NGS-PrimerPlex

NGS-PrimerPlex designs primers for high-throughput multiplex PCR to support amplicon-based genome target enrichment and multiple PCR methodologies including nested and anchored multiplex PCR.

Key Features:

• Target specification: Allows specification of targets using gene lists or specific gene parts (e.g., exons, codon numbers) for automated primer design.
• Multiplex PCR support: Supports amplicon-based genome target enrichment strategies and multiple PCR methodologies, including nested and anchored multiplex PCR.
• Secondary structure avoidance: The algorithm considers and prevents primer secondary structures during design.
• Non-target amplicon avoidance: The algorithm avoids design of primer pairs that would generate non-target amplicons between primers.
• SNP overlap avoidance: The algorithm detects and avoids high-frequency single nucleotide polymorphisms (SNPs) overlapping primer sequences.

Scientific Applications:

• LRRK2 coding-region NGS panel: Used to develop an NGS-panel for sequencing LRRK2 coding regions, achieving a median coverage of 97.4% of target regions with at least 30 reads in a study of 354 DNA samples.

Methodology:

The algorithm evaluates primer secondary structures, avoids non-target amplicons between primers, and detects high-frequency SNPs overlapping primers; the implementation comprises four Python scripts executable as a Docker image.