Primer design

# Made a blastn db of the TrR.v5.fasta reference genome file
makeblastdb -in ../Reference_genomes/Trifolium_repens/Trifolium_repens/TrR.v5.fasta -parse_seqids -dbtype nucl -out blastdb_TrR_v5

# Make a fasta file of all the primers you want to use
# Then blastn this against the TrR.v5.fasta reference genome using the blastdb_TrR_v5
blastn -task blastn-short -db /Volumes/archive/userdata/student_users/olivernewman/2023/TrR_v5_blastdb/blastdb_TrR_v5_2 -query Chr8_23223144_SNP_primers.fasta -outfmt "6 qseqid sseqid pident length mismatch gapopen qstart qend sstart send evalue bitscore salltitles" -max_target_seqs 5 -num_threads 20 -evalue 1e-5 -out blastn_chr8_23223144_primers_result.txt

# The above blastn command gave a result that only showed hits for one of the 5 primers in the fasta file
# Thus I decided to tweak things a little;

# The multifasta looks like this:
  >2536_R
  CATACACAACAGGTTACATTTCTCC
  >2572_R
  CATCTTTTGCTGGAGCGTTACA

SUBJ=$(echo "/Volumes/archive/userdata/student_users/olivernewman/2023/TrR_v5_blastdb/blastdb_TrR_v5_2")
# Use this if you want a table format:
blastn -db ${SUBJ} -query Chr8_23223144_SNP_primers.fasta -task blastn-short -outfmt 6 -out blastn_chr8_23223144_primers2_result.txt
# Use this if you want an alignment format:
blastn -db ${SUBJ} -query Chr8_23223144_SNP_primers.fasta -task blastn-short -out blastn_chr8_23223144_primers2_align_result.txt

# Make a database of just the ROI
makeblastdb -in ../Tr_RED_LEAF_entire_region_7kb.fasta -parse_seqids -dbtype nucl -out blastdb_TrRED_LEAF_region
SUBJ2=$(echo "/Volumes/archive/userdata/student_users/olivernewman/2023/Primer_and_REDLEAF_ROI_fastas/Tr_RED_LEAF_exons_cds/blastdb_TrRED_LEAF_region")