Peak ratio bar is representing, for each assay of a primer pair, relative expression of all detected PCR amplicons. For an assay, relative expression is shown as a stacked bar graph. Bar colors are matched to the amplicon's associated length (see legend below figure). The assay reaction total expression value (molarity) is shown at the right of each bar.

In the bar color legend, a solid color is used when the detected amplicon was matched to the length expected by primer design. If the color is cross-hatched, then this amplicon could not match any expected by primer design. This unexpected amplicon length is aligned across all assay reactions.

In alternative splicing RT-PCR assays, we are monitoring the expression shift between different isoforms of an alternative splicing event. Percent Spliced-In (PSI) is a metric based on the long and short isoforms, respectively representing the alternative portion inclusion and exclusion. PSI is calulated as L/(L+S) where L and S are the measured expression of the long and short isoforms, respectively. Thus PSI is interpreted as the expression portion taken by the event inclusion part. PSI are reported as a ratio value between 0 and 1, or as a percentage.

PSI is defined using two isoforms. When there is more than two amplicons detected, the PSI algorithm automatically associates long and short isoforms to the two most abundantly expressed amplicons in all assays for this event (primer pair).

PSI is represented here as an histogram for all assays done with this primer pair.

Peaks detected by the electrophoresis machine are associated to PCR amplicons expected from primer design using a peak alignment algorithm. The algorithm first cluster together all detected peaks from all assay of a primer pair using k-means. Resulting peak centroids are used as reference for individual peak association by matching them to amplicon expected from primer design. Centroids not matching any expected amplicon are kept and labelled as unexpected amplicons for downstream analysis. The table below summarizes alignment between centroids and expected amplicons, and between all detected peaks. A centroid not aligned to an expected amplicon is labelled as unexpected.

Electropherogram from the electrophoresis capillary machine, with peak annotations. On top are length and molarity of peaks detected by the machine. At bottom are the peak association to amplicon as expected from primer design and gene transcript information:

• Expected: length of amplicon as expected from primer design. In italic are amplicon that are unexpected by primer design. Long and short labels are added on amplicons used for PSI calculation. The letter u is added when this PSI amplicon is unexpected from primer design.
• Hint: length of associated peak centroid. No value when peak is not associated to any expected or unexpected amplicon.
• Matched: Lentgh of detected peak associated. No value when peak is not associated to any expected or unexpected amplicon.
• Molarity: Peak molarity as calculated by electrophoresis machine (from area under the curve and amplicon length). No value when peak is not associated to any expected or unexpected ampicon.

Electropherogram from the electrophoresis capillary machine, with peak annotations. On top are length and molarity of peaks detected by the machine. At bottom are the peak association to amplicon as expected from primer design and gene transcript information:

• Expected: length of amplicon as expected from primer design. In italic are amplicon that are unexpected by primer design. Long and short labels are added on amplicons used for PSI calculation. The letter u is added when this PSI amplicon is unexpected from primer design.
• Hint: length of associated peak centroid. No value when peak is not associated to any expected or unexpected amplicon.
• Matched: Lentgh of detected peak associated. No value when peak is not associated to any expected or unexpected amplicon.
• Molarity: Peak molarity as calculated by electrophoresis machine (from area under the curve and amplicon length). No value when peak is not associated to any expected or unexpected ampicon.

Electropherogram from the electrophoresis capillary machine, with peak annotations. On top are length and molarity of peaks detected by the machine. At bottom are the peak association to amplicon as expected from primer design and gene transcript information:

• Expected: length of amplicon as expected from primer design. In italic are amplicon that are unexpected by primer design. Long and short labels are added on amplicons used for PSI calculation. The letter u is added when this PSI amplicon is unexpected from primer design.
• Hint: length of associated peak centroid. No value when peak is not associated to any expected or unexpected amplicon.
• Matched: Lentgh of detected peak associated. No value when peak is not associated to any expected or unexpected amplicon.
• Molarity: Peak molarity as calculated by electrophoresis machine (from area under the curve and amplicon length). No value when peak is not associated to any expected or unexpected ampicon.

Electropherogram from the electrophoresis capillary machine, with peak annotations. On top are length and molarity of peaks detected by the machine. At bottom are the peak association to amplicon as expected from primer design and gene transcript information:

• Expected: length of amplicon as expected from primer design. In italic are amplicon that are unexpected by primer design. Long and short labels are added on amplicons used for PSI calculation. The letter u is added when this PSI amplicon is unexpected from primer design.
• Hint: length of associated peak centroid. No value when peak is not associated to any expected or unexpected amplicon.
• Matched: Lentgh of detected peak associated. No value when peak is not associated to any expected or unexpected amplicon.
• Molarity: Peak molarity as calculated by electrophoresis machine (from area under the curve and amplicon length). No value when peak is not associated to any expected or unexpected ampicon.

Electropherogram from the electrophoresis capillary machine, with peak annotations. On top are length and molarity of peaks detected by the machine. At bottom are the peak association to amplicon as expected from primer design and gene transcript information:

• Expected: length of amplicon as expected from primer design. In italic are amplicon that are unexpected by primer design. Long and short labels are added on amplicons used for PSI calculation. The letter u is added when this PSI amplicon is unexpected from primer design.
• Hint: length of associated peak centroid. No value when peak is not associated to any expected or unexpected amplicon.
• Matched: Lentgh of detected peak associated. No value when peak is not associated to any expected or unexpected amplicon.
• Molarity: Peak molarity as calculated by electrophoresis machine (from area under the curve and amplicon length). No value when peak is not associated to any expected or unexpected ampicon.

Electropherogram from the electrophoresis capillary machine, with peak annotations. On top are length and molarity of peaks detected by the machine. At bottom are the peak association to amplicon as expected from primer design and gene transcript information:

• Expected: length of amplicon as expected from primer design. In italic are amplicon that are unexpected by primer design. Long and short labels are added on amplicons used for PSI calculation. The letter u is added when this PSI amplicon is unexpected from primer design.
• Hint: length of associated peak centroid. No value when peak is not associated to any expected or unexpected amplicon.
• Matched: Lentgh of detected peak associated. No value when peak is not associated to any expected or unexpected amplicon.
• Molarity: Peak molarity as calculated by electrophoresis machine (from area under the curve and amplicon length). No value when peak is not associated to any expected or unexpected ampicon.

Electropherogram from the electrophoresis capillary machine, with peak annotations. On top are length and molarity of peaks detected by the machine. At bottom are the peak association to amplicon as expected from primer design and gene transcript information:

• Expected: length of amplicon as expected from primer design. In italic are amplicon that are unexpected by primer design. Long and short labels are added on amplicons used for PSI calculation. The letter u is added when this PSI amplicon is unexpected from primer design.
• Hint: length of associated peak centroid. No value when peak is not associated to any expected or unexpected amplicon.
• Matched: Lentgh of detected peak associated. No value when peak is not associated to any expected or unexpected amplicon.
• Molarity: Peak molarity as calculated by electrophoresis machine (from area under the curve and amplicon length). No value when peak is not associated to any expected or unexpected ampicon.

Electropherogram from the electrophoresis capillary machine, with peak annotations. On top are length and molarity of peaks detected by the machine. At bottom are the peak association to amplicon as expected from primer design and gene transcript information:

• Expected: length of amplicon as expected from primer design. In italic are amplicon that are unexpected by primer design. Long and short labels are added on amplicons used for PSI calculation. The letter u is added when this PSI amplicon is unexpected from primer design.
• Hint: length of associated peak centroid. No value when peak is not associated to any expected or unexpected amplicon.
• Matched: Lentgh of detected peak associated. No value when peak is not associated to any expected or unexpected amplicon.
• Molarity: Peak molarity as calculated by electrophoresis machine (from area under the curve and amplicon length). No value when peak is not associated to any expected or unexpected ampicon.

Electropherogram from the electrophoresis capillary machine, with peak annotations. On top are length and molarity of peaks detected by the machine. At bottom are the peak association to amplicon as expected from primer design and gene transcript information:

• Expected: length of amplicon as expected from primer design. In italic are amplicon that are unexpected by primer design. Long and short labels are added on amplicons used for PSI calculation. The letter u is added when this PSI amplicon is unexpected from primer design.
• Hint: length of associated peak centroid. No value when peak is not associated to any expected or unexpected amplicon.
• Matched: Lentgh of detected peak associated. No value when peak is not associated to any expected or unexpected amplicon.
• Molarity: Peak molarity as calculated by electrophoresis machine (from area under the curve and amplicon length). No value when peak is not associated to any expected or unexpected ampicon.

Electropherogram from the electrophoresis capillary machine, with peak annotations. On top are length and molarity of peaks detected by the machine. At bottom are the peak association to amplicon as expected from primer design and gene transcript information:

• Expected: length of amplicon as expected from primer design. In italic are amplicon that are unexpected by primer design. Long and short labels are added on amplicons used for PSI calculation. The letter u is added when this PSI amplicon is unexpected from primer design.
• Hint: length of associated peak centroid. No value when peak is not associated to any expected or unexpected amplicon.
• Matched: Lentgh of detected peak associated. No value when peak is not associated to any expected or unexpected amplicon.
• Molarity: Peak molarity as calculated by electrophoresis machine (from area under the curve and amplicon length). No value when peak is not associated to any expected or unexpected ampicon.

Electropherogram from the electrophoresis capillary machine, with peak annotations. On top are length and molarity of peaks detected by the machine. At bottom are the peak association to amplicon as expected from primer design and gene transcript information:

• Expected: length of amplicon as expected from primer design. In italic are amplicon that are unexpected by primer design. Long and short labels are added on amplicons used for PSI calculation. The letter u is added when this PSI amplicon is unexpected from primer design.
• Hint: length of associated peak centroid. No value when peak is not associated to any expected or unexpected amplicon.
• Matched: Lentgh of detected peak associated. No value when peak is not associated to any expected or unexpected amplicon.
• Molarity: Peak molarity as calculated by electrophoresis machine (from area under the curve and amplicon length). No value when peak is not associated to any expected or unexpected ampicon.

Electropherogram from the electrophoresis capillary machine, with peak annotations. On top are length and molarity of peaks detected by the machine. At bottom are the peak association to amplicon as expected from primer design and gene transcript information:

• Expected: length of amplicon as expected from primer design. In italic are amplicon that are unexpected by primer design. Long and short labels are added on amplicons used for PSI calculation. The letter u is added when this PSI amplicon is unexpected from primer design.
• Hint: length of associated peak centroid. No value when peak is not associated to any expected or unexpected amplicon.
• Matched: Lentgh of detected peak associated. No value when peak is not associated to any expected or unexpected amplicon.
• Molarity: Peak molarity as calculated by electrophoresis machine (from area under the curve and amplicon length). No value when peak is not associated to any expected or unexpected ampicon.

Electropherogram from the electrophoresis capillary machine, with peak annotations. On top are length and molarity of peaks detected by the machine. At bottom are the peak association to amplicon as expected from primer design and gene transcript information:

• Expected: length of amplicon as expected from primer design. In italic are amplicon that are unexpected by primer design. Long and short labels are added on amplicons used for PSI calculation. The letter u is added when this PSI amplicon is unexpected from primer design.
• Hint: length of associated peak centroid. No value when peak is not associated to any expected or unexpected amplicon.
• Matched: Lentgh of detected peak associated. No value when peak is not associated to any expected or unexpected amplicon.
• Molarity: Peak molarity as calculated by electrophoresis machine (from area under the curve and amplicon length). No value when peak is not associated to any expected or unexpected ampicon.

Electropherogram from the electrophoresis capillary machine, with peak annotations. On top are length and molarity of peaks detected by the machine. At bottom are the peak association to amplicon as expected from primer design and gene transcript information:

• Expected: length of amplicon as expected from primer design. In italic are amplicon that are unexpected by primer design. Long and short labels are added on amplicons used for PSI calculation. The letter u is added when this PSI amplicon is unexpected from primer design.
• Hint: length of associated peak centroid. No value when peak is not associated to any expected or unexpected amplicon.
• Matched: Lentgh of detected peak associated. No value when peak is not associated to any expected or unexpected amplicon.
• Molarity: Peak molarity as calculated by electrophoresis machine (from area under the curve and amplicon length). No value when peak is not associated to any expected or unexpected ampicon.

Electropherogram from the electrophoresis capillary machine, with peak annotations. On top are length and molarity of peaks detected by the machine. At bottom are the peak association to amplicon as expected from primer design and gene transcript information:

• Expected: length of amplicon as expected from primer design. In italic are amplicon that are unexpected by primer design. Long and short labels are added on amplicons used for PSI calculation. The letter u is added when this PSI amplicon is unexpected from primer design.
• Hint: length of associated peak centroid. No value when peak is not associated to any expected or unexpected amplicon.
• Matched: Lentgh of detected peak associated. No value when peak is not associated to any expected or unexpected amplicon.
• Molarity: Peak molarity as calculated by electrophoresis machine (from area under the curve and amplicon length). No value when peak is not associated to any expected or unexpected ampicon.