Display of SNPs which can be used to differentiate between groups.

<p>SNPs which can be used to differentiate between the defined groups must be perfectly conserved within each group (green column in the group’s consensus graph) and must differ between the groups (orange or red column in the global consensus graph at the top). These positions can be automatically identified and are marked by red columns in the alignment.</p

Principle of multiplex pyrosequencing.

<p>In multiplex pyrosequencing, several primers are used simultaneously in the sequencing reaction so that their signals overlap. A1: In this example, primer 1 (upper part) reads the sequence TTAACCT and primer 1 (middle part) reads the sequence CGCCGTC. Since the signals overlap, the fingerprint (lower part) represents the sequence TTCAAGCCCCGTTC. It is important to note that in this fingerprint, it is not possible to tell which base was read by which primer. A2: The T→C mutation after primer 1 and the C→T mutation after primer 2 are used as targets for differentiating between two species. However, they cancel each other out, causing the fingerprints for A1 and A2 to be identical. B: Moving primer 1 one base to the left alleviates this problem: the fingerprints for B1 and B2 are now different. This demonstrates the importance of correct pyrosequencing primer positioning relative to all utilized SNPs.</p

Design of multiplex pyrosequencing assay with display of predicted pyrograms.

<p>Display of an alignment with two pyrosequencing primers, one of which is visible on the screen (green annotation), and four PCR primers, one of which is also visible (blue annotation). For each primer, the melting temperature is displayed at the 5′ end and the length is displayed at the 3′ end. A line connects the forward PCR primer with its reverse counterpart (not visible, offscreen). The product size is shown in the middle of the connecting line, and the red color warns of a high difference in predicted melting temperature. In subfigure A, the predicted pyrograms from the two pyrosequencing primers are shown for each group – with just 5 cycles of the pyrosequencing machine, a unique pyrogram can be obtained for each of the groups. In subfigure B, the pyrosequencing primer has been moved one base to the left, thus preventing sequencing of one SNP. This leads to the predicted pyrograms for advE and advB being identical.</p

Analysis of putative OPV mature virion-associated cellular proteins.

<p>The distribution of the protein copy numbers of viral and human proteins is shown in A. In order to discriminate between putative MV-associated human proteins and contaminants, the protein abundances within the MV preparations and the abundances in HepG2 cells were used for 1D and 2D annotation enrichment analysis. For the 2D analysis, normalized iBAQ values of the OPV MV and HepG2 cells were used. The score distribution of enriched GO terms with a FDR below 0.01 is plotted in C. Terms along the diagonal (black) are enriched or depleted in the MV and the cells in the same way and represent cellular background. The GO terms on the right side of the diagonal are enriched in the MV relative to the cell, and therefore represent putative MV-associated human proteins. If the proteins behind the GO terms are associated with increasing abundance in the OPV MV the terms are labelled blue, if they are associated with increasing abundance in the MV and associated with low abundance in the cells the terms are labelled red and if they are associated with low abundance in the cells the terms are labelled green. For 1D annotation enrichment analysis, iBAQ ratios for the human proteins in the MV preparations relative to the abundance in HepG2 cells were calculated (B). The score distribution of GO terms enriched in the MV preparations with a FDR below 0.01 is shown in D, redundant terms were removed from the Fig but can be found in Table L in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141527#pone.0141527.s003" target="_blank">S1 File</a>.</p

Hierarchical clustering and PCA of viral protein copy numbers.

<p>The protein copy numbers of all OPV proteins without missing values were used for hierarchical clustering (A) and principal component analysis (PCA) (B) of the mature virion samples. The biological replicates of each strain cluster together in both analysis and the OPV species, CPXV and VACV, can be discriminated by the mature virion proteome composition. Species-specific proteins were identified from the quantitative comparison using ANOVA tests with a permutation based FDR of 0.01 (Table F-G in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141527#pone.0141527.s003" target="_blank">S1 File</a>). The main proteins for species classification in the PCA, CPXV-GRI A27, A59 and D1 (C), were among these proteins with significant differences in MV abundance.</p

MultiPSQ: A Software Solution for the Analysis of Diagnostic n-Plexed Pyrosequencing Reactions

<div><p>Background</p><p>Pyrosequencing can be applied for Single-Nucleotide-Polymorphism (SNP)-based pathogen typing or for providing sequence information of short DNA stretches. However, for some pathogens molecular typing cannot be performed relying on a single SNP or short sequence stretch, necessitating the consideration of several genomic regions. A promising rapid approach is the simultaneous application of multiple sequencing primers, called multiplex pyrosequencing. These primers generate a fingerprint-pyrogram which is constituted by the sum of all individual pyrograms originating from each primer used.</p> <p>Methods</p><p>To improve pyrosequencing-based pathogen typing, we have developed the software tool MultiPSQ that expedites the analysis and evaluation of multiplex-pyrograms. As a proof of concept, a multiplex pyrosequencing assay for the typing of orthopoxviruses was developed to analyse clinical samples diagnosed in the German Consultant Laboratory for Poxviruses.</p> <p>Results</p><p>The software tool MultiPSQ enabled the analysis of multiplex-pyrograms originating from various pyrosequencing primers. Thus several target regions can be used for pathogen typing based on pyrosequencing. As shown with a proof of concept assay, SNPs present in different orthopoxvirus strains could be identified correctly with two primers by MultiPSQ.</p> <p>Conclusions</p><p>Software currently available is restricted to a fixed number of SNPs and sequencing primers, severely limiting the usefulness of this technique. In contrast, our novel software MultiPSQ allows analysis of data from multiplex pyrosequencing assays that contain any number of sequencing primers covering any number of polymorphisms.</p> </div

GO term distribution of strain- and species-specific OPV proteins.

<p>GO term distribution of strain- and species-specific OPV proteins.</p

Strain-specific OPV mature virion proteins.

<p>The proteome composition of the mature virions was analyzed for strain-specific proteins using ANOVA tests with a permutation based FDR of 0.01 separately for the LFQ intensities and the normalized protein copy numbers. The abundance of strain-specific proteins identified by LFQ-based quantification is displayed in heatmap A, while strain-specific proteins according to protein copy numbers are shown in heatmap B. Red displays proteins whose abundance is above the mean protein abundance of the OPV homologues, green means abundance is below the mean and grey are missing values in the copy number approach. The proteins are named after the CPXV-GRI 90 homologue.</p

Common VACV and CPXV mature virion proteome.

<p>The common VACV and CPXV mature virion proteome consists of 133 proteins. The gene ontology terms of these proteins are grouped according to their frequency (A). The relative amount of protein copy numbers of the ten most abundant viral proteins named after their CPXV GRI-90 homologue is shown in (B). The protein copy numbers of the common proteins are plotted on a logarithmic scale (basis = 2) against the gene position within the genome of the reference sequence CPXV strain GRI-90 (C). Below the x-axis, the distribution of essential (green), conserved (blue) and host-virus interactors (orange, host range factors are black) as well as uncharacterized proteins (red) within the linear refernce genome is displayed.</p

Number of identified proteins.

<p>Number of identified proteins.</p