How to get all Blastn hits

Question

[mschmid]

I am performing the following workflow:

1. I start with a bunch of nucleotide sequences as a FASTA
2. I blast them against a custom blast database using standalone blastn (-task blastn)
3. Then I cut out all the blastn hits from the initial FASTA nucleotide sequences. This leaves me with supposedly "clean" nucleotide sequences regarding the blast DB used in step 2.
4. I check if the sequences are now "clean" regarding the Blast database used in step 2

The thing is that I get hits in the second Blast run which did not pop up in the first run.

I tried to tweak my settings so that I find all hits in the first run. With limited success. I tried:

1. Setting -max_target_seqs extremely high. So this should not be the problem
2. Setting evalue extremely high. Should also not be limiting
3. Disabled dust and soft masking of low complex. regions
4. lowered the parameter -best_hit_overhang
5. and finally made value -best_hit_score_edge bigger

But I still get hits in the second Blast run after cutting out the hits from the first run. What am I missing?

[GenoMax]

Did you also try setting num_descriptions and num_alignments to an equally high number? Default hits that are returned I think are capped at 500. I don't remember if these apply if you were using -outfmt 6.