Tutorial:Prediction of Ribosomal RNA Genes Using RNAmmer Software

Question

[Dr.Animo]

Introduction

Ribosomal RNA (rRNA) genes are known to be an integral part of ribosome synthesis machinery hence been studied extensively. Due to their repetitive nature, evolutionary converseness, and ubiquitous distribution /omnipresence, these genes are playing a key role in varying functions and mechanisms including maintenance of genome integrity, control of cellular aging, and serving as standard phylogenetic markers in molecular taxonomic studies, etc. Considering their significance, several databases and computational tools have been developed to organize, annotate and analyze rRNA gene data. Of these, only RNAmmer accurately predicts the location of 5s/8s, 16s/18s, and 23s/28s rRNA in full genome sequences. It made predictions using Hidden Markov Model (HMM) and to design this tool training data sets from the 5S ribosomal RNA database and the European ribosomal RNA database project was used.

Tutorial

1. RNAmmer is freely available for academic users at the following link: Download

2. After filling out the form, you will receive an email with the download link.

3. Now put the *.tar.gz file in an appropriate folder. like /home/user/tool/RNAMMER/.

4. Run the following command to extract the files.

   tar -xzf rnammer-1.2.src.tar.gz

5. RNAmmer dependencies:

   It required the following three dependencies:

   a. Perl

   b. Hmmer2

   c. libxml-simple-perl

6. After installation of the above dependencies, change line 35 of rnammer script

   Default line my $INSTALL_PATH = "/usr/cbs/bio/src/rnammer-1.2"; After changing my $INSTALL_PATH = "/home/user/tool/RNAMMER";

7. Now also change line 50 of rnammer script:

   Default line

   $HMMSEARCH_BINARY = "/usr/cbs/bio/bin/linux64/hmmsearch";

   After changing

   $HMMSEARCH_BINARY = "/home/user/tool/HMMER/hmmsearch2";

1. Now RNAmmer can be used.

2. It requires the following arguments to predict rRNAs from the genome:

-S: This option is used to specify the kingdom of our genome whether it is bacteria, archaea, or eukaryotes. For bacteria, archaea, and eukaryotes we use “bac”, “arc” and “euk” respectively.

-m: Molecule type can be tsu for 5/8s rRNA, ssu for 16/18s rRNA, lsu for 23/28s rRNA, or any combination separated by a comma.

-multi: If you want to run it parallel then use the -multi argument.

-gff: Specifies a filename for output in GFF version 2 output.

-xml: Specifies a filename for output in XML output.

-h: hmmreoport: is used to specify the filename for the output HMM report.

-f: Specifies filename for output FASTA sequence of predicted rRNA genes.

1. Now here are the commands to run RNAmmer for different kinds of organisms. Before that first export it in the $PATH variable

   export PATH="/home/user/tool/RNAMMER/:$PATH"

For Bacteria:

perl rnammer -S bac -m lsu,ssu,tsu -multi -gff ecoli.rrna.gff -xml ecoli.rrna.xml -h ecoli.rrna.hmm -f ecoli.rrna.fasta ecoli.genome.fasta

For Archaea:

perl rnammer -S arc -m lsu,ssu,tsu -multi -gff A_pernix.rrna.gff -xml A_pernix.rrna.xml -h A_pernix.rrna.hmm -f A_pernix.rrna.fasta A_pernix.genome.fasta

For Eukaryote:

perl rnammer -S euk -m lsu,ssu,tsu -multi -gff H_sapien.rrna.gff -xml H_sapien.rrna.xml -h H_sapien.rrna.hmm -f H_sapien.rrna.fasta H_sapien.genome.fasta

1. After running this command, RNAmmer produces four files GFF file, XML file, HMM report file, and FASTA sequence.

   a. GFF file (genomic feature format) containing the genomic coordinate information that describes the starting and end of the gene at which strand that gene is present and which subunit is coded by that gene. It also gives the alignment score for that particular gene.

   b. FASTA file is containing the nucleotide sequence of the predicted rRNAs.

   c. The HMM report file contains all statistical and detailed information of the search which is done by the HMMER program. It contains cutoff values, alignment detail information, E-value, and this kind of detail information.

   d. In the XML file, the output is generated in the XML file format.

Critical Parameters/Troubleshooting

Problem 1

Can't open Perl script "rnammer": No such file or directory (or when you are not in the directory where rnammer is installed)

Reason:

Perl is not installed or you are not in the directory where RNAmmer is installed.

Solution:

Perl is not installed or you are not in the directory where RNAmmer is installed.

Problem 2

No output file generated

Reason:

Hmmer2 is not installed or Hmmer 3 is installed.

Solution:

RNAmmer use HMMER2, install HMMER2, and set the path accordingly in rnammer script.

Problem 3

Can't locate XML/Simple.pm in @INC (you may need to install the XML::Simple module) (@INC contains: /etc/perl /usr/local/lib/x86_64-linux-gnu/perl/5.22.1 /usr/local/share/perl/5.22.1 /usr/lib/x86_64-linux-gnu/perl5/5.22 /usr/share/perl5 /usr/lib/x86_64-linux-gnu/perl/5.22 /usr/share/perl/5.22 /usr/local/lib/site_perl /usr/lib/x86_64-linux-gnu/perl-base.) at /home/user/Desktop/rnammer-1.2/xml2gff line 4.
BEGIN failed--compilation aborted at /home/user/Desktop/rnammer-1.2/xml2gff line 4.
error converting xml into gff

Reason:

XML::Simplemodule is not installed

Solution:

Install the XML::Simple module

Problem 4

FATAL: Failed to open HMM file /home/user/Desktop/rnammer-1.2/lib/bac.tsu.rnammer.hmm

Reason:

lib folder is deleted.

Solution:

Download the RNAmmer package again and get the lib folder.

[JackAttack]

This is great! But it looks like that link to RNAmmer is already broken. Is this software no longer available?

Edit: Nevermind-I was able to get a new link from the creators. Note: There is a typo in the command for eukaryotes. It should be "-S euk" instead of "-S bac"

[Dr.Animo]

Thanks, I updated the post.

Answer 1

[colindaven]

Another alternative is the truly free implementation barrnap https://github.com/tseemann/barrnap. This is also installable via conda so quite easy to run.