Microbial Genetics Laboratory Report No.

Name:
Yr/Cr/Sec:

Manuel, Kristie Kaye S.

4BSBio-A

Score: _______________
Date: December 05, 2016

IDENTIFICATION OF UNKNOWN ISOLATES AND

PHYLOGENETIC TREE CONSTRUCTION USING MEGA 6.0
INTRODUCTION
Many suggest that all organisms are genetically related. These relationships can
be presented by a phylogenetic tree. A phylogenetic tree represents the evolutionary
relationship between various species based on their genetic closeness. Basically, it
shows the evolutionary history of life forms and how theyre related to each other
(Mahapatro, et al., 2012).
Species diversity within a community and how distantly-related the constituent
species are from one another affect phylogenetic diversity (Chamberlain et al., 2014).
The key tasks for many molecular evolution analyses are multiple alignment and
phylogenetic tree reconstruction from molecular sequence data. There are various ways
of constructing a phylogenetic tree, and these involve the use of several programs in
sequences that perform series of slow and error- prone data reformatting sequences
and trees between these programs (Gouy et al., 2009).
According to Donkor et al. (2014), there are several tools in evaluating similarities
among biological species sequences is extremely important to our understanding of
evolution, and one of this can be achieved by basic local alignment search tool
(BLAST). BLAST compares all combinations of nucleotide or protein queries with
nucleotide or protein databases and finds short matches between two sequences and
attempts to start alignments from these hot spots. Also, it provides statistical
information about an alignment (Ye et al., 2006). The National Center for Biotechnology
information (NCBI) maintains BLAST server for easy access.
There are softwares that provide a clear graphical user interface for multiple
sequence alignment and distance or phylogenetic tree construction and display. One of
these is the MEGA software (Tamure et al., 2007). MEGA has lots of versions from 1.0
to 7.0. It is developed for comparative analyses of DNA and protein sequences that are

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aimed at interfering the molecular evolutionary patterns of genes, genomes, and

species over time (Tamura et al., 2013).
In this laboratory activity, we will use the MEGA 6.0 version for construction of
phylogenetic tree and NCBI BLAST for identifying unknown isolates.

OBJECTIVES

To identify unknown isolates using NCBI BLAST

To construct phylogenetic tree using MEGA 6.0 software.

MATERIALS
NCBI BLAST
MEGA 6.0 software
METHODS
NCBI BLAST
1. Make a random sampling and pick 10 out of 15 unknown isolates given.
2. Go to http://www.ncbi.nlm.nih.gov/BLAST/.
3. Enter the sequence of the given isolates in the FASTA box.

4. Copy and paste the sequence. Make sure your sequence will start with > sign.

5. Then click BLAST.

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6. Wait for the result to load then the list of sequences producing significant
alignments will appear. The top result will be your identified isolate which depicts
the nearest sequences of your unknown.
MEGA 6.0
1. Open the MEGA 6.0, then click Align and click edit/build alignment.
2. Under the alignment editor, select create a new alignment, then click OK. Then
choose DNA.
3. Now a new window called alignment explorer will appear. Then copy and paste
your sequences.
4. Highlight all the sequences you selected to align. Click alignment and click
ClustalW, then click OK. Wait until the pairwise and multiple alignments are
5.
6.
7.
8.

complete.
Once the alignment is finished, the nucleotide sequences will appear.
Save your file and choose MEGA format.
Close the alignment editor and open again the file you saved.
Under analysis, select substitution and click Best DNA/Protein Model. Then click

YES.
9. Select Maximum Likelihood as your statistical method, and then click compute.
10. The results will appear as shown. Choose the model with the lowest BIC scores
(Bayesian Information Criterion) because they are considered to describe the
best substitution pattern the best.
11. Afterwards, select Phylogeny under Analysis and click Construct Maximum
Likelihood tree. Click YES.
12. Under the analysis preference, then change number of boostrap to 1000. Then
run your analysis.
13. When your analysis is finished, the original tree will appear.
14. Root the tree to your outgroup. The final tree will be shown in the results.

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RESULTS

Fig. 1. Randomly picked isolates. Numbers 3,4,5,6,7,8,9,10,11 and 14 were selected.

Fig. 2. Isolate 3 identified as Uncultured Flavobacteria bacterium gene for 16s rRNA,
isolate S10.

Fig. 3. Alignments of Uncultured Flavobacteria bacterium gene for 16s rRNA, isolate S10.

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Fig. 4. Isolate 4 identified as Vitellibacter aestuarii strain 0193 rRNA gene.

Fig. 5. Alignments of Vitellibacter aestuarii strain 0193.

Fig. 6 Isolate 5 has no match in NCBI BLAST.

Fig. 7. Isolate 6 identified as Uncultured actinobacterium clone ZJ1002B110 16S rRNA

gene.

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Fig. 8. Alignments of Uncultured actinobacterium clone ZJ1002B110.

Fig. 9. Isolate 7 identified as Uncultured actinobacterium gene for 16S rRNA, isolate M15.

Fig. 10. Alignments of Uncultured actinobacterium gene for 16S rRNA, isolate M15.

Fig. 11. Isolate 8 identified as Uncultured bacterium clone Z1-55 16S rRNA gene.

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Fig. 12. Alignments of Uncultured bacterium clone Z1-55 16S rRNA gene.

Fig. 13. Isolate 9 has no match in NCBI BLAST.

Fig. 14. Isolate 10 identified as Uncultured cyanobacterium gene for 16S rRNA, isolate
N12

Fig. 15. Alignments of Uncultured cyanobacterium gene for 16S rRNA, isolate N12.

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Fig. 16. Isolate 11 identified as Uncultured cyanobacterium isolate DGGE band 8 16S
rRNA gene.

Fig. 17. Alignments of cyanobacterium isolate DGGE band 8 16S rRNA gene.

Fig. 18. Dinophysis tripos chloroplast gene for 16S rRNA, isolate N21.

Fig. 19. Alignments of Dinophysis tripos chloroplast gene for 16S rRNA, isolate N21.

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Fig. 20. The outgroup Halobacterium salinarum strain S-1 16S rRNA gene has already
been identified and was confirmed in the NCBI BLAST.

Fig. 21. Alignments of Halobacterium salinarum strain S-1 16S rRNA gene.

Fig. 22. Nucleotide sequences after the alignment in MEGA 6.0 software.

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Fig. 23. Final phylogenetic tree created using MEGA 6.0 software.

Fig. 24. The number of base substitution per site from between sequences.

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DISCUSSION
As shown in the results in NCBI BLAST from the 10 randomly selected given
isolates, isolates 5 and 9 has no matches. Other isolates has been identified as shown
in the figures above.
In the phylogenetic tree shown in figure 22, the line segment with the number
0.1 shows the length of branch that represents an amount genetic change of 0.1. The
numbers next to each isolates that represents a measure of support in which a high
value means that there is strong evidence that the sequences to the right cluster
together (Rambaut, 2013).
It can be inferred that Uncultured actinobacterium isolate M15 and Uncultured
bacterium clone Z1-55 are more closely related to each other and equally related to the
Uncultured actinobacterium clone ZJ1002B110. In fact, Uncultured actinobacterium
isolate M15 and Uncultured bacterium clone Z1-55 are the same.

Fig. 25. Nucleotide alignments using Mega 6.0 software, showing the same sequences.

However, the common ancestors of Uncultured actinobacterium isolate M15 and

Uncultured bacterium clone Z1-55 lie within the diversity of Uncultured actinobacterium
clone ZJ1002B110. Same as with Dinophysis tripos isolate N21, Uncultured
cyanobacterium isolate N12 and Uncultured cyanobacterium isolate DGGE. Vitellibacter
aestuarii strain 0193 exists prior to the others.
Based on figure 23, showing number of base substitution per site from between
sequences that the higher the number, the greater is the divergence between the
sequences. As what the values have shown, have not diverged extremely from each
other but instead on a very minimal divergence.
Uncultured actinobacterium isolate M15 and Uncultured bacterium clone Z1-55
are basically the same with p- distance 0.000, and can also be observed in the

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phylogenetic tree. Same with Uncultured cyanobacterium isolate N12 and Uncultured
cyanobacterium isolate DGGE with a p- distance of 0.059. The outgroup Halobacterium
halobium strain S-1 and Uncultured actinobacterium clone ZJ1002B110 have the
highest value of divergence among the other isolates, with a p-distance value of 0.898.

Literature Cited
Chamberlain, S., Vazquez D., Carvalheiro, L., Elle, E.,and Vamosi, J. (2014).
Phylogenetic tree shape and the structure of mutualistic networks. Journal of
Ecology. v. 102, i.5, p. 1234- 1243.
Donkor, E.S., Dayie, N., and Adiku, T.K. (2014). Bioinformatics with basic local
alignment search tool (BLAST) and fast alignment (FASTA).
Gouy, M., Guindon, S., and Gascuel, O. (2009). Multiplatform Graphical User Interface
for Sequence Alignment and Phylogenetic Tree Building. Mol Biol Evol v. 27, i.2,
p. 221- 224.
Mahapatro, G., Mishra, D., Shaw, K., Mishra, S., and Jena, T. (2012). Phylogenetic Tree
Construction for DNA sequences using Clustering Methods. International
Conference on Modelling Optimization and Computing. Elsevier. v. 38, p. 13621366.
Rambaut, A. (2013). How to read a phylogenetic tree. Epidemc: Molecular Epidemiology
and Evolution of Viral Pathogens.
Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007). MEGA: Molecular Evolutionary
Genetics Analysis (MEGA) Software. Mol Biol Evol v. 24, p. 1596- 1599.
Tamura, K., Stecher, G., Peterson, D., Flipski, A., and Kumar, S. (2013). MEGA6:
Molecular Evolutionary Genetics Analysis Version 6.0. Mol Biol Evol v. 30, p.
2725- 2729.
Ye, J., McGinnis, S., and Madden, T.L. (2006). BLAST: Improvements for better
sequence analysis. Nucleic Acid Res v. 34. NCBI.

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