PCRinSilico 1.1.3

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