NCBI BLAST+ blastn misses target sequence when searching multiple sequence, but not individually

Hi all!

I’ve been recently using the BLAST+ blastn tool to search my de novo assembled contigs for sequences of interest. I’ve done this successfully with both gene and primer sequences.

However, I queried these same contigs (which had been made into a database with “NCBI BLAST+ makeblastdb”) with primer sequences I expected to find in a subset of the contigs and the blastn output was blank. When I pull out an individual contig that I suspected to have the sequence, turn it into a database, and run the blastn, the result shows a perfect match.

It seems like the issue may lay somewhere with BLASTing multiple sequences at once with these particular primers (since I’ve done this same workflow with identical settings successfully with other sequences including the gene that the primers were designed for).

Anyone run into this same issue/have any advice?

I apologize if any important details are missing, please let me know what else would be needed.

Thank you!

Follow up:

I filtered my contigs for those that contained my gene of interest and threw those into a separate file and then ran the same workflow mentioned above. I was able to find the primers in these contigs easily. But if there are additional contigs in the same database, then something happens and I get zero output.

In this case it works out since I happen to have this additional information, but in cases where I didn’t have a way to filter my contig list down, this could give inaccurate results. I’m assuming the issue lies with my settings somehow. Anyone have any ideas?