60-61 / 134
Draft, Do Not Distribute
Annex 1: Bioinformatics Analyses of Signature Sequences Underlying
Venezuelan Equine Encephalitis Virus
Assays
226
227
In silico
screening will be performed on signature sequences (e.g., oligo primers/probes) to predict specificity to
Variola virus
and inclusivity across all
228
sequenced
Variola virus
strains.
229
230
In silico
results are suggestive of potential performance issues, so will guide necessary additions to the wet screening panels.
In silico
identification of
231
potential cross-reactions (false positives) or non-verifications (false negatives) would identify the relevant strains to be included in the exclusivity or
232
inclusivity panels, respectively, if available.
233
234
A method developer-selected tool to carry out the bioinformatics evaluation should be able to predict hybridization events between signature
235
components and a sequence in a database including available genomic sequence data, using public Genbank nucleotide
236
[http://www.ncbi.nlm.nih.gov/genbank/]. The selected tool should be able to identify predicted hybridization events based on platform annealing
237
temperatures, thus ensuring an accurate degree of allowed mismatch is incorporated in predictions. The program should detect possible amplicons from
238
any selected database of sequence.
239
240
Potential tools for
in silico
screening of real-time PCR signatures include:
241
242
•
Simulate_PCR:
http://sourceforge.net/projects/simulatepcr/files/?source=navbar243
o
This program will find all possible amplicons and real time fluorescing events from any selected database of sequence.
244
245
•
NCBI Tools:
246
247
•
FastPCR:
http://primerdigital.com/fastpcr.html248
249
The method developer submission should include:
250
251
•
Description of sequence databases used in the
in silico
analysis
252
•
Description of tool used for bioinformatics evaluation
253
o
Data demonstrating the selected tool successfully predicts specificity that has been confirmed by wet-lab testing on designated isolates
254
This data can be generated retrospectively using published assays
255
•
List of additional strains to be added to the inclusivity or exclusivity panels based on the bioinformatics evaluation
256
14 Draft EEVs SMPR V4