Multiplex Pyrosequencing®: Simultaneous genotyping based on SNPs from distant genomic regions

Abstract

Pyrosequencing ® is a technique that allows the quick sequencing of short stretches of DNA, and results can be inspected in real time as they are generated. As such, it is a highly useful tool for the typing of organisms, based on single-nucleotide polymorphisms (SNPs). However, if a single polymorphism is not suffi - cient for typing and several distant genomic regions need to be examined, multiple Pyrosequencing reactions have to be performed for each organism. This strongly increases both workload and reagent costs. Alternatively, multiplex Pyrosequencing can be performed, in which the multiple sequencing reactions for all analyzed genomic regions are performed in a single reaction. However, when using this method, special care has to be taken while designing the assay and analyzing the results. Here we present a detailed protocol using our newly developed software mPSQed for assay design and MultiPSQ for data analysis.