Workflow for identification of Primer/Marker

Summary

The workflow is for identification of primer sequence. The input nucleotide sequence is subjected to tools like Primer3 for prediction of primers. The primers predicted are then subjected to search against the database of choice using BLASTN and to RNA secondary structure prediction. Outputs from BLASTN search and RNA secondary structure prediction are then combined to give an output, suggesting the most probable primer sequence pair. 

Input files required for this workflow:

1. Nucleotide sequence file for which primer needs to be designed (can be a multi fasta formatted sequence)
2. BLAST database (if user defined) against which BLASTN is to be performed

Fig:Translated implementation

Standard Tools

·        BLAST:

Find regions of similarity between biological sequences.

·        Primer3

Picks PCR primers and hybridization oligos

Primer3 picks primers for PCR reactions, considering as criteria:

• oligonucleotide melting temperature, size, GC content, and primer-dimer possibilities,
• PCR product size,
• positional constraints within the source sequence, and
• Miscellaneous other constraints.

All of these criteria are user-specifiable as constraints.

·        RNAfold

It predicts secondary structures of single stranded RNA or DNA sequences.

Custom Tools

·        Predict Primer

It is a custom tool that reports a summarized output of the predicted primers by Primer3 after performing a similarity search against a desired database using BLASTn search and secondary structure formation using RNAfold. It takes as input two files, the BLASTn output file after running BLAST and the output of RNAfold. It reports the summarized output of atleast two hits reported by BLASTN for a query and its corresponding secondary structure prediction at a given temperature.

Usage of this tool:

./predict_primer.pl -iBLAST <BLASTn_output_filename> -ifold <RNAfold_output_filename>  -[temperature] <temprature_at_which_RNAFold_has_run> -output <output_file_name>

Where, predict_primer.pl is the tool

BLASTn_output_filename is the BLASTn output file of the primers predicted with the desired database provided

RNAfold_output filename is the RNAfold output of the predicted pimers at a given desired temperature

Temperature is an optional parameter and is to be given for the desired temperature at which secondary structures are to be predicted

Output filename is the desired output filename for the tool

Output of this tool:

·        a textual output file which gives a summarized output of BLASTN and secondary structure predictions of the predicted primers at a given temperature. Only first two BLASTN hits are reported, if found and their corresponding secondary structure predictions are also reported.

Parsers

·        Primer to BLAST parser and Primer to RNAfold parser:

Both the parsers perform a similar task. They report the predicted primers in a multi-fasta formatted file. The unique id of the primer is taken and flags (whether forward (F) or reverse (R)) are concatenated to its unique id and reported as the header followed by the predicted primer by Primer3. This multi-fasta formatted file goes as input to both the tools like BLASTN and RNAfold.