Hello,

I'm using EMBOSS for the first time and I have several doubts. I have got a short sequence of RNA. I need to find out if there is unintentional hybridisation with RNA regions similar to my sequence (Hybridisation-mediated off-targets).

To do this what I have done is:

1. Installing emboss package on Ubuntu running the following command on terminal:

    sudo apt-get update
    sudo apt-get install emboss
   

2. Download human genome for Searching for potential off-target liabilities in mature human RNAs:

    wget ftp://ftp.ncbi.nlm.nih.gov/refseq/H_sapiens/annotation/GRCh38_latest/refseq_identifiers/GRCh38_latest_rna.fna.gz
   
    gzip -d GRCh38_latest_genomic.fna.gz
   

3. Make an index bd with emboss

    dbifasta -dbname GRCh38_latest_genomic.fna -idformat simple -directory Database directory -filenames newname -release 0 -date date -outfile refMrna1.dbifasta
   

4. To identify potential off-target genes, similarity searches were performed using the pattern match search (fuzznuc):

    fuzznuc -complement -pmismatch 5 -pattern query -sequence refMrna1.fa
   

   I know that the results of that command should show 4 alignments with mature human RNAs, 3 with 100% identity and one with 87.5% identity but what I see in the report is not that, it's a bunch of alignments with 4 or 5 mismatches and none with no mismatches.

I am probably doing something wrong.

Thank you!