and use function search “Gene” and put keywords “cytochrome b and species name” for example “cytochrome b human” in search box.

Lists of species in this study

1.Human / Homo sapiens
2.Dog / Canis familiaris
3.Rabbit / Oryctolagus cuniculus
4.Rhinoceros / Rhinoceros unicornis
5.Dugong / Dugong dugon
6.Mouse / Mus musculus
7.Whale / Balaena mysticetus (bowhead whale)
8.Bovine / Bos Taurus
9.Sicklebill / Epimachus meyeri (brown sicklebill)
10.Chicken / Gallus gallus
11.Magpie / Anseranas semipalmata
12.Frog / Rana nigromaculata (black-spotted frog)

The results of searching

---

---

Getting sequence in FASTA format

---

Copy Cytochrome B sequence

---

Paste every sequence to Notepad, save in same file, for example "cytochrome b.txt"

By this way, we can get almost all species except for "sicklebill" which is searched by using function "Nucleotide" instead of “Gene” in NCBI.

---

After we get all cytochrome b sequence, we can start phylogenetic tree construction. The necessary bioinformatics tools in this study are ClustalX, BioEdit, Phylip and Treeview32.

1.Align all sequences in ClustalX
: File > Load Sequences > Open file “cytochrome b.txt”
the result is in picture below

: Edit > Select All Sequences
: Alignment > Out put Format Options > Select PHYLIP format > Close

: Alignment > Do complete Alignment
the result is in picture below

From alignment result, we can get the position of dinosaur sequence that might come close to other species that is at position about 880 bp to 1010 bp of other sequences.

2. Edit sequences by using BioEdit
Because of different length of sequences, we have to choose position that nucleotides exist in all sequences, so we choose sequence at about position 880 bp to 1010 bp.
: Open BioEdit >Mode >Edit > Delete irrelevant sequences

After edit sequences, save file into FASTA, for example " cytochrome b edit.fas".
Align all sequences again by ClustalX, get result as picture below.

From alignment result, we will get new order of species which 1st specie is Chicken and last (13th) is Frog. The number of species is important for picking the outgroup in PHYLIP.

3. Prepare file for PHYLIP analysation
By rename file “cytochrome b edit.phy” to “infile”. Then move “infile” to folder “exe” in PHYLIP

4. PHYLIP : Seqboot
: double click on icon "seqboot.exe" in exe folder of PHYLIP
: Press y > enter > Random number seed > 777 > enter. Then wait until this window disappear.

---

---

---

5. PHYLIP : Dnadist
: delete or move “infile” to other place
: rename “outfile” to “infile”
: double click on icon “dnadist.exe”

---

: change Distance from F84 to Kimura 2-parameter by press D > enter
: change Form matrix from Square to Lower-triangular by press L > enter
: set mutiple data sets by press M > enter > D (to choose data set) > enter > 100 (as set in last step) > enter > Y > enter. Then wait until this window disappear.

---

6. PHYLIP : Neighbor
: delete or move “infile” to other place
: rename “outfile” to “infile”
: double click on icon “neighbor.exe”

: Set outgroup from 1 to 13 by press O > 13 >enter (in this study, I set Frog as outgroup because there are 2 main species group which are mammalian and avian, so frog is most different specie. The number of Frog specie is 13 as the result from ClustalX.)
: Set Lower-triangular data matrix by Press L > enter
: Set mutiple data sets by press M > 100 > enter > Random number seed > 777 > enter > Y > enter .Then wait until this window disappear.

---

7. PHYLIP : Consense
: delete or move “infile” to other place
: rename “outfile” to “infile” and “outtree” to “intree”
: double click on icon “consense.exe”

: Set outgroup from 1 to 13 by press O > 13 > enter
: Set root tree by press R > enter > Y > enter. Then wait until this window disappear.

---

8. Get Tree
: Open file “outtree” in TreeView32, get 4 types of tree
1.Unrooted tree
2.Slanted cladogram
3.Rectangular cladogram
4.Phylogram

From phylogram, there are 3 main branches
1.Amphibian (frog)
2.Mammalian (mouse, dinosaur, human, dugong, rhinoceros, bovine, dog, rabbit and whale)
3.Avian (magpie, chicken and sicklebill)

In mammalian group, we can differentiate into 4 subgroups
1.Mouse
2.Dinosaur, Human
3.Dugong, Rhinoceros and Bovine
4.Dog, Rabbit and Whale

so from all results of phylogenetic analysis, we can conclude that
1.Human and Dinosaur have same ancestors
2.The most related specie to dinosaur is human.
3.The most different specie from dinosaur is Avian (sicklebill or chicken).

However, the results of this study may not be the exact one because we did only with short sequences of cytochrome b (only 133 bp).
So if we want to get better tree, we have to do with longer sequences and get more information from Dinosaur.

Hosted by www.Geocities.ws