title: 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 sufficient 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.
authors.affiliation: ZBS 1: Highly Pathogenic Viruses/ZV4: Information Technology, Robert Koch Institute, Nordufer 20, D-13353, Berlin, Germany, dabrowskiw@rki.de.
authors.forename: Piotr Wojciech
authors.initials: PW
authors.lastname: Dabrowski
authors.forename: Kati
authors.initials: K
authors.lastname: Bourquain
authors.forename: Andreas
authors.initials: A
authors.lastname: Nitsche
mesh_terms: D053000:Analytic Sample Preparation Methods; D001483:Base Sequence; D019921:Biotinylation; D017931:DNA Primers / Q000235:genetics / Q000378:metabolism; D004587:Electrophoresis, Agar Gel; D023281:Genomics; D060005:Genotyping Techniques / Q000379:methods*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*; D013997:Time Factors
chemical_list: D017931:DNA Primers