Melting Peak Specificity

Gate amplicon specificity by significant melting-curve peak count.

Description

Melting peaks can screen for multiple amplification products.

Melting peaks can screen for multiple amplification products. Significance here means height relative to the largest supplied peak, not a statistical test.^[1]

Gate amplicon specificity by significant melting-curve peak count.

When to use Melting Peak Specificity

Use this tool to gate amplicon specificity by significant melting-curve peak count.

Prepare the upstream counts, expression values, sequences or record lists requested by this tool. Keep corresponding features in the same order and confirm count units, transformations and caller-selected criteria.

How to use Melting Peak Specificity and understand its results

Prepare the upstream counts, expression values, sequences or record lists requested by this tool. Keep corresponding features in the same order and confirm count units, transformations and caller-selected criteria.
Enter peakTemperaturesC, peakHeights, minimumRelativePeakHeight using the field table and calculation rules, then run the tool.
Read significantPeakCount, primaryPeakC, specific. The explanation below defines the units, signs, ordering and any flags or placeholder values.
If the tool reports an input issue, correct the values and run it again. Apply the stated assumptions when interpreting a successfully returned result.

Input field	Meaning	Value type; see rules below	Requirement
peakTemperaturesC	Peak temperatures c	list of number	Required
peakHeights	Peak heights	list of number	Required
minimumRelativePeakHeight	Minimum relative peak height	number	Required

Enter aligned nonempty peak temperatures and nonnegative heights with at least one positive height, plus a relative cutoff in 0–1. Count inclusively qualifying peaks and report the first maximum-height peak temperature. Cutoff zero counts even zero-height supplied peaks.

significant if height≥maxHeight×minimumRelativeHeight; specific=(significantCount=1)

Units, indexing, boundary conditions and exceptional cases are defined in the calculation rules above.

Output field	Meaning	Value type
significantPeakCount	Significant peak count	number
primaryPeakC	Primary peak c	number
specific	Specific	boolean

json

{
  "input": {
    "peakTemperaturesC": [
      80,
      85,
      95
    ],
    "peakHeights": [
      5,
      100,
      4
    ],
    "minimumRelativePeakHeight": 0.1
  },
  "output": {
    "significantPeakCount": 1,
    "primaryPeakC": 85,
    "specific": true
  }
}

Limitations and assumptions

Peak detection and curve processing occur upstream. A single significant peak does not identify an amplicon or prove assay specificity, and tied primary maxima choose the first.

Numeric inputs must be finite. Follow the calculation-specific rules for overflow, underflow and exceptional cases. Lists are limited to 100,000 entries; the rules above may require smaller limits. Missing required fields, extra fields, wrong value types and violations of the calculation rules are input errors. A successfully returned false flag is an evaluated outcome; its meaning is explained above.

Alternative or Complementary methods

Use NTC Contamination Gate: Flag early no-template-control amplification against caller-supplied Ct limits. For a complementary task, use MIQE Coverage Audit: Audit caller-supplied MIQE reporting checklist coverage.

References

Melting Peak Specificity — tool.io

Similar or alternative tools

NTC Contamination Gate
Flag early no-template-control amplification against caller-supplied Ct limits.
MIQE Coverage Audit
Audit caller-supplied MIQE reporting checklist coverage.