A comparative method for finding and folding RNA secondary structures within protein-coding regions
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README

README

                      RNA-decoder README

INTRODUCTION:

This file briefly documents RNA-decoder v.1.0 - a program for making
comparative predictions of RNA secondary structure in regions which
may be protein coding. The program has two main uses: (1) Scanning for
novel structures in alignments of genomic regions. (2) Predicting the
fold of regions which are known to form RNA structures. RNA-decoder
takes an alignment with coding annotation as well as a phylogenetic
tree relating the individual sequences as input. The predictions are
based on an analysis of the substitution pattern along the
alignment. A stochastic context free grammar (SCFG) is used to
evaluate all possible secondary structure conformations. A detailed
description of the methodology can be found in:

    Pedersen, J.S., Meyer, I.M., Forsberg, R., Simmonds, P. and Hein,
    J.. A comparative method for finding and folding RNA secondary
    structures in protein-coding regions. Submitted.

The program takes a single XML file as input. Sections of the XML file
are common to every analysis and others are data specific. The common
sections of the XML file contains a complete specification of the
employed model as well as a specification of the type of analysis to
perform. The data specific sections contain a pointer to the alignment
file as well as a specification of the phylogenetic tree and an
ordered list of sequence names. The specific sections have to be
replaced for each new data analysis.

RNA-decoder is shipped with alignment files and XML files to perform
some of the analysis reported in the above mentioned paper. This
documentation will show how to run these examples and describe the
structure of the data specific sections of the XML files.


DIRECTORY STRUCTURE:

The directory structure of the RNA-decoder tar ball is as follows:

--------------------------------------------------------------------
Structure                   Contents
--------------------------------------------------------------------
.                         : main directory 
|-- bin			  : RNA-decoder executable for LINUX OS
|-- python_scripts	  : RNA-decoder-extended script and 
|   |			    RNA-decoder-two-step script.
|   `-- py_modules	  : python modules needed by scripts
|-- XML_files		  : XML-file examples for scanning and folding
|-- data		  : 
|   |-- cross_validation  : 
|   |   |-- test	  : alignments used for cross-validation 
|   |   `-- train	  : alignments used for cross-validation
|   |-- full_train	  : complete training set used to train final
|   |-- folds		  : test files for predicting folds
|   `-- scans		  : alignments used for scans reported in article
|   			    scan and fold models
|-- tree                  : examples of newick tree files
|-- scan_results	  : 
|    |-- COL-files	  : Raw output from scanning examples
|    `-- eps-figures	  : visualization of scan output
`-- output		  : empty, used for storing output files


DATA SPECIFIC XML-FILE ELEMENTS:

The top sections of the XML-files contain the three data specific
elements. They are preceded by short comments in the provided
examples, which should make them easy to find and modify. Their
structure is described below:

  
THE ALIGNMENT FILE POINTER:

The element looks as follows:

      <observationSet id="input_alg" format="col">
	<file name="<path and name sting>" />
      </observationSet>

The path and name string should point to the file containing the
alignment to be analyzed. The alignment must be in COL format (see
below).
  
  
THE PHYLOGENETIC TREE:

The phylogenetic tree defines how the sequences of the multiple
alignment are related to each other. It is specified by the following
element:

      <tree id="T_all">
	<newick>
	   
	   ... tree in newick format ...

	</newick>
      </tree>

The phylogenetic tree must be specified in NEWICK format, and should
be based on third codon positions (see article for details). The
following files, which can be found in the tree directory, contain the
phylogenetic trees employed in the article:
  
  HCV_all_type_1a_and_1b.tree  
  HCV_all_type_1a.tree         
  polio.tree
   
A description of the newick format can be found here:
  
  http://evolution.genetics.washington.edu/phylip/newicktree.html
  http://evolution.genetics.washington.edu/phylip/newick_doc.html
  
A net-based viewer can be found at:

  http://iubio.bio.indiana.edu/treeapp/treeprint-form.html


THE SEQUENCE NAME LIST:
  
The sequence-name list defines a mapping between the leaves of the
tree and the sequences of the alignment. The list specifies the order
in which the sequences appears in in the alignment (it is somewhat
redundant since this information could be extracted from the alignment
- but its currently not). The element looks as follows:

      <taxa id="TA_all">
	<taxon id="<first sequence name>" />
	<taxon id="<second sequence name>" />
			.
			.
			.
	<taxon id="<last sequence name>" />
      </taxa>



DESCRIPTION OF COL FORMAT:

RNA-decoder currently only accepts alignments in a COL format. A
general description of the COL format can be found here:

  http://www.bioinf.au.dk/colformat/

The data directory holds several examples of our specific COL format.
A col file contains a number of entries, each having a header section
and a column section. The header must specify the contents of each
column. The main keywords used in our col format are:

  symbols, taxa, labels, type

The 'symbols' and 'taxa' keywords are used to define a sequence as
follows:

  ; COL 2	symbols taxa="<name of sequence>"

Where the name of the sequence must be identical to the name given to
the corresponding leaf node.

The 'labels' and 'type' keywords are used to define a label-column as
follows:

  ; COL 0	labels type="<label type>"

A label column specifies some structural annotation of the alignment.
The label type designates which type of annotation is being
described. Two different label types are employed by RNA-decoder:
'codonmask' and 'pairingmask'.

The codonmask defines the codon position within the coding regions
(denoted by '1','2', and '3'), non-coding regions should be denoted as
third codon positions. The input alignment most have a codonmask.

The pairingmask defines the RNA secondary structure. Right and left
stem-pairing positions are denoted by a right and left parenthesis
respectively (i.e. by '(' and ')'). Loop positions and non-structural
positions are denoted by '.'. The paringmask is outputted together
with the original alignment when RNA-decoder is used for predicting
folds.

The COL format can also include a number of columns which are given
some descriptive name, e.g:

  posteriorProbabilities

The posteriorProbabilities column state the posterior probability of
the assigned annotation, and is outputted when predicting folds.


OUTPUT:

The output of the analysis also consists of COL files. The provided
XML-files will by default write their results to the output directory,
this can be changed by modifying the analysis section at the bottom of
the files.

The scanning XML files will by default create a file called:

  ./output/scan_PP.col

This COL file will include two columns called 'left' and 'left_NS',
which designate the posterior probability of a given position being
part of a loop or a non-structural region, respectively. Their sum
therefore defines the posterior probability that the given position
forms part of a stem-pair. This can be used to scan for RNA-secondary
structures (see the article for details).

The folding XML files will by default create a file called.

  ./output/fold_cyk.col

This file will contain the original input COL file with a couple of
added columns. The "pairingmask" columns defines the predicted RNA
secondary structure and the 'posteriorProbabilities' column will give
a measure of reliability of the predictions. Both are described in the
'COL FORMAT SECTION' above.


RUNNING THE EXAMPLES:

Examples of both scanning and folding XML files can be found in the
'XML-files' directory. Each contain a path to an input alignment and to
the output directory. They should therefore by default be run from the
main directory as follows:

   ./bin/RNA-decoder <path to XML file>


The following files will perform a scan along genomic sequences:

./XML_files/scanning_scfg_HCV_1a_and_1b.xml
./XML_files/scanning_scfg_HCV_1a.xml
./XML_files/scanning_scfg_polio.xml

and will analyze the following alignments (stated in the same order):

./data/scans/HCV_all_type_1a_and_1b_no_anno_overlap_split.col
./data/scans/HCV_all_type_1a_no_anno_overlap_split.col
./data/scans/polio_no_anno_overlap_split.col

The computational complexity of the method used prohibits calculations
on long alignments. The above files therefore contain sets of
overlapping alignment fragments (each of length 300). The final
prediction therefore needs to be assembled from these.


The following files will predict a specific fold:

./XML_files/folding_scfg_HCV_1a_and_1b.xml
./XML_files/folding_scfg_HCV_1a.xml
./XML_files/folding_scfg_polio.xml 

and will analyze the following alignments (stated in the same order):

./data/folds/HCV_all_type_1a_and_1b_TEST_SET_all.col
./data/folds/HCV_all_type_1a_TEST_SET_all.col
./data/folds/polio_TEST_SET_1.col


DOWNLOAD:

A tar-ball containing a RNA-decoder executable compiled for Linux
together with the above mentioned examples can be downloaded from:

http://www.stats.ox.ac.uk/~meyer/RNA-decoder


COPYRIGHT AND NON-WARRANTY:

Copyright (c) 2002 by Jakob Skou Pedersen and Irmtraud Meyer
All rights reserved.

This program is free software; you can redistribute it and/or modify
it under the terms of the GNU General Public License as published by
the Free Software Foundation; either version 2 of the License, or
(at your option) any later version.

This program is distributed in the hope that it will be useful,
but WITHOUT ANY WARRANTY; without even the implied warranty of
MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE.  See the
GNU General Public License for more details.

You should have received a copy of the GNU General Public License
along with this program; if not, write to the Free Software
Foundation, Inc., 59 Temple Place, Suite 330, Boston, MA  02111-1307  USA


AUTHORS:

Jakob Skou Pedersen,  jsp@daimi.au.dk
Irmtraud Meyer,       meyer@stats.ox.ac.uk