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virtualPCR

In silico PCR for simple and complex tasks

Java Platform License Online Tool DOI

Table of Contents

Overview

virtualPCR is a versatile tool for conducting in silico PCR analysis, designed to ensure primer/probe specificity across a wide range of applications. It enables researchers to predict primer/probe binding sites, assess mismatch tolerance, evaluate DNA duplex stability, and perform genome-wide searches.

Applications

  • Gene discovery via homology analysis
  • Molecular diagnostics and primer validation
  • Genome profiling and repeat sequence identification
  • CRISPR-Cas gRNA target evaluation
  • miRNA and probe specificity testing

Key Capabilities

  • Genome-wide primer/probe specificity analysis
  • Linear and circular DNA support (plasmids, mitochondria, plastids)
  • Bisulfite-treated DNA simulation (C→T conversion for methylation studies)
  • Linked search mode for complex primer arrangements
  • Degenerate nucleotides (IUPAC), LNA, and inosine support
  • Batch file processing and automation

Citation

Kalendar R, Shevtsov A, Otarbay Z, Ismailova A. (2024). In silico PCR analysis: a comprehensive bioinformatics tool for enhancing nucleic acid amplification assays. Frontiers in Bioinformatics, 4:1464197. DOI:10.3389/fbinf.2024.1464197

Requirements

Requirement Details
Java Version 24 or higher
OS Windows, Linux, or macOS
RAM Default is sufficient; increase for large genomes (see Memory)

Download Java: https://www.oracle.com/java/technologies/downloads/ Set Java Path: https://www.java.com/en/download/help/path.html

Installation

Option 1: Direct Download

  1. Download virtualPCR.jar from the dist directory
  2. Place it in your preferred location
  3. Ensure Java 24+ is installed and available in your PATH

Option 2: Install Java via Conda

# Add conda-forge channel and set priority
conda config --add channels conda-forge
conda config --set channel_priority strict

# Create environment with OpenJDK 25
conda create -n java25 openjdk=25

# Activate environment
conda activate java25

# Verify installation
java -version

Genome Downloads

Download individual chromosome FASTA files from NCBI Datasets. For example, the human genome (T2T-CHM13v2.0) is available at NCBI FTP.

Quick Start

# Basic usage
java -jar virtualPCR.jar config.file

# With increased memory for large genomes
java -Xms4g -Xmx16g -jar virtualPCR.jar config.file

Large Genomes

For genome-wide analyses, allocate additional memory using JVM flags:

java -Xms4g -Xmx16g -jar C:\virtualPCR\dist\virtualPCR.jar C:\virtualPCR\test\config.file
  • -Xms — initial heap size (memory allocated at startup)
  • -Xmx — maximum heap size (upper memory limit)

Configuration File

All parameters are specified in a plain text file (e.g., config.file):

targets_path=C:\virtualPCR\test\ch02.fasta
output_path=
primers_path=C:\virtualPCR\test\rt.txt
type=primer/probe
linkedsearch=false/true
molecular=linear/circle
primerstatistic=true
number3errors=1
minlen=200
maxlen=500
FRpairs=false/true
CTconversion=false/true
SequenceExtract=true
ShowPrimerAlignment=true
ShowOnlyAmplicons=true/false
ShowPCRProducts=true/false
ShowPrimerAlignmentPCRproduct=true/false

Paths

Parameter Description
targets_path Path to the target sequence file (FASTA or plain text)
primers_path Path to the primer/probe file (FASTA or tab-delimited)
output_path Output directory (leave empty for same directory as input)

Option Reference

type — Search Mode

Value Description
primer Standard PCR primer search (default)
probe Search for binding sites of primers, probes, and short sequences — TaqMan, Molecular Beacons, miRNA, CRISPR-Cas gRNA, microarray oligos, etc. Recommended when primer binding sites are not found, or when complementarity is expected for only part of the sequence (e.g., Molecular Beacon stems)

molecular — Template Topology

Value Description
linear Linear DNA (default)
circle Circular DNA (plasmids, mitochondrial/plastid genomes). Primers may produce one or two amplicons spanning the origin

minlen / maxlen — Amplicon Size Filter

Defines the expected PCR product size range (in bp). Amplicons outside this range are filtered out.

  • Default: 5000 bp maximum
  • Example: minlen=200 and maxlen=500 restricts output to 200–500 bp products

CTconversion — Bisulfite Conversion Simulation

Simulates bisulfite conversion for methylation studies. Only cytosines not followed by guanine (non-CpG) are converted to thymine on both strands:

5'-aaCGaagtCCCCa-3'        5'-aaCGaagtTTTTa-3'
   |||||||||||||     →         ||||||:|::::|
3'-ttGCttCaggggt-5'        3'-ttGCttTaggggt-5'

FRpairs — Restrict to Defined Primer Pairs

When enabled, only defined forward/reverse primer pairs are analyzed. Each line in the primer file defines one pair (tab-separated):

1020	aggcctgtgatgctgatgat	cccaacaccaaagaggaaag
1021	gctctgacctattgtctgtctgtct	cagtctccacagcagcagag
1022	gtcctgctgacacacaccact	cgcaggagtagaagaaaga

A single forward primer can be paired with multiple reverse primers (and vice versa).

ShowPrimerAlignment — Display All Binding Sites

When true (default), displays all stable primer-to-target alignments, including sites that may not produce PCR products under current conditions. Useful for examining binding site stability, orientation, and coordinates.

ShowPrimerAlignmentPCRproduct — Alignments for PCR Products Only

When true, restricts alignment output to only those primer binding sites that contribute to predicted PCR products.

ShowPCRProducts — PCR Product Prediction

When true (default), outputs predicted PCR products. Set to false to search for primer binding sites only, without amplicon prediction.

ShowOnlyAmplicons — Amplicon Lengths Only

When true, outputs only amplicon lengths without detailed alignment analysis. Recommended for genome-wide in silico PCR with highly abundant repeat-based markers (iPBS, IRAP, ISSR, RAPD).

LinkedSearch — Linked/Associated Search

A programmable search mode where binding sites are found for linked sequences within a specified distance. Linked searching supports tasks ranging from conventional sequence matching to in silico PCR with approximate matching.

Syntax: The forward primer sequence is followed by the expected distance [min-max] and the second sequence:

>RT+(QMDVK)_RT-(YVDDML)
CARATGGAYGTNAARAC[200-300]TAYGTNGAYGAYATG

Variants:

# Fixed distance (no range)
CARATGGAYGTNAARAC[300]TAYGTNGAYGAYATG

# Force second sequence to be reverse-complemented (@ prefix)
CARATGGAYGTNAARAC[300]@CATRTCRTCNACRTA

number3errors — 3' Mismatch Tolerance

Number of allowed mismatches at the 3' end of the primer.

Input Formats

Target Sequences

Sequence files can be plain text, FASTA, or multi-entry FASTA. No file extension is required. Template length is not limited.

FASTA format consists of a header line starting with > followed by one or more lines of sequence:

>sequence_id Optional description
ATCGATCGATCGATCGATCG...

Primer/Probe Files

Primers can be provided in tab-delimited or FASTA format. The software reads the first two columns (name and sequence) or three/four columns for primer-probe sets.

Tab-delimited format:

ITS1	TCCGTAGGTGAACCTGCGG
ITS2	GCTGCGTTCTTCATCGATGC
ITS3	GCATCGATGAAGAACGCAGC
ITS4	TCCTCCGCTTATTGATATGC

FASTA format:

>ITS1
TCCGTAGGTGAACCTGCGG
>ITS2
GCTGCGTTCTTCATCGATGC
>ITS3
GCATCGATGAAGAACGCAGC
>ITS4
TCCTCCGCTTATTGATATGC

Primer names can contain any characters (including spaces). Names may be identical across entries. Sequences must not contain spaces or non-nucleotide characters.

Supported Nucleotide Codes

Code Bases Code Bases
A Adenine M A/C
T Thymine R A/G
G Guanine W A/T
C Cytosine S G/C
U Uracil Y C/T
I Inosine K G/T
N A/G/C/T V A/G/C
H A/C/T
D A/G/T
B C/G/T

LNA (Locked Nucleic Acid) codes: dA=E, dC=F, dG=J, dT=L

Output

Results are saved as tab-delimited plain text files, containing (depending on options enabled):

  • Primer binding site coordinates and orientations
  • Primer-target alignment details and mismatch analysis
  • Predicted PCR product sizes
  • Extracted amplicon sequences

Author & Contact

Ruslan Kalendar 📧 ruslan.kalendar@helsinki.fi

Online Version: https://primerdigital.com/tools/epcr.html