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Name:
Yr/Cr/Sec:
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Date: December 05, 2016
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OBJECTIVES
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.
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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. 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. 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. 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.
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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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