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Description:

PCRinSilico 1.1.3

README
In-Silico PCR tool

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

Python 3
Biopython
pandas
BLAST+

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, Ignored if Q5 True).
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"

--Q5
Use Q5 approximation settings for Tm calculations?
"True"

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