I have some 16S RNA sequence with which I want to do the phylogenetic analysis, so Initially I did BLAST against SILVA db and NACBI 16S db and went for MSA in MEGA-X using Muscle and Clustal both but it seems with the nearby sequences and one outgroup my query sequence is placed near the out group or if using without outgroup still it is out grouped.
So is there any specific MSA outline regarding 16S ??
Are you sure your organism is not truly an "outgroup" a new type of organism not yet in the databases? If not, the method of alignment for 16S should not matter much, but there can be many different reasons why your sequence did not align well to the other sequences. You should always look at the alignment with an alignment editor such as AliView (for Mac) or BioEdit (for Windows) to check the alignment for quality and any possible problems.
http://ormbunkar.se/aliview/
http://www.mbio.ncsu.edu/BioEdit/bioedit.html
Brian Thomas Foley sir, it’s an unknown sequence so it’s not in the database, but as I am getting close to 97-98% similarity in the blast result then shouldn’t it be close to those species? and come to their nearest branch?
It depends on what type of organism you are studying. For some organisms such as enterobacteria found in mammalian intestines and feces, humans have put a lot of effort into studying them and we have a very different data set, than for other organisms such as hot springs microbiomes, or the ocean floor. I am attaching a sample from SILVA, of Clostridium tetani which is of huge human interest because it kills humans and their livestock. As you can see from the alignment, the evolution of 16S rRNA is not uniform, there are many regions of 100% identity and other regions where one or more isolates is very different.
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