primerJinn 2.0.3

In silico PCR tool

PrimerJinn

primerJinn has two main functions: it designs primers for multiplex PCR of given target regions in a DNA sequence (FASTA file) and performs in silico PCR given a list of primers and a reference FASTA file.

Features

• Multiplex Primer Design: Design optimal primer sets for multiple target regions
• In Silico PCR: Simulate PCR reactions with your primers against a reference sequence
• Primer Interaction Analysis: Check for primer dimers and cross-reactions
• Q5 Polymerase Support: Optimized settings for NEB Q5 hotstart polymerase
• Illumina Compatibility: Option to add Illumina partial adapters
• Comprehensive Output: Excel workbooks with detailed primer information and interactions

Installation

pip install primerJinn

System Requirements

• Python 3.6+
• BLAST+

Usage

1. Multiplex Primer Design

getMultiPrimerSet \
    --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 \
    --output "example" \
    --output_fasta \
    --exclude_primers

2. In Silico PCR

PCRinSilico \
   --primer_seq ./example/primers.txt \
   --target_tm 50 \
   --input_file ./example/ref.fasta \
   --output "in_silico_PCR" \
   --output_fasta \
   --exclude_primers

Parameters

Common Parameters (Both Tools)

Parameter	Required	Description	Default
--input_file	Yes	Reference FASTA file path	NA
--target_tm	No	Target melting temperature (°C)	60
--salt_concentration	No	Salt concentration (nM, ignored if Q5=True)	50
--output	No	Output file prefix	'MultiPlexPrimerSet' or 'in_silico_PCR'
--Q5	No	Use Q5 polymerase settings	False
--output_fasta	No	Output amplicon sequences in FASTA format	False
--exclude_primers	No	Exclude primer sequences from FASTA output	False

Primer Design Specific Parameters

Parameter	Required	Description	Default
--region_file	Yes	TSV/XLSX file with regions (name, start, end)	NA
--primer_len	No	Primer length	20
--product_size_min	No	Minimum amplicon size	400
--product_size_max	No	Maximum amplicon size	800
--ret	No	Maximum primer pairs to return	100
--background	No	Mispriming library FASTA	None
--ill_adapt	No	Add Illumina partial adapters	False
--clamp	No	Require GC clamp	0
--poly	No	Max mononucleotide repeat length	3
--no_self_background	No	Skip self-mispriming check	False

In Silico PCR Specific Parameters

Parameter	Required	Description	Default
--primer_seq	Yes	File with primer sequences	NA
--product_size_max	No	Maximum amplicon size	2000
--req_five	No	Require 5' end binding	True
--output_fasta	No	Output amplicons in FASTA	False
--exclude_primers	No	Exclude primers from FASTA output	False

Output Files

Multiplex Primer Design

• MultiPlexPrimerSet.xlsx: Contains primer pairs, Tm values, and product sizes
• MultiPlexPrimerSet_amplicons.fasta: (Optional) FASTA file of amplicon sequences

In Silico PCR

• in_silico_PCR.xlsx: Excel workbook with multiple sheets:
  • amplicons: Predicted PCR products
  • dimers: Potential primer-dimer interactions
  • interactions: Cross-reactions between amplicons
• in_silico_PCR_amplicons.fasta: (Optional) FASTA file of predicted amplicons

The FASTA output files contain:

• Headers in format: >target_name_start_end_length
• Sequences either:
  • Include primers (default)
  • Exclude primers (with --exclude_primers)

Testing Recommendations

When validating primers for diagnostic assays:

1. Perform qPCR with individual primer pairs
2. Use EVA Green plus (20X in water) and ROX (50X)
3. Analyze melt curves
4. Confirm single bands via agarose gel electrophoresis

Online Version

A web version of primerJinn's getMultiPrimerSet is available at DrDx.Me

Citation

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

Author

Jason Limberis ([e-mail])

License

This project is licensed under the MIT License.