Extract ncRNA sequencesΒΆ

All Rfam ncRNA sequences become available on the ftp with every new release. The following is a tutorial on how to extract sequences using the public instance of the MySQL database and esl-sfetch tool.

Requirements:

  1. MySQL Community Server, freely available here
  2. esl-sfetch from Infernal’s easel miniapps

  1. Download and install the Infernal software. You can find additional information in the Infernal User’s Guide.

See also

Genome annotation section

2. Add Infernal tools to your $PATH using the following command:

> export PATH="/path/to/infernal-1.1.x/bin:$PATH"

3. Create a new directory and download all fasta files from the FTP using wget :

> mkdir rfam_sequences && cd rfam_sequences
> wget ftp://ftp.ebi.ac.uk/pub/databases/Rfam/CURRENT/fasta_files/* .

4. Decompress all sequence files and merge them in a single fasta file:

> gunzip *.gz
> cat *.fa > Rfam.fa
  1. Index the unified sequence file using esl-sfetch :
> esl-sfetch --index Rfam.fa

Note

If the above command is successfull, you should see a .ssi file generated in your current directory

  1. Create a .sql file with a SQL command that fetches the regions of interest.

Example query to retrieve all human ncRNAs:

select concat(fr.rfamseq_acc,'/',fr.seq_start,'-',fr.seq_end)
from full_region fr, genseq gs
where gs.rfamseq_acc=fr.rfamseq_acc
and fr.is_significant=1
and fr.type='full'
and gs.upid='UP000005640' -- human upid
and gs.version=14.0;

Example query to retrieve all human snoRNAs:

select concat(fr.rfamseq_acc,'/',fr.seq_start,'-',fr.seq_end)
from full_region fr, genseq gs, family f
where gs.rfamseq_acc=fr.rfamseq_acc
and f.rfam_acc=fr.rfam_acc
and fr.is_significant=1
and fr.type='full'
and gs.upid='UP000005640' -- human upid
and f.type like '%snoRNA%'
and gs.version=14.0;

Example query to retrieve all Mammalian 5S ribosomal RNAs (RF00001):

select concat(fr.rfamseq_acc,'/',seq_start,'-',seq_end)
from full_region fr, rfamseq rs, taxonomy tx
where fr.rfamseq_acc=rs.rfamseq_acc
and tx.ncbi_id=rs.ncbi_id
and fr.rfam_acc='RF00001'
and tx.tax_string like '%Mammalia%'
and is_significant=1;

Note

In order for esl-sfetch to work with the Rfam fasta file, the regions need to be in the format: rfamseq_acc/seq_start-seq_end.

  1. Fetch a list of accessions to extract from the database and save them in a .txt file using the MySQL database :
> mysql -urfamro -hmysql-rfam-public.ebi.ac.uk -P4497 --skip-column-names --database Rfam < query.sql > accessions.txt
  1. Extract the ncRNA sequences in the .txt file generated in step 7 from the unified Rfam fasta file from step 4 using esl-fetch:
> esl-sfetch -f Rfam.fa /path/to/accessions.txt > Rfam_ncRNAs.fa