MultiPSQ: a software solution for the analysis of diagnostic n-plexed pyrosequencing reactions

Area of Science:

• Molecular Biology
• Bioinformatics
• Genomics

Background:

• Pyrosequencing is used for Single-Nucleotide-Polymorphism (SNP)-based pathogen typing and DNA sequencing.
• Complex pathogens require analysis of multiple genomic regions, which is challenging with standard pyrosequencing.
• Multiplex pyrosequencing, using multiple primers simultaneously, generates a combined pyrogram fingerprint.

Purpose of the Study:

• To develop a software tool, MultiPSQ, for improved analysis of multiplex pyrosequencing data.
• To demonstrate the utility of MultiPSQ for pathogen typing using a proof-of-concept orthopoxvirus assay.

Main Methods:

• Development of the MultiPSQ software for analyzing multiplex pyrosequencing data.
• Creation of a multiplex pyrosequencing assay for orthopoxvirus typing.
• Analysis of clinical samples diagnosed at the German Consultant Laboratory for Poxviruses.

Main Results:

• The MultiPSQ software successfully analyzed multiplex pyrograms from multiple pyrosequencing primers.
• The software enabled the identification of SNPs in different orthopoxvirus strains using a two-primer assay.
• MultiPSQ demonstrated the feasibility of using several genomic regions for pyrosequencing-based pathogen typing.

Conclusions:

• Existing software for pyrosequencing is limited by the number of SNPs and primers.
• The novel MultiPSQ software overcomes these limitations, allowing analysis of multiplex pyrosequencing assays with any number of primers and polymorphisms.
• MultiPSQ significantly enhances the utility of multiplex pyrosequencing for molecular typing of pathogens.