This pyrogram cannot be used directly for sequence analysis as it is unknown from which of the sequencing reactions the respective light signal originates. However, the resulting single pyrogram can be used as a unique fingerprint which is representative of a specific combination of sequences. Using software currently commercially intracellular pcb distribution investigated primary adipocytes unilocular lipid droplets available, analysis of multiplex pyrograms is still challenging. Therefore, an in-house software tool called MultiPSQ was developed to analyse and evaluate multiplex pyrosequencing results. To verify 
this software, we developed a multiplex pyrosequencing assay identifying all human-pathogenic orthopoxviruses. In addition to the causative agent of smallpox, variola virus, the genus OPV comprises diverse species including those transmitted zoonotically and causing infections in humans, like monkeypox virus, cowpox virus and vaccinia virus. Further OPV that do not infect humans are camelpox virus, mousepox virus, raccoonpox virus and taterapox virus as well as various unclassified OPV. As genomic sequences are well conserved among OPV, virus type differentiation by only sequence variations or SNPs within a single stretch of sequence is often impossible. This makes OPV a good candidate for the demonstration of the utility of multiplex pyrosequencing and our tool’s capabilities. Critical steps in designing a multiplex pyrosequencing assay were the identification of suitable sequence variations and the respective selection of sequencing primers in combination with an optimal nucleotide dispensation order. Suitable SNPs could be identified by the following strategy: First, all relevant known sequences were aligned. Then the sequences were grouped by species. Potentially interesting SNPs could be found at those positions in the alignment which are fully conserved within each of the individual groups but not fully conserved across the groups to be differentiated. Ideally, regions in the alignment should be identified which contain several such representative SNPs within a stretch of only a few bases, as several SNPs could then be sequenced with a single primer. In contrast to laborious and time-consuming Sanger sequencing which is usually done to identify and type pathogens following PCR, pyrosequencing represents a fast and reliable alternative method. It allows sequence generation and pathogen identification in less than one hour from preparation of PCR products to final sequence. However, due to the underlying mechanism of sequencing-by-synthesis, sequences generated by pyrosequencing are usually short. Therefore, discrimination of more than two closely related organisms that differ only in a few but non-conserved SNPs is not possible with conventional pyrosequencing. To circumvent this problem, the generation of sequences starting from more than one primer leads to specific sequence fingerprints, enabling pathogen typing despite non-conserved SNPs that are representative for a respective group. Recently, such a multiplex pyrosequencing assay has been developed e.g. for genotyping hepatitis C virus.