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README

In-Silico PCR tool

DOI

This script takes a text file with primer sequence (one per line,optinal name in column 2, tab-seperated) and a reference FASTA file as input and identifies primer pairs which amplify a DNA sequence of length less than or equal to a user-specified maximum, at a given Tm and salt concentration. The script outputs the sequences of the primers, the portion of the primer that binds, the number of mismatches, as well as the start and end coordinates of the amplified sequence. It also outputs the Tm of the amplicons and the Tm of amplicon pairs.

Dependencies

These may be installed by running

pip install PCRinSilico

Usage

PCRinSilico [options]
   --primer_seq [path to primer sequence file, one primer per line]
   --ref_fasta_file [path to reference FASTA file]

Options

Argument Description Default
--annealing_temp Annealing temperature (in Celsius). 60.0
--salt_concentration Salt concentration (in nM). 50
--max_amplicon_len Maximum length of PCR products in nucleotides. 2000
--req_five Require the 5' end of the primer to bind? True
--out_file Output file name. "in_silico_PCR"

Example

PCRinSilico \
   --primer_seq ./example/primers.txt \
   --ref_fasta_file  ./example/ref.fasta

in_silico_PCR.tsv

qseq1 qseq2_input qstart1 qend1 direction1 mismatch2 qseq2 qseq1_input qstart2 qend2 direction2 mismatch1 binding_pos_diff reference ref_region
kkd_F_2 gaacaccggcagtggttc 1 18 + 0 sge_R ctgccgcagcggt 1 13 - 0 300 example 772, 1072
kkd_R accgagctgccggacggcac 1 20 + 0 sge_R ctgccgcagcggt 1 13 - 0 318 example 772, 1090

in_silico_PCR_amplicon_interactions.tsv

amplicon1_PF amplicon1_PR amplicon2_PF amplicon2_PR tm
kkd_F_2 sge_R kkd_R sge_R 90.22726832

in_silico_PCR_primer_dimears.tsv

Sequence1 Sequence2 MeltingTemp
sge_F sge_R 75
sge_F kkd_F 72
sge_F kkd_F_2 73
sge_F kkd_R 79
sge_R kkd_F 74
sge_R kkd_F_2 74
sge_R kkd_R 80
kkd_F kkd_F_2 72
kkd_F kkd_R 78
kkd_F_2 kkd_R 78

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