Blast Large and small fasta, which to be database and which to be query?

Question

[Dgg32]

Dear all, say I have a small 16S reference database and a large amplicon fasta and I want to classify the amplicons. If I use blast or usearch, do you think if I make the amplicon file into a blast database and blast the 16s reference against it is Ok? Does it make any difference than the other way around? In terms of Speed and Results? Thank you!

Answer 1

[Istvan Albert]

The choice of target vs query seems like a "commuting" operation a vs b is the same as b vs a but that is not the case. One of your datasets has high information content (reference) whereas the other may be a highly redundant and noisy measurement. The information content in the two files is radically different.

You almost always want to take each one of your measurements and query with it against a reference database. Among many reasons also because blast E-values will be computed relative to database size.