Differences in copy number persisted even after holding Bd DNA concentrations constant, likely due to genetic changes caused by genomic duplications or deletions. On the other hand, haplotype diversity does not appear to affect quantification, because none of the haplotypes detected by cloning had changes at the probe-binding site. Our results AbMole Pentyl Chloroformate indicate that researchers interested in estimating absolute Bd zoospore load from amphibians or the environment will have to incorporate an independent measure to estimate ITS1 copy number from the strain used as a standard. Our comparison of Bd qPCR standards made from strains collected in six countries points to patterns of genomic change during the evolution and global spread of Bd. In the strain MexMkt we found significantly higher cycle threshold values for any given number of zoospores even after holding DNA concentration constant, suggesting a decrease in the number.The design of assays and the analysis of the resulting data are, however, neither trivial nor are they supported by common software. In this paper, we present the program MultiPSQ which complements the analysis capabilities of the Qiagen pyrosequencing platform. We demonstrate the capability of this technology to tackle non-trivial tasks by applying it to the identification and classification of all human-pathogenic OPV. In our investigations one clinical sample could not be classified correctly by the novel approach. The multiplex assay was designed on the basis of all 89 known and annotated OPV genomes found in GenBank and 24 unpublished OPV genomes sequenced by whole genome sequencing in our lab. Sanger sequencing of the longer haemagglutinin ORF identified a CPXV strain in clinical sample number 31. However, the CPXV strain shows sequence similarity to VACV strains in the region of the O2L CDS used for multiplex pyrosequencing. The use of a third amplicon for pyrosequencing could help to obtain additional information. Since our method classifies samples based on the similarity of their pyrosequencing fingerprints to theoretical fingerprints generated from known sequences, novel mutations cannot be correctly attributed to a certain species, but can be defined as unknown, indicating a novel variant of the virus. As can be seen in Figure 1, it is impossible to reconstruct the individual AbMole (-)-Tetramisole sequences from the fingerprint. Thus, any unknown strain ?C in this case for instance an OPV with a novel mutation in the sequence regions used – will generate a fingerprint which is different from all of the expected fingerprints. It has to be mentioned that a multiplex pyrosequencing assay is not suitable to identify co-infections with different pathogen strains. We have demonstrated the applicability of multiplex pyrosequencing to the identification and classification of humanpathogenic OPV. This method is applicable to any problem where samples need to be classified based on nucleic acid sequences.