BIRCH

Workflow example: Use BLASTP to search for sequences related to a query protein, within a specific taxonomic group


The pea defense gene DRR206 confers strong resistance to fungal pathogens when transformed into Brassica napus. Although no Brassica homologue of this gene has been found by hybridization, we would like to see if a homologous sequence can be found in species closely-related to B. napus. Since protein searches are more sensitive than DNA searches, we need to get the amino acid sequence from the genomic sequence. This involves one step to extract the protein coding sequence (CDS), and another to translate the CDS into protein.

The first step is to read in GenBank file for the genomic sequence. Save the file PSU1716l.gen  in your current working directory. In bldna, choose File --> Open, and read in the file.



Next, we need to extract the protein coding sequence from the genomic sequence. In GenBank files, coding sequences are annotated as CDS features. Select the sequence and choose Database --> FEATURES - Extract by feature keys:



Set the feature key to CDS, Database to "Selected sequences", and send output to bldna. A new bldna window pops up with the coding sequence which was extracted from the larger genomic sequence.

To translate into protein, select PSU11716:CDS1, and choose DNARNA --> Ribosome. The translated protein will be sent to a new blprotein window.



To run the BLASTP search, select the amino acid sequence and choose Database --> NCBI BLASTP. Choose the GenBank Nonredundant Protein database. To limit the search to relatives of Brassica napus, click "Yes" for Restrict search to entries containing string:, and type "Brassicaceae" as the search string. To eliminate poor matches, set the # matches expected by random chance to 1.0e-8 (ie. 10-8). Click on Run to start the search.



The query sequence is sent to the NCBI Blast server, and the results are returned when complete. The results appear in several windows. The Blast report appears in a text editor


and the Accession numbers of the hits are sent to another text editor window:


The Accession number file can be saved, and used to retrieve proteins from the NCBI Batch Entrez service at   http://www.ncbi.nlm.nih.gov/entrez/batchentrez.cgi?db=Protein.