Open-MSSPE-Design

Open-MSSPE-Design is a Rust-based pipeline for designing primers for Metagenomic Sequencing with Spiked Primer Enrichment (MSSPE). This approach supports viral diagnostics and genomic surveillance by enriching viral sequences during sequencing, as described in Deng et al. (2020), and openly implemented in nf-msspe by Simon Maestri. This implementation introduces significant revisions to optimize and automate the primer design process.

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Overview

Key features:

• Fully automated primer design workflow.
• Fully customizable kmer selection process.
• Uses nearest-neighbor thermodynamic models from the standalone Primer3 package ntthal
• Enhanced filtering for:
  • nucleotide repeats & homopolymers
  • specific minimum and maximum temperature of melting (tm) values
  • strict tm value ranges (within 2 standard deviations of mean)
  • hairpins (with same primer)
  • cross-dimers (across multiple primers/entire pool)
  • check strength of secondary structures via DeltaG calculations
• Rust implementation for performance and reliability.

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Getting Started

Prerequisites

• Rust (latest stable version)
• Cargo (Rust package manager)
• mafft for multiple sequence alignment
• Primer3 for designing PCR primers.

Installation

Clone the repository and navigate to the project directory:

git clone https://github.com/opendream/open-msspe-design.git
cd open-msspe-design/od-msspe

Install dependecies:

macOS

brew install rust
brew install mafft
brew install primer3

Input Requirements

Prepare a FASTA file containing viral genome sequences. This will serve as the input for primer design.

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Usage

Build the pipeline:

cargo build --release

To run the pipeline:

./target/release/od-msspe

OR build & run:

cargo run

Required Config

• --input: Path to the input FASTA file containing viral genome sequences.
• --output: Directory where the designed primers will be saved.

Optional Config

The following arguments control various aspects of the primer design process:

Primer Design Parameters

• --kmer-size: Size of k-mers used in primer design (default: 13).
• --window-size: Window size for genome scanning (default: 500).
• --overlap-size: Overlap size between adjacent windows (default: 250).
• --max-mismatch-segments: Maximum number of mismatched segments allowed (default: 1).
• --max-iterations: Maximum number of iterations for primer optimization (default: 1000).
• --search-windows-size: Size of search windows for primer candidates (default: 50).

Thermodynamic Parameters

• --mv-conc: Monovalent cation concentration in mM (default: 50.0).
• --dv-conc: Divalent cation concentration in mM (default: 3.0).
• --dntp-conc: dNTP concentration in mM (default: 0.0).
• --dna-conc: Primer concentration in nM (default: 250.0).
• --annealing-temp: Annealing temperature in °C (default: 25.0).

Temperature Thresholds

• --min-tm: Minimum melting temperature allowed (default: 30.0).
• --max-tm: Maximum melting temperature allowed (default: 60.0).
• --tm-stddev: Set the number of standard deviations away from the mean of the tm values (default: 2).
• --max-self-dimer-any-tm: Maximum Tm for self-dimer at any position (default: 10°C below max-tm).
• --max-self-dimer-end-tm: Maximum Tm for self-dimer at 3' end (default: 10°C below max-tm).
• --max-hairpin-tm: Maximum Tm for hairpin structures (default: 10°C below min-tm).
• --max-delta-g: Maximum delta G value for secondary structures (default: -9).

Boolean Flags

• --keep-all: Ignore all filtering criteria and keep all primers.
• --check-cross-dimers: Enable cross-dimer checking between all primer pairs.
• --check-self-dimer: Enable self-dimer checking for individual primers.
• --check-hairpin: Enable hairpin structure checking for individual primers.
• --disable-tm-stddev: Turns off tm-stddev config. Use if you do not want strictly similar tm values across all primers.
• --do-align: Perform MAFFT multiple sequence alignment if true. Set to false if sequence already aligned.

Example

cargo run -- --input data/viral_genomes.fasta --output results/msspe_primers.csv --kmer-size=15

Debugging

RUST_LOG=info cargo run -- --input data/viral_genomes.fasta --output results/msspe_primers.csv

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Contributions

Contributions, issues, and feature requests are welcome! Please open an issue or submit a pull request to improve the project.

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License

This project is licensed under the MIT License.

Maintainers of this project are grateful for the support of the Skoll Foundation.