GORAP - Genomewide ncRNA Annotation Pipeline

Gorap screens genomic sequences for all non-coding RNAs present in the Rfam database using either a generalized strategy which includes multiple filters or utilizing specialized software. Furthermore, Gorap provides ncRNA based reconstruction of phylogenetic trees and is able to perform de novo predictions including TPM calculations from RNA-Seq experiments. RNA family specific screening options like command lines, threshold and constrains can be easily setupped.

Gorap is shipped with a setup routine which installs the Perl library Bio::Gorap, all necessary third-party software and databases without any requirements. Bio::Gorap is a distribution of Perl modules to provide software wrappers and functions for efficient Fasta, GFF, Stockholm alignment file and taxonomic tree manipulation.

Outline

1. Used software and databases
2. Installation
3. Update Gorap
4. Run Gorap
5. Results
6. Configure Gorap
7. Contact

Used software and databases

Tool	Source
Infernal	http://eddylab.org/infernal
Blast	https://blast.ncbi.nlm.nih.gov
tRNAscan-SE	http://lowelab.ucsc.edu/tRNAscan-SE
Barrnap	https://github.com/tseemann/barrnap
CRT	http://room220.com/crt
RAxML	https://cme.h-its.org/exelixis/web/software/raxml
htslib	https://htslib.org
BioPerl	https://bioperl.org

Database	Source
Rfam DB	http://rfam.xfam.org
NCBI Taxonomy	https://ncbi.nlm.nih.gov/taxonomy
Silva DB	https://arb-silva.de

Installation

Set the GORAP environment variable, assigned to its installation directory

export GORAP=/path/to/install/dir

Optionally, Store the GORAP environment variable permanently to your bashrc

echo "export GORAP=$GORAP" >> ~/.bashrc

Download Gorap

git clone --recursive https://github.com/koriege/gorap.git
git checkout $(git describe --tags)

Enter the source directory

cd gorap

Install Bio::Gorap, Rfam and Silva DB plus third-party tools

./setup.sh -d $GORAP

Download latest NCBI Taxonomy

$GORAP/bin/gorap -update ncbi

Update Gorap

Download Gorap

git clone --recursive https://github.com/koriege/gorap.git
git checkout $(git describe --tags)

Enter the source directory

cd gorap

Update Gorap and replace databases plus configurations from enclosed data archive

./setup.sh -u -d $GORAP

Optionally, download latest (bleeding edge) databases. This requires manual config file adaptions!

$GORAP/bin/gorap -update all

Run Gorap

Run Gorap using command line parameters

Screen E. coli genome for 6S (Rfam entry 13), and RNaseP (Rfam entries 9-11) on 8 threads given taxonomic information

• Hint 1: Multiple runs can be saved into the same output directory
• Hint 2: Restart a run with same parameters plus -skip option fastforwards to downstream analysis
• Hint 3: Restart a run with same output directory and label plus -refresh option will update annotation files (GFF,FASTA) according to present alignments and sequences

$GORAP/bin/gorap -q 13 -c 8 -k bac -s 562 -r 543 -i $GORAP/db/example/ecoli.fa -a ecoli -l ecoli_ncrna -o results
$GORAP/bin/gorap -q 9:11 -c 8 -k bac -s 562 -r 543 -i $GORAP/db/example/ecoli.fa -a ecoli -l ecoli_ncrna -o results
$GORAP/bin/gorap -i $GORAP/db/example/ecoli.fa -a ecoli -l ecoli_ncrna -o results -refresh

Run Gorap using a parameter file

• Hint 1: See template parameter.txt file
• Hint 2: Command line parameters priorize parameter file settings

$GORAP/bin/gorap -file parameter.txt
$GORAP/bin/gorap -file parameter.txt -skip -sort -l ecoli_sorted

Run Gorap and perform phylogenetic tree reconstruction

Example for annotation and SSU/RNome based phylogeny reconstruction in one step

• Hint 1: A given outgroup genome will be screened only for ncRNAs predicted in genome files given by -i
• Hint 2: For phylogeny reconstruction on curated alignments see below

$GORAP/bin/gorap -i <FASTA>,<FASTA>,<FASTA>,<FASTA> -k bac -og <FASTA>

Example for annotation and SSU/RNome based phylogeny reconstruction on curated alignments

• Hint 1: Using labels is highly recommended to update initially created HTML output
• Hint 2: Manual curation of alignments can be done after step 1

$GORAP/bin/gorap -i <FASTA>,<FASTA>,<FASTA>,<FASTA> -k bac -l mylabel
$GORAP/bin/gorap -i <FASTA>,<FASTA>,<FASTA>,<FASTA> -k bac -l mylabel -refresh
$GORAP/bin/gorap -i <FASTA>,<FASTA>,<FASTA> -og <FASTA> -k bac -l mylabel -skip

Results

• index.html file
  inspect results using a webbrowser

• annotations directory
  *.orig - GFF and FASTA files with original sequence ids
  *.passed - GFF and FASTA files with predictions that passed all filters
  * - GFF and FASTA files with predictions which failed at least one filter

  GFF filter tags - sorted in order of application

  • L - Length filter
    Applied if a predicted gene is shorter than 40% of the seed consensus sequence.
  • B - Bitscore filter
    Applied if the score of a prediction gene is below the minimum of scores of taxonomically related species. If no information is passed to Gorap or genes of related species can be found in the seed alignemnt, Gorap uses thee Rfam suggested noise cutoff.
  • S - Structure filter
    Applied if a predicted gene supports less than 50% conserved seed hairpins.
  • P - Primary sequence filter Applied if a predicted gene either supports less than 70% of nucleotides which are strongly conserved (>=90%) within the seed alignment. User defined constrains replace the default conservation based filter strategy.
    Exception: C/D-Box snoRNA familiy constrains must cover C and D box motifs which are subsequently screened for a proper kink-turn motif.
  • C - Copy number filter
    Applied according to the number of allowed pseudogenes as setupped in the RNA family specific configuration files. The primary genes are seleted by their Bitscores.
  • O - Overlap filter
    Applied if multiple ncRNA predictions intersect each other with more than 50%. All but the highest scored ncRNAs will be filtered.
  • ! - All filters passed
  • X - User filter
    Introduced upon executing Gorap with -refresh paramter to flag manually deleted sequences.

• alignments directory
  Alignments in Stockholm format which hold all predicted genes (! - tagged) plus Rfam seed sequences

• phylogeny directory
  phylogenetic trees in newick and PDF format

  • RNome - all ncRNA predictions (except rRNAs and tRNAs), using a super-matrix approach
  • core50RNome - ncRNA predictions (except rRNAs and tRNAs), present in >=50% of given input FASTA files, using a super-matrix approach
  • coreRNomeSTK - ncRNA predictions (except rRNAs and tRNAs) present in all input FASTA files, using a super-matrix approach
  • *.stk - super-matrix approach contrained by secondary structure based on horizontally concatenated Stockhol alignments
  • *.mafft - super-matrix approach from aligned and horizontally concatenated ncRNA seqeunces

• meta directory
  Stockholm alignments for each Gorap filter applied

Configure Gorap

Configuration files are located in the $GORAP/db/config directory and enable RNA family specific definitions of command line(s), thresholds and constrains.

Custom constrains

Define consensus sequence dependend mismatch constrains as shown below.

Excpetion: C/D-Box snoRNA familiy constrains must cover C and D box motifs by four region constrains. Gorap comes with pre-defined constrains for many C/D-Box snoRNAs.

Example:

[constrains]
conserved=uuGCAAUGAUGuUAagAAUUUCUUcacCUGAAuuaaaCcuUGAaGuucAAAaauCGAGCUUUUUAACaCUGAGCaaa
constrain=.....|..1...|......................................................|.1..|....
constrain=......|0.|...........................................................|0.|....

Use constrains to analogously set the number of allowed mismatches (..|.<number>.|..) in a specific region of non-snoRNAs. The given STK consensus can be modified according to IUPAC definition, but must not change in length.

Custom tools

Requirement: The output format of yet unsupported tools must be GFF3

The command line can be defined using this list of available placeholders

• $genome - internally substituted by a fasta file path
• $kingdom - internally substituted by bac, arc, euk, fungi or virus
• $cpus - internally substituted by number of defined worker threads
• $output - internally substituted by a temporary file path
  without $output , STDOUT will be parsed

Example:

[cmd]
tool=barrnap
parameter=--threads $cpus --kingdom $kingdom $genome

Custom RNA families

Create a new configuration file in the $GORAP/db/config directory with a file name that follows this naming scheme.

<RFxxxxx_description.cfg>

To use the default screening methods Infernal and/or Blast, setup the following tool definitions in your config file.

[cmd]
tool=Infernal
tool=Blast

According to the chosen naming scheme, place the folling files into a new directory under $GORAP/db/data/rfam/<RFxxxxx_description>

• A Stockholm alignment file <RFxxxxx_description.stk>
• A covariance model <RFxxxxx_description.cm>
• A FASTA file <RFxxxxx_description.fa>

Contact

Suggestions? Bugs? Troubles? Please let me know!

konstantin.riege@leibniz-fli.de

See also https://www.rna.uni-jena.de