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Draft, Do Not Distribute
Annex V: Bioinformatics Analyses of Signature Sequences Underlying
Variola virus
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Assays
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In silico
screening will be performed on signature sequences (e.g., oligo primers/probes) to
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predict specificity to
Variola virus
and inclusivity across all sequenced
Variola virus
strains.
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In silico
results are suggestive of potential performance issues, so will guide necessary additions
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to the wet screening panels.
In silico
identification of potential cross-reactions (false positives)
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or non-verifications (false negatives) would identify the relevant strains to be included in the
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exclusivity or inclusivity panels, respectively, if available.
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A method developer-selected tool to carry out the bioinformatics evaluation should be able to
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predict hybridization events between signature components and a sequence in a database
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including available genomic sequence data, using public Genbank nucleotide
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[
http://www.ncbi.nlm.nih.gov/genbank/]. The selected tool should be able to identify predicted
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hybridization events based on platform annealing temperatures, thus ensuring an accurate
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degree of allowed mismatch is incorporated in predictions. The program should detect possible
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amplicons from any selected database of sequence.
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Potential tools for
in silico
screening of real-time PCR signatures include:
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•
Simulate_PCR:
http://sourceforge.net/projects/simulatepcr/files/?source=navbar307
o
This program will find all possible amplicons and real time fluorescing
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events from any selected database of sequence.
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•
NCBI Tools:
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•
FastPCR:
http://primerdigital.com/fastpcr.html313
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The method developer submission should include:
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•
Description of sequence databases used in the
in silico
analysis
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•
Description of tool used for bioinformatics evaluation
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o
Data demonstrating the selected tool successfully predicts specificity that has
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been confirmed by wet-lab testing on designated isolates
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This data can be generated retrospectively using published assays
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•
List of additional strains to be added to the inclusivity or exclusivity panels based on the
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bioinformatics evaluation
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11 Approved
Variola
SMPR v7.5