Location: Geospatial and Environmental Epidemiology Research Unit
Project Number: 6064-32000-001-009-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 15, 2024
End Date: Aug 14, 2026
Objective:
Veterinary Diagnostics Laboratories generally use sequence-independent, single-primer amplification (SIPSA) for most viral pathogen sequencing. SIPSA amplifies everything, meaning they can’t assemble low-level infections or pool many samples on the sequencer. Multiplex Polymerase Chain Reaction (PCR) amplification of viral fragments increases sensitivity and reduces sequencing costs, But the design of large numbers of amplification primers is difficult. For SARS-CoV-2, low-cost, multiplex PCR panels were designed that amplify long overlapping regions which are then amplicon or shotgun sequenced. This project will Develop software to easily develop and validate multiplex PCR primer sets from a small set of training genomes. The tool will increase the amount of data available for the molecular epidemiology of animal disease.
Approach:
Multiplex primers are normally designed manually, but recently an algorithm to optimize multiplex primer sets was published (Xie et al. 2022). The SADDLE algorithm takes a set of target points in the genome and designs primers that reduce dimerization and off-target binding. This algorithm could be used to write full multiplex genome design software. Complete design software would take a set of viral genomes and first align them. Then, conserved anchor regions would be identified and used as candidate regions to design multiplex primers using the SADDLE algorithm. The Cooperator will write software that takes a set of existing virus genomes and automatically designs a set of multiplex primers for sequencing a new virus.
