Blast for 16Sr Dna

Question

[carlos.bolanos]

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).

Answer 1

[h.mon]

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?

[carlos.bolanos]

Thanks for your response:

These results were obtained using the nt database. The idea was to download and make a blast db using 16S sequences of Phytoplasma (from sequences deposited in the gene bank) or using the draft genome of phytoplasma (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4661319/). In the sample potentially infected with Clavibacter, the idea was to use the draft genome of Clavibacter michiganensis (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4498123/) The primers used for the amplicons were 27F and 1525R. I suppose there are 16S bacteria specific. The similarity was: sample seq. length ID Max score Total score Query cover E value Identity accession

P1 1183 S.nigrum chloroplast 2067 1119 1183 0 99% KM489055.2

SQ1 1183 S.nigrum chloroplast 2067 1119 1183 0 99% KM489055.2 SQ2 1123 S. nigrum chloroplast 1803 976 1123 0 99% KM489055.2 SQ3 1137 S. nigrum chloroplast 1772 959 1137 0 99% KM489055.2 SQ4 1209 S. nigrum chloroplast 2056 1113 1209 0 99% KM489055.2 SQ5 1198 S. nigrum chloroplast 2074 1123 1198 0 99% KM489055.2 . . SQ10 1172 S. nigrum chloroplast 2071 1121 1172 0 99% KM489055.2

Samples came from potato (P1) and a wild Solanum fruit (Solanum quitoense) (SQ1-10) = lulo. Something that seems strange is that we obtained the same values (max score, total score and query cover) from P1 and SQ1, though there are different species (same genus Solanum) but P = (S. tuberosum) and SQ = (S. quitoense)

[h.mon]

I don't know for this particular pair of primers, but amplifying chloroplasts is a common issue in 16S amplicon studies. With 99% similarity to Solanum nigrus chloroplasts, your amplicon are Solanum chloroplasts.

we obtained the same values (max score, total score and query cover) from P1 and SQ1, though there are different species

Align both sequences and check if they are identical. Even if they aren't, I don't think having identical blast stats is strange, as they are probably very close.