I want to run blast using the line of command (standalone blast). I have some sequences of plant material potentially infected with bacteria (clavibacter and phytoplasma). When running Blast in the ncbi page, the result showed homology with solanum nigrum chloroplast. I wonder if creating a database of 16S rDNA with makeblast database and running bkast of my samples versus the database of the potential causal agent i will have different results in my blast homology? I have homology for aprox. 1100 bp to solanum nigrum, but my sequence amplicon is 1498 bp (the whole 16S).
I am supposing you ran online blast using nt database, right? What will you include in your custom 16S database? If you include all 16S sequences, then the result would probably be very similar - chloroplast from some plant. If you only include bacterial 16S (which you can do online, by the way), the results would change - you would get similarity to some bacteria - but for the wrong reason, that is, the most similar sequences are now excluded from the database.
Which primers did you use for the amplicon? Are they bacteria-specific? And how much similarity between your amplicon and the database sequence?