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NAME

validate_gff3.pod

SYNOPSIS

GFF3 Validation Tutorial

DESCRIPTION

This package contains code for validating a GFF3 file. Validation can be performed using the command-line script or the web script.

In either case, analysis is performed using the GFF3::Validator module.

This module, parses the GFF3 file, loads data into a MySQL/SQLite database, performs a number of analysis steps and produces a report file.

This document details the analysis steps performed. The command-line script and the web script generate a Validator object, managing I/O and run Validator object methods, and provide the results to the user.

Analysis and Validation Steps

Parsing and Format Validation

In this step, the GFF3 file is read from beginning to end, lines are parsed and formatting errors are recorded. Feature lines and directives are stored in two separate tables for further processing. Malformed directives, comments and FASTA lines are not loaded into the database. Parseable parts of feature lines are loaded into the database. The implication of this is that, if a line is not parseable, it will be disregarded in further analysis. For example, (i) if feature A has a parent feature A and (ii) feature A cannot be parsed and loaded, (iii) in downstream analysis, since feature A points to an invalid feature, this will be raised as an error.

The following points are validated at this step:

 - spaces at the end of lines are allowed and disregarded
 - empty lines, comments (starting with a single #) are disregarded
 - '>' implies a '##FASTA' directive, following lines that follow are disegarded

 - the following directives are recognized and checked for format
   multiple spaces in the format are treated as single space

    - ##gff-version version
      - must be "##gff-version 3"
      - must be present and be the first line

    - ##sequence-region seqid end
      - must have the format "##sequence-region \S+ \d+ \d+"

    - ##feature-ontology URI

    - ##attribute-ontology URI
      - cannot be used now, reserved for future use

    - ##source-ontology URI
      - cannot be used now, reserved for future use

    - ###

    - ##FASTA
      - lines following this directive are disregarded

 - the following checks are performed on feature lines:

    - must be exactly 9 fields
    - fields are split on single tabs
    - empty fields must have a dot

    - individual fields are processed as follows:

      - field 1: seqid
        - only following characters are allowed [a-zA-Z0-9\.\:\^\*\$\@\!\+\_\?\-\|\%]
        - cannot be empty (dot)

      - field 2: source
        - only following characters are allowed [a-zA-Z0-9\.\: \^\*\$\@\!\+\_\?\-\%]
        - can be empty (dot)

      - field 3: type
        - only following characters are allowed [a-zA-Z0-9\.\: \^\*\$\@\!\+\_\?\-]
        - cannot be empty (dot)

      - fields 4 and 5: start and end
        - must be integers
        - must be in 1-based coordinate system
        - end must be greater than or equal to start
        - cannot be empty (dot)

      - field 6: score
        - must be a floating number (e.g. "1.2", "5", "1e-10", "-1e+10")
          (for scientific notation: /^[\+\-]{0,1}\d+(e|E)[\+\-]\d+$/ is used)
        - can be empty (dot)

      - field 7: strand
        - must be + or -
        - can be empty (dot)

      - field 8: phase
        - must be 0 or 1 or 2
        - can be empty (dot)
        - must be 0, 1 or 2 for CDS features

      - field 9: attributes
        - can be empty (dot)
        - attributes are in tag=value format, separated by semicolon (tag=value pairs split on semicolon)
        - tag=value pairs are parsed based on first equals sign
        - value cannot contain an unescaped equals sign
        - each tag should must appear only once, multiple values can be provided to a tag
          separated by comma
        - tag cannot have a preceding or leading space and cannot be empty
        - value cannot be empty
        - first-upprcase tags are reserved, only the following are recognized and checked as follows

          - tag: ID
            - there can be only one ID in a single line

          - tag: Name

          - tag: Alias

          - tag: Parent
            - parents are captured for further analysis

          - tag: Target
            - format must be target_id start end [strand]
            - multiple spaces can be used

          - tag: Gap
            - format checked, e.g. Gap=M8 D3 M6 I1 M6
            - the following operations are allowed: [MIDFRmidfr]
            - multiple spaces can be used

          - tag: Derives_from
            - derives_from parents are captured for further analysis
          - tag: Note

          - tag: Dbxref
            - format checked for DBTAG:ID (split on first colon)

          - tag: Ontology_term
            - format checked for DBTAG:ID (split on first colon)

Unique ID Validation

ID attributes provided for features must be unique throughout the gff3 file. These ID's are used to make "part_of" (Parent) associations between features. Each line, if contains an ID, must be unique. For multi-feature features, such as alignments, multiple lines can share the same ID. If this is the case, the seqid, source, type (method), strand, target name and all other attributes other than target must be the same.

This step goes through the features that have an ID and checks for uniqueness, taking into account multi-feature cases.

Unique features are marked as unique. Features that illegally share the same ID are marked as non-unique. This information is later used for further processing.

Loading Ontology

GFF3 requires types of features to be coming from an ontology and Parent relationships to be valid "part_of" relationships based on the ontology. In this step, the ontology that will be used as the basis of this analysis is loaded.

GFF3 specs allow the ontology to be loaded by the ##feature-ontology directive. Multiple ontologies can be loaded, in which case, they need to be merged and used for the analysis. If they are not provided, the ontology defaults to SOFA.

In this validation implementation, the following rule is used:

 - Ontology files provided in the command line and ##feature-ontology directives are
   retrieved. If at least one of these files are retrieved, that ontology file is loaded in memory
   and used for the analysis.

 - If no ontology files are provided in the command-line and ##feature-ontology directives, or
   if they are provided but none of them are accesible, default ontology file provided in the
   configuration file is retrieved and used for the analysis. If it is not accessible either,
   an error is recorded and loading ontology, type field validation, parent and derives_from
   validation steps are skipped.

For retrieval of ontologies, the following rule is used:

 - the URIs that begin with http:, ftp:, file: are retrieved
   by LWP
 - the URIs that begin with other <protocol>: are not retrieved
 - other URIs are treated as local file names and ther existence is checked

"type" Field Validation

GFF3 file type fields must be valid ontology terms (names or accession numbers). In this step, the type fields are validated against the ontology, as loaded in the previous step. This step is skipped if no ontology can be loaded.

Features are marked as valid or invalid. This information is later used for further processing.

"part_of" Relationship Validation

For features in a GFF3 file, Parent attribute, if provided, indicates that the feature is "part_of" the feature, assigned as Parent. The feature type of the parent and the feature type of the child must be terms that can have a part_of relationship based on the ontology. Circular assignments are checked at this step as well (e.g. if C is parent of B, B is parent of A, then A cannot be parent of B or C). or the parent have in-valid types, or parent has a non-unique ID.

These checks are not performed if either the child or the parent have invalid types, or parent has a non-unique ID or does not exist (These cases are recorded separately as erros).

This step is skipped if no ontology can be loaded.

"derives_from" Relationship Validation

Similar to Parent attribute, Derives_from attribute, if provided, indicates that the feature "derives_from" the feature, assigned as Derives_from. The feature type of the parent and the feature type of the child must be terms that can have a derives_from relationship based on the ontology.

Unlike Parent assignments, Derives_from assignments are checked for one level only.

*** Currently type validation for Derives_from assignments is disabled. ***

These checks are not performed if either the child or the parent have invalid types, or parent has a non-unique ID or does not exist (These cases are recorded separately as erros).

This step is skipped if no ontology can be loaded.

Report Generation

At the end of analysis, errors and warnings are compiled into a single list, sorted by their line numbers.

Installation

Command-line tool

- Copy over lib/GFF3/Validator.pm. Adjust your Perl library paths to make it accesible.

- Create a writable temporary directory and include path to it in the configuration file.

- Use validate_gff3.pl validation script with its configuration file, validate_gff3.cfg.

- For usage information on validate_gff3.pl, use:

  perldoc validate_gff3.pl

- For configuration information, check documentation embedded in validate_gff3.cfg .

Online tool

- Copy over lib/GFF3/Validator.pm and lib/GFF3/Online.pm. Adjust your Perl library paths to make them accesible.

- In your web site's document root, create the following directory (you can use another web accesible directory, if this is the case configure the paths in the configuration files accordingly):

  validate_gff3_online

Copy over contents of html directory (images, documentation, stylesheet) into this directory, adjust permissions.

- Copy over validate_gff3_online (CGI script) into a CGI-executable directory, adjust permissions.

- Create a writable temporary directory and include path to it in the configuration files (see configuration files).

- Copy over validate_gff3.cfg and validate_gff3_online.cfg (configuration files) into $ENV{DOCUMENT_ROOT}/../conf directory. Configure the application using these files (files contain embedded documentation). Start with validate_gff3.cfg. Note: If you want to place validate_gff3_online.cfg in a different directory, modify the location of this file in validate_gff3_online script. The location of validate_gff3.cfg can be set in the validate_gff3_online.cfg file.

Notes

- The default source for an ontology file is to retrieve it from the Sequence Ontology site. However, if you intend to make many runs locally, or if you install an online version, please download the ontology file and change the configuration to point to the local file.

SEE ALSO

AUTHOR

Payan Canaran <canaran@cshl.edu>

VERSION

$Id: validate_gff3.pod,v 1.1 2007/12/03 14:20:23 canaran Exp $

CREDITS

- SQLite support adapted from patch contributed by Robert Buels <rmb32@cornell.edu>.

COPYRIGHT AND LICENSE

Copyright (c) 2006-2007 Cold Spring Harbor Laboratory

This program is free software; you can redistribute it and/or modify it under the same terms as Perl itself. See DISCLAIMER.txt for disclaimers of warranty.