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mPSQed: a software for the design of multiplex pyrosequencing assays.

Dabrowski PW, Nitsche A - PLoS ONE (2012)

Bottom Line: While PCR can be designed to be specific for a certain pathogen, a subsequent sequence analysis is frequently required for confirmation or typing.One extremely useful but underused method for this purpose is the multiplex pyrosequencing technique.Here, we present mPSQed (Multiplex PyroSeQuencing EDitor), a program targeted at closing this gap.

View Article: PubMed Central - PubMed

Affiliation: Central Administration 4, IT, Robert Koch Institute, Berlin, Germany. DabrowskiW@rki.de

ABSTRACT
Molecular-based diagnostic assays are the gold standard for infectious diseases today, since they allow a rapid and sensitive identification and typing of various pathogens. While PCR can be designed to be specific for a certain pathogen, a subsequent sequence analysis is frequently required for confirmation or typing. The design of appropriate PCR-based assays is a complex task, especially when conserved discriminating polymorphisms are rare or if the number of types which need to be differentiated is high. One extremely useful but underused method for this purpose is the multiplex pyrosequencing technique. Unfortunately there is no software available to aid researchers in designing multiplex pyrosequencing assays. Here, we present mPSQed (Multiplex PyroSeQuencing EDitor), a program targeted at closing this gap. We also present the design of an exemplarily theoretical assay for the differentiation of human adenovirus types A-F using two pyrosequencing primers on two distinct PCR products, designed quickly and easily using our software.

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Display of SNPs which can be used to differentiate between groups.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.
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pone-0038140-g003: Display of SNPs which can be used to differentiate between groups.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.

Mentions: The identification of discriminating SNPs is an important step in the creation of diagnostic assays. To facilitate the selection of a set of discriminative SNPs several steps are required to be performed. After an alignment containing all relevant sequences has been loaded into the program the sequences can be grouped, and consensus sequences can be calculated both for the alignment globally and for each group individually. Groups can be collapsed, leaving only the conservation graph and the consensus sequence visible. This allows the user to work with a significantly reduced amount of visible data while still retaining easy access to all relevant information (see Figure 2). SNPs which are conserved within a single group and are thus candidates for use in a differentiation assay can be automatically detected and highlighted (see Figure 3). Some basic primer design functionality (such as Tm calculation, product size calculation, degeneration etc.) is supported. Novel functionality is provided for the design of multiplex pyrosequencing assays. Primers can be marked as pyrosequencing primers and the predicted pyrograms which would be generated using these primers in a multiplex pyrosequencing assay can be displayed. When pyrosequencing primers are moved, the predicted pyrograms are updated in realtime, allowing for a quick optimisation of primer positioning. If the expected pyrogram is not unique for each of the defined groups, a warning is displayed (see Figure 4).


mPSQed: a software for the design of multiplex pyrosequencing assays.

Dabrowski PW, Nitsche A - PLoS ONE (2012)

Display of SNPs which can be used to differentiate between groups.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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366982&req=5

pone-0038140-g003: Display of SNPs which can be used to differentiate between groups.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.
Mentions: The identification of discriminating SNPs is an important step in the creation of diagnostic assays. To facilitate the selection of a set of discriminative SNPs several steps are required to be performed. After an alignment containing all relevant sequences has been loaded into the program the sequences can be grouped, and consensus sequences can be calculated both for the alignment globally and for each group individually. Groups can be collapsed, leaving only the conservation graph and the consensus sequence visible. This allows the user to work with a significantly reduced amount of visible data while still retaining easy access to all relevant information (see Figure 2). SNPs which are conserved within a single group and are thus candidates for use in a differentiation assay can be automatically detected and highlighted (see Figure 3). Some basic primer design functionality (such as Tm calculation, product size calculation, degeneration etc.) is supported. Novel functionality is provided for the design of multiplex pyrosequencing assays. Primers can be marked as pyrosequencing primers and the predicted pyrograms which would be generated using these primers in a multiplex pyrosequencing assay can be displayed. When pyrosequencing primers are moved, the predicted pyrograms are updated in realtime, allowing for a quick optimisation of primer positioning. If the expected pyrogram is not unique for each of the defined groups, a warning is displayed (see Figure 4).

Bottom Line: While PCR can be designed to be specific for a certain pathogen, a subsequent sequence analysis is frequently required for confirmation or typing.One extremely useful but underused method for this purpose is the multiplex pyrosequencing technique.Here, we present mPSQed (Multiplex PyroSeQuencing EDitor), a program targeted at closing this gap.

View Article: PubMed Central - PubMed

Affiliation: Central Administration 4, IT, Robert Koch Institute, Berlin, Germany. DabrowskiW@rki.de

ABSTRACT
Molecular-based diagnostic assays are the gold standard for infectious diseases today, since they allow a rapid and sensitive identification and typing of various pathogens. While PCR can be designed to be specific for a certain pathogen, a subsequent sequence analysis is frequently required for confirmation or typing. The design of appropriate PCR-based assays is a complex task, especially when conserved discriminating polymorphisms are rare or if the number of types which need to be differentiated is high. One extremely useful but underused method for this purpose is the multiplex pyrosequencing technique. Unfortunately there is no software available to aid researchers in designing multiplex pyrosequencing assays. Here, we present mPSQed (Multiplex PyroSeQuencing EDitor), a program targeted at closing this gap. We also present the design of an exemplarily theoretical assay for the differentiation of human adenovirus types A-F using two pyrosequencing primers on two distinct PCR products, designed quickly and easily using our software.

Show MeSH
Related in: MedlinePlus