Hi,
I need to create a table with how e-values are distributed for some sequences, as a way of reporting how conserved the sequences are.

I got some inconsistent results, and boiled it down to if I query a sequence alone, or if I query it together with other sequences. The result page has a drop-down menu where you can only pick a single query sequence. So I assume that it is independent of the other query sequences?

Here is an example to show it. 
In the first test, I query ">1" alone, and the top hits are 4e-9.
In the second test, I query ">1" together with ">0" and ">2", and when I look at only ">2", the top hits are "3e-9"

First test set-up: ![https://i.imgur.com/E3xJqOj.png][1]

First test results: ![https://i.imgur.com/AOccFBj.png][2]

Second test set-up: ![https://imgur.com/uYYnnkC][3]
Second test results: ![https://imgur.com/v3SJUZ7][4]

I just did the same test with other sequences, and I got either 0.35 or 0.1 as the top hits. All settings are identical between the two searches. I just go to nucleotide BLAST, enter my queries, enter an organism, change to "blastn", and change the number of hits to 20.000. All other settings are the defaults.

So what is the **correct** way of doing this search? I'm so confused at the moment :<


  [1]: https://i.imgur.com/E3xJqOj.png
  [2]: https://i.imgur.com/AOccFBj.png
  [3]: https://i.imgur.com/uYYnnkC.png
  [4]: https://i.imgur.com/v3SJUZ7.png
Update. The NCBI help desk responded quickly, was able to replicate the bug, and quickly also addressed the bug.

For others that may have performed similar BLAST searches I paste their response below.

> The developers have addressed this issue. 
> 
> In summary:
> 
>• The problem only occurred for BLASTN/megaBLAST searches. 
>
>• It only happened if multiple queries were submitted at once. Results for the
> first query would be correct, but all other searches would use the
> search space for the first query instead of for each individual query.
>
> • It only affected the web page. Stand-alone BLAST+ does not have this
> issue. 
>
>• Also are you aware of this paper:
> https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6662297/ This does have
> implications for E-values in some situations.