Package: primerJinn
primerJinn has two main functions, it designs primers for a multiplex PCR of given target regions of a DNA sequence in a FASTA file and it performs in silico PCR given a list of primers and a reference FASTA file.The scripts takes several arguments:ProgramRequiredParameterDescriptionDefaultBothYinput_fileThe path to the input FASTA file.NAprimer designYregion_fileThe path to the primer design region file. This file should have three columns: name, start position, and end position (1-based). It can be in either tsv or xlxs format.NABothNsalt_concentrationSalt concentration (in nM, Ignored if Q5 True).50BothNtarget_tmThe desired melting temperature (Tm) for the primers60CBothNoutputThe name of the output file.'MultiPlexPrimerSet' or 'in_silico_PCR'primer designNprimer_lenThe desired length of the primers.20primer designNproduct_size_minThe desired min size for the PCR product.400primer designNproduct_size_maxThe desired max size for the PCR product.800primer designNretThe maximum number of primer pairs to return100primer designNQ5A boolean indicating whether to use NEB Q5 hotstart polymerase settings for primer3TRUEprimer designNbackgroundThe path to the mispriming library FASTA fileprimer designNill_adaptAdd Illumina partial adaptersFALSEprimer designNclampRequire GC clamp0primer designNpolyMaximum allowable length of a mononucleotide repeat (poly-X) in the primer sequence3in silico PCRNproduct_size_maxMaximum length of PCR products in nucleotides.2000in silico PCRNreq_fiveRequire the 5' end of the primer to bind?TRUEDependenciesPython 3BLAST+Installationpip install primerJinnMultiplex primer generation example usagegetMultiPrimerSet \
    --region_file "./example/primer_regions.tsv" \
    --input_file "./example/ref.fasta" \
    --target_tm 65 \
    --primer_len 20 \
    --product_size_min 400 \
    --product_size_max 800 \
    --ret 100 \
    --Q5 \
    --background "" \
    --output "example"###MultiPlexPrimerSet.xlxsForward PrimerReverse PrimerForward tmReverse tmProduct SizeNameForward Primer TM NEBReverse Primer TM NEBGAGGAACACCACTAGTACCGCTCGATGACTTTACGGCCAT65.0833464.69482675Target 1461045-14612916464CATGGGATATGGAGCGATCGGGGGTCGTAGGAGATCTTGA65.9558665.59987791Target 490900-4914166565CCGGTTGTCCATTCCGTTTACTGTACGTATTTGGGTTGCG64.1796765.57867454Target 1303831-13039116564GGATGCGAGCTATATCTCCGAATACGCCGAGATGTGGATG65.1566964.9819458Target 1674182-16742226565CAACAGTTCATCCCGGTTCGGACGGATTTGTCGCTCACTA64.8888966.57304759Target 2288681-22892426664GCCACCATCGAATATCTGGTGCTCCAGGAAGGGAATCATC65.6373665.01728778Target 761007-7612776465GCATACCGAACGTCACAGATACGGTCACCTACAAAAACGG65.6894165.23466665Target 778990-7794886565GCTCTTAAGGCTGGCAATCTCGGTCACACTTTCGGTAAGA64.8204765.53067577Target 2154831-21548736564There is an online version of primerJinn getMultiPrimerSet available atDrDx.MeNotesWhen testing the primers for a targeted diagnostic assay, we would recommend doing a qPCR using individual primers and reagents intended for the PCR, and adding in EVA Green plus (20X in water) and ROX (50X). This will allow you see the relative efficiency of each primer, and investigate the melt curves, the products can also be run on an agarose gel to confirm single bands.PCR in silico example usagePCRinSilico \
   --primer_seq ./example/primers.txt \
   --target_tm 50 \
   --input_file ./example/ref.fastain_silico_PCR.tsvqseq1qseq1_inputqstart1qend1direction1mismatch1qseq2qseq2_inputqstart2qend2direction2mismatch2binding_pos_diffreferenceref_regionp1GAGGAACACCACTAGTACCG120+0p9CTCGATGACTTTACGGCCAT120-0655NC_000962.314616371460982p2CATGGGATATGGAGCGATCG120+0p10GGGGTCGTAGGAGATCTTGA120-0771NC_000962.3491474490703p3CCGGTTGTCCATTCCGTTTA120+0p11CTGTACGTATTTGGGTTGCG120-0434NC_000962.313041111303677p4GGATGCGAGCTATATCTCCG120+0p12AATACGCCGAGATGTGGATG120-0438NC_000962.316745581674120p5CAACAGTTCATCCCGGTTCG120+0p13GACGGATTTGTCGCTCACTA120-0739NC_000962.322893912288652p6GCCACCATCGAATATCTGGT120+0p14GCTCCAGGAAGGGAATCATC120-0758NC_000962.3761564760806p7GCATACCGAACGTCACAGAT120+0p15ACGGTCACCTACAAAAACGG120-0645NC_000962.3779595778950p8GCTCTTAAGGCTGGCAATCT120+0p16CGGTCACACTTTCGGTAAGA120-0557NC_000962.321552892154732in_silico_PCR_amplicon_interactions.tsvamplicon1_PFamplicon1_PRamplicon2_PFamplicon2_PRtmkkd_F_2sge_Rkkd_Rsge_R90.22726832in_silico_PCR_primer_dimears.tsvSequence1Sequence2MeltingTempp1p273p1p374p1p473p1p573p1p673p1p772p1p873p1p973p1p1074Citation(Limberis, J.D., Metcalfe, J.Z. primerJinn: a tool for rationally designing multiplex PCR primer sets for amplicon sequencing and performing in silico PCR. BMC Bioinformatics 24, 468 (2023).)[https://doi.org/10.1186/s12859-023-05609-1]