Need step by step guideline to construct a 16SrRNA phylogenetic tree using MEGA /NCBI-BLAST.
Dear Ekramul,
There are many software to construct phylogenetic tree. Usually, MEGA is a free software and good for the beginners. But the basic steps are same.
1. Check the sequence similarity in NCBI
2. Select and download the sequences that you want to use for your analysis.
3. Do Multiple sequence alignment (MSA) using Clustal or Mafft or MUSCLE or other MSA algorithm.
4. Editing and refining the alignment
5. Use your final MSA file for Model test
6. Make NJ tree (Distance tree), MP (Maximum parsimony ), ML (Maximum likelihood ), using Bootstrap analysis and BI ( Bayesian inference) tree.
7. Save the tree and can be visualized in the MEGA or Fig tree software.
8. After making all trees combine the topologies and put the nodal values in one tree using photo editor software. And finally it can be used for publication
Please also find the attached files, it will be helpful for you.
Best of luck
Dear Md. Ekramul Karim,
select your sequence assembly, open it with MEGA, after opening in MEGA, search option for alignment and align sequences with any one program. Usually we use Muscle alignment algorithm. After alignment, export your assembly as MEGA file and save it on your PC. Close program and again open newly saved file with MEGA. After opening, search phylogeny option and click, you will get various phylogeny building options. Depending on your need, select options (like Boot Strap, etc) click on one and wait. Depending on your system configuration it may take few minutes to few days even. After display of tree, you can adjust topology or even edit branch, fonts, colors etc. This assembly can be exported as JPEG, PDF or other formats for publishing in manuscripts.
Dear Ekramul,
There are many software to construct phylogenetic tree. Usually, MEGA is a free software and good for the beginners. But the basic steps are same.
1. Check the sequence similarity in NCBI
2. Select and download the sequences that you want to use for your analysis.
3. Do Multiple sequence alignment (MSA) using Clustal or Mafft or MUSCLE or other MSA algorithm.
4. Editing and refining the alignment
5. Use your final MSA file for Model test
6. Make NJ tree (Distance tree), MP (Maximum parsimony ), ML (Maximum likelihood ), using Bootstrap analysis and BI ( Bayesian inference) tree.
7. Save the tree and can be visualized in the MEGA or Fig tree software.
8. After making all trees combine the topologies and put the nodal values in one tree using photo editor software. And finally it can be used for publication
Please also find the attached files, it will be helpful for you.
Best of luck
MEGA is friendly and free. Simply download and install it. Read the manual and understand the concept. You are good to go.
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