Add Primer-BLAST

Description:

Primer-BLAST feature is Primer3 + BLAST. It uses Primer3 to design PCR primers and then uses BLAST and global alignment algorithm to screen primers against user-selected database in order to avoid primer pairs that can cause non-specific amplifications.

GUI:

Add a new menu called "Primer-BLAST" to "Tools -> Primers". Clicking on "Primer-BLAST" opens the new dialog with following fields.

Input fields (should be united into a group box):

• "Forward primer (5'->3' on direct strand)" line edit (from 10 to 36 bases; A, C, G, T and N only).
• "Reverse primer (5'->3' on reverse-complementary strand)" line edit (from 10 to 36 bases; A, C, G, T and N only).
• "Database" - combobox with the same meaning as in the "Query NCBI BLAST database", but has only three optins: "Nucleotide collection (nt)", "NCBI Transcript Reference Sequences (refseq_rna)" and "Custom".
• "Custom database (sequences or BLAST database directory)". This option is enabled only if "Database" has "Custom" option. Here you have two radio buttons - "Use sequence files" and "Use NCBI BLAST database":

• • Use sequence files (default) - if this button is checked, then you as many sequences as you need (FASTA, Genbank, etc - any valid sequence format).
  • Use NCBI BLAST database - if this button is checked, you should use a directory and a basename, which contain NCBI BLAST database (as we have in "Query with local BLAST...")

Output fields:

• "Primers info" - table widget
• "Products on target templates" - table widget

Other buttons:

• "Help" - open help page.
• "Start" - starts execution with the set parameters.
• "Close" or "Hide":
  • "Hide" - if execution task is running (read note below).
  • "Close" - if execution task is not running.

Algorithm:

1. Run "Primer3" for the set primers.
   • "Forward primer" should be used as SEQUENCE_PRIMER.
   • "Reverse primer" as SEQUENCE_PRIMER_REVCOMP,
   • Use melting temperature settings from the attached file primer-blast-melting-temp.txt. 
   • Set PRIMER_PICK_ANYWAY to 1.
   • Important: SEQUENCE_TEMPLATE should not be set, because the point is we do not know a sequence this primers are located at, we need to find possible sequences using BLAST.

The result of Primer3 work should be shown in the "Primers info" table widget as follows:

1. Run BLAST for both primers. Important: run BLAST once for both sequences, not twice for each of them! If "Database" is:
   • "Nucleotide collection (nt)" or "NCBI Transcript Reference Sequences (refseq_rna)" set - use "Query with NCBI BLAST". You shoul have the following requst parameters:
     • CMD=Put
     • PROGRAM=blastn
     • DATABASE=nr
     • QUERY=join forward and reverse primers.
   • "Custom database" set - use "Query with local BLAST". (TODO - another issue)
2. Check request and show sequence as result to "BLAST info". BLAST result is filtered if:
   • One or both primers have 6 or more mismatches with BLASTed sequence.
   • One or both primers have 2 or more mismatches with BLASTed sequence within the last 5 characters at the 3' end.
3. The "Products on target templates" table widget should be filled with nt fillered results (10 max). Table should have the following view:

Additional note: BLAST may be very time consuming. In this case, "Close" button switceswitches to "Hide". If "Hide" is clicked, the dialog hides and opens again when calculation is finished.