SPADA Book - April 11, 2017
DRAFT ENVIRONMENTAL ORGANISMS PANEL, VERSION 5
2.2 73 74 In silico screening shall be performed on all nucleic acid signature sequences used in the assay (e.g., 75 primers, probes, amplicons, etc.) to demonstrate specificity to the target biological threat agent. 76 77 In silico results are suggestive of potential performance issues, so will guide necessary additions to the 78 wet screening panels. In silico identification of potential cross‐reactions (false positives) or non‐ 79 verifications (false negatives) would require the affected organism/strain be included in the exclusivity 80 or inclusivity panels, respectively, if the strains are available. 81 82 A method developer‐selected tool to carry out the bioinformatics evaluation should be able to predict 83 hybridization events between signature components and a sequence in a database including available 84 genomic sequence data, databases and/or published documents describing the genetic sequences found 85 in soils that are representative of the regions of operation. The selected tool should be able to identify 86 predicted hybridization events based on platform annealing temperatures, thus ensuring an accurate 87 degree of allowed mismatch is incorporated into predictions. The program should detect possible 88 amplicons from any selected database of sequences. 89 90 Potential tools for in silico screening of nucleic acid sequences include: 91 92 http://sourceforge.net/projects/simulatepcr/files/?source=navbar 93 o This program will find all possible amplicons and real time fluorescing events from any 94 Bioinformatics Analyses
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selected database of sequences.
NCBI tools
The method developer submission should include:
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Description of sequence databases used in the in silico analysis
Description of conditions used for in silico analysis
o Stringency of in silico analysis must match bench hybridization conditions
Description of the tool(s) used for bioinformatics evaluation
o Data demonstrating the selected tool(s) successfully predicts specificity that has been
confirmed by wet‐lab testing on designated isolates
These data can be generated retrospectively using published assays
List of additional organisms and/or strains to be added to the inclusivity (Annex II) or exclusivity
(Annex III) panels based on the bioinformatics evaluation
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