Ferramentas (Utilities) por João Feio de Almeida

Foreword

This tool was developed in order to allow users to compare ORFomes themselves under their own understanding of stringency. The question of stringency is especially important for BLAST searches, where threshold values (e-value) are usually under evaluated, and when similarity may be low (e.g. paralogs).

Although a few bidirectional BLAST pipelines may be publicly available, they are usually implemented in PERL and designed for UNIX family environments. These issues make their transfer for desktop computers at least demanding.

After the bi-directional matching hits are subjected to further analyses, namely straight global alignment of nucleotide sequences, global alignment based on the products, calculation of molecular evolution rates, etc.

Tecnhical Specifications

Input Data

Input files should be placed in the 'input' folder.

CDS/ORF files should be supplied in FASTA/Pearson format, one for each ORFome. Two examples are supplied (target_cds.fasta and test_cds.fasta) and users should be warned about the need to avoid special characters in the annotation line.

These same files should be used to produce BLAST databases with suitable acronyms. The user may command this application to build them or may choose to build them. In the latter case the resulting files should be placed in the '/blast/db' folder.

(..\bin\formatdb -i <path\orfome filename> -p F -o T -n <blast database acronym> )

GO terms file (e.g. go_terms.tab) were obtained from SGD (http://downloads.yeastgenome.org/literature_curation/go_terms.tab) and updated versions should be downloaded regularly. For organisms other than fungi the user are encouraged to get hold of more adequate versions.

GO Slim mapping files should be provided with identical structure to the one available at SGD (http://downloads.yeastgenome.org/literature_curation/go_slim_mapping.tab). Any other file using the same column arrangement will be suitable. The reference field values must be the ones used for the reference ORFome.

A set of test files are supplied and the user encouraged to use them in a test run of this application.

Start Program

In the 'biDiBlast' folder locate and execute 'start.bat'.

Graphic User Interface

The application is started executing the 'start.bat' batch file in the main folder. This file should be edited to force special memory configurations or particular Java Virtual Machines.

The GUI is divided in two panels: a parameter value panel at left, and a execution control panel at right.

• 'Databases already populated?'
• Checked - Uploads the ORF files into the databases (redundant entries are filtered)
• Unchecked - Assumes the databases are already populated
  
• 'Last query seq. browsed' - <accession nr.>/none
• <accession nr.> - Restarts the bi-directional BLAST comparisons from the referred entry
• none/no - This parameter value will be overlooked
  
• 'Bi-Directional BLAST already done?'
  
• Checked - In the next run the bi-directional BLAST comparison is assumed as done
• Unchecked - The comparisons are yet to be done
• 'GO DB's already populated?'
  
• done - The Slim GO objects were already created and linked to the putative homologue ORFs found
  
• yes - The GO-Slim objects were already created in the database
• no - The GO-Slim information is yet to be uploaded
  
• 'Orthologs already refined?'
• Checked - The sequence of the putative homologous ORFs and their conceptual translation were globally aligned (stretcher, EMBOSS). The substitution matrix employed is the 'SubstMat' at 'data' folder.
• Unchecked - Forces global alignment procedure
  
• 'Paralog clusters already built?'
  
• Checked - The putative paralogs were subjected to global alignment in the same terms as happened for the orthologs. The orthologues were used as reference for clustering paralogs.
• Unchecked - The procedure is yet to be executed

• 'Query CDS Fasta file' (../input)
  
• The name of the text file containing the uncharacterized CDSs
  
• 'Reference CDS Fasta file' (../input)
• The name of the text file containing the CDSs from the reference genome
  
• 'Query Blast Database'
• The acronym given to the BLAST database built upon the query CDSs. Avoid names with more than eight characters.
  
• 'Reference Blast Database'
• The acronym given to the BLAST database built upon the reference CDSs
  
• 'Query CDS Database (*.yap)'
• The name of the file to be given to the internal database (DB4O format) built upon the query CDSs. The name should be informative as this file can be used for different comparisons
  
• 'Reference CDS Database (*.yap)'
• The name of the file to be given to the internal database (DB4O format) built upon the reference CDSs
• 'GO-Slim mapping file (SGD)' (../input)
• The name given to a file with the same structure and data as http://downloads.yeastgenome.org/literature_curation/go_slim_mapping.tab
  
• 'GO terms list (SGD)' (../input)
• The name given to a file with the same structure and data as http://downloads.yeastgenome.org/literature_curation/go_terms.tab

• Query/Reference (BioJava) Genetic Code
• The genetic code for each set of coding sequences should be given in order to translate and align the conceptual products. The allowed values are defined through BioJava, and provided through a drop-down menu:
• UNIVERSAL
  BACTERIAL
  YEAST_MITOCHONDRIAL
  VERTEBRATE_MITOCHONDRIAL
  MOLD_MITOCHONDRIAL
  INVERTEBRATE_MITOCHONDRIAL
  ECHINODERM_MITOCHONDRIAL
  ASCIDIAN_MITOCHONDRIAL
  FLATWORM_MITOCHONDRIAL
  CILIATE_NUCLEAR
  EUPLOTID_NUCLEAR
  ALTERNATIVE_YEAST_NUCLEAR
  BLEPHARISMA_MACRONUCLEAR