Greetings,
I want to construct phylogenetic trees for the identification of my bacteria (16s rRNA sequences). In order to do so, i need to select refrence sequences from the BLAST list.
Among the 100 sequences displayed in the BLAST list, how many sequences should i use and which ones should i select (what are the creteria for selection)?
Best regards.
Hi Emm.
If you want just to identify the organism, I think it is enough to perform Blast and the closest organisms will be showed in the blast results with the highest identity and coverage. Assuming you choose a universal primer which can amplify all the target bacteria and your PCR product is variable enough to identify your isolates.
You can search for you organism refseq, if there is any, in https://www.ncbi.nlm.nih.gov/refseq/rsg/
Hi Emm W Kay ,
For phylogenetic inference of strains/genotypes I would recommend the following:
1. Include reference sequences for the most common known strains/genotypes isolated in your area/country (This should include a few, say about 5-10, from your BLAST search).
2. Include reference sequences for the most common known strains/genotypes from your area isolated in other countries.
3. Include reference sequences for well established strains/genotypes that occur frequently globally - particularly if you are incorporating a phylogeographic approach to your analyses.
4. Include a few strains/genotypes that are the "most distant" from your study strains/genotypes (You will probably use them to "root" your tree or as an outgroup).
I usually wouldn't do a phylogenetic analysis with fewer than 30-50 sequences (although you can do much more than that, easily more than 100). I recommend you look at papers published on your topic and the trees they did to have a better idea of which ones to include. Using only sequences that are close BLAST matches may result in a tree that isn't very informative, at the same time using only randomly selected sequences (and no BLAST result) may give you a tree that makes your isolates look "unique" or "rare" when there may have been many previously published ones that are fairly similar. Ideally your alignments should include enough similar ones to "match" your isolates to while having enough different ones to contextualize where they fit in, in the bigger picture.
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