
Create blast database and perform blastn alignment ...
perl est_alignment.pl -b blastall -f formatdb -r rice_ref.fasta -i test.fasta -o test.fasta_report.txt

Performing blast againt database sequences rice_ref.fasta...
blastall -p blastn -d rice_ref.fasta -i test.fasta -o test.fasta_report.txt -b 10 -e 1e-10

Intron-exon annotation and create hit file and N-marked sequences for conserved primer design ...
 java -jar IntronExonAnalysis.jar test.fasta_report.txt test.fasta_hits.txt 1500
Total query sequences = 11
Total sequence with hits = 19
Non-unique microcolinerity: 1
Unique microcolinerity: 1
Total time used in Step 1: 0.0833333333333333 minutes

Batch primer design using Primer3 core program ...
perl pick_primers.pl -i test.fasta_hits.txt_seqs.fasta_cleaned.fasta -o test.fasta_primer_report.txt
test.fasta_primer_report.txt already exists!  Overwrite (Y/N)? test.fasta_primer_report.txt_seq.fasta already exists!  Overwrite (Y/N)? Total sequences: 11
Total sequences with sucessful primers: 11
Total time used in Step 2: 0.433333333333333 minutes

Primer analysis...
perl primer_analysis.pl -b blastall -f formatdb -d test.fasta -i test.fasta_primer_report.txt_seq.fasta -j test.fasta_primer_report.txt -s test.fasta_hits.txt_summary.txt_cleaned.txt

Create blast database for database sequence test.fasta ...
formatdb -p F -i test.fasta


Performing blast againt database sequences test.fasta...
blastall -p blastn -d test.fasta -i test.fasta_primer_report.txt_seq.fasta -o test.fasta_primer_report.txt_seq.fasta_blast_report.txt -b 10 -W 7 -e 1e-1
Total primer sets: 0
No homology hits are found in the designed primers.
No primer sets which are located on the same exons are found in the designed primers.
Total time used in Step 3: 0.0166666666666667 minutes
Total time used in whole pipeline: 0.533333333333333
