ProbePy

Fluorescent in situ Hybridization Probe Design Tool

A comprehensive Python package for designing custom fluorescent in situ hybridization (FISH) probes.

Features

• Multi-species support: Works with any organism with genome and transcriptome annotations
• Compatible probe design: Probes are compatible with Molecular Instruments HCR v3.0 B-system amplification
• Facilitates Multiplexing: Probes can be ordered with B1-B5 amplifier recognition motifs
• BLAST-based specificity: Ensures unique binding by blasting against the transcriptome
• Comprehensive visualization: Generate publication-ready plots showing probe locations on gene structures
• Flexible output formats: Export probes in IDT-compatible Excel format for direct ordering

Installation

git clone https://github.com/Social-Evolution-and-Behavior/ProbePy.git
cd ProbePy 
poetry install

Requirements: Python 3.11+, Poetry, NCBI BLAST+ command line tools

Install NCBI BLAST+ tools

The package facilitates the installation of NCBI BLAST+ command line tools.

import probepy
probepy.install_blast_tools()

Alternatively, you can install it yourself:

• macOS: brew install blast
• Ubuntu/Debian: sudo apt-get install ncbi-blast+
• Windows: Download from NCBI BLAST+

Quick Start

1. Basic Probe Design Workflow

import probepy

# Check BLAST tools availability
probepy.check_blast_tools()

# Load transcriptome object (pre-built or create new)
transcriptome = probepy.load_transcriptome_object("species_transcriptome")

# Get gene of interest
gene = transcriptome.get_gene("Or9a")
transcript = gene.get_transcript_longest_cds()

# Design HCR probes
sequence = transcript.mrna_sequence
probes, regions, positions = probepy.design_hcr_probes(sequence, "B3")

print(f"Designed {len(probes)} probe pairs for {gene.name}")

2. Complete Pipeline Example

Visit the docs for a complete walkthrough including:

• Downloading data with rsync
• Building BLAST databases
• Transcriptome loading and gene selection
• BLAST-based specificity checking
• Probe design and filtering
• Visualization and export

Probe Design Algorithm

The probe design follows these key principles:

1. Target sequence processing: Uses mature mRNA (exons only, no introns)
2. Probe pair generation: Designs complementary 25-nt probe pairs with optimal spacing
3. Quality filtering:
   • GC content between 25-75%
   • No homopolymer runs >4 bases
   • No gaps from unique region masking
4. Specificity verification: BLAST against transcriptome with/without introns
5. Visualization: Maps probe locations to genomic coordinates

Output Formats

IDT Order Sheets

• Excel format compatible with IDT oPools bulk ordering
• Includes pool names and probe sequences
• Ready for direct upload to IDT website

Probe Binding Regions

• Detailed information about where each probe binds
• Useful for troubleshooting and analysis

Visualization

• Gene structure plots with probe locations

Acknowledgments

• HCR v3.0 system developed by Molecular Instruments
• Built with Python scientific computing stack (NumPy, Pandas, Matplotlib)
• BLAST integration via NCBI BLAST+ suite
• Visualization powered by pygenomeviz