Validation of TaqMan® SNP genotyping specificity for rs12979860 of IL-28B: Modeling primer specificity in vitro
Bibliographic record
Abstract
Members of the type III interferon gene family arose by gene duplication events and have retained a high percent identity both in their coding and non-coding regions. In this study, the specificity of a widely used TaqMan(®) SNP genotyping assay for rs12979860 is validated. The 66 bp template for SNP genotyping has only 3 bp at one 5' end that vary between IL-28B and IL-28A; excluding the rs12979860 SNP itself. Conflicting annealing temperatures were found for the mismatched 19 bp primer to IL-28B and IL-28A with in silico melting temperature algorithms, or with in vitro dissociation curves. In order to prove specificity for IL-28B, an in vitro competition assay was setup with genomic DNA and synthetic oligonucleotides. When genomic DNA, containing equimolar concentrations of rs12979860 and the homologous region of IL-28A are present, no off-target amplification was observed. This SNP genotyping assay is therefore specific for rs12979860 and all previously reported results are valid. Finally, using a completely synthetic in vitro competition assay it was possible to calculate the amount of off-target template that will produce 1/2 the maximum on-target (VIC) fluorescent signal, a value that is between a C/C genotype and a T/T genotype. This value is defined in the manuscript as the half maximum positive value, KHPV, and in the present assay KHPV is 15.75±0.0721, represented as the relative fold increase in the amount of IL-28A over rs12979860. This method will be of interest to those performing genotyping on highly conserved gene families.
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How this classification was reachedexpand
Full frame distilled prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. Learned from the 10,348 direct Codex labels and 10,348 direct Gemma labels. Candidate is the union of thresholded teacher heads; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels or direct frontier model labels.
Codex and Gemma teacher scores by category
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.004 | 0.002 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.001 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.000 |
| Insufficient payload (model declined to judge) | 0.000 | 0.000 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one teacher head, not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".