SPADA Meeting Book

all of the desired target organism sequences (i.e. inclusivity panel) and does not detect most or all 956 non-target sequences (exclusivity panel, near neighbors, and environmental panel). This 957 definition deviates from the traditional definition of a signature as a single oligonucleotide 958 sequence that is “present” in all targets and “absent” in all non-targets. This traditional definition 959 is problematic due to the various definitions of “presence” and “absence” depending on the 960 threshold parameters set for example, 1) Edit distance; 2) BLAST alignment score; 3) Melting 961 temperature or delta G; 4) Impact of 3’ terminal primer mismatches. 962 ( hh ) Signature erosion .—Emergence of mutations in the sequences of the signature (primers 963 and probes)that may lead to assay failure. This may happen via the natural course of evolution, 964 especially in viruses, due to genetic drift or shift or deliberate acts. 965 ( ii ) Specificity .—The true negative rate is the fraction of tests that are true negative divided 966 by the actual number of negative samples (i.e., true negative plus false positive). Assays with 967 high specificity have a low occurrence of false positives. The equation for specificity is given by: 968

Commented [LN(25]: It isn’t clear to me how this list ties  into the “presence” and “absence” – is it just a list of  example methods to quantify presence?  Please clarify  Commented [SS26R25]: Gans

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where TN = true negatives and FP = false positives.

( jj ) Target Selection .—Entails identifying suitable “unique regions” in the genome of interest

970 for assay design. These regions may be longer than the actual PCR amplicon. 971

( kk ) Test Portion .—A quantity of subsample or member of a sample set that is taken for

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973

analysis by the method.

( ll ) Test and evaluation (T&E) .—A process by which a system or components are tested and 974 results analyzed to provide performance related information. The results provide information to 975 identify risks, empirical data for validation, and assessment of technical performance, 976 specifications, system maturity, and suitability for intended use. 977

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