Peak Ratio Data Analysis

Overview

Computes molarity ratios in PCR reaction and identifies unexpected peaks (using unexpected peak alignment analysis), missing peaks, or dubious peak matching (using peak matching analysis).

Background

This analysis is useful to quantify splicing shifts in a reaction sets for a primer pair. For example, a set of 4 reactions for a primer pair with 2 expected peaks can present molarity ratios of 0.25 and 0.75 in two reactions for peak 1 and 2, respectively, and of 0.80 and 0.20 in the two other reactions.

Interpretation

The result of this analysis is presented as a data sheet in CSV format (currenlty tab-separated). Each row contains the data for a single PCR peak; the whole sheet holds all peaks from all the reactions in the group, and is available for online view or download at the PCR reaction group page. Here is the definition of each column in the data sheet:

gene

Accession name of the gene targeted by the primer pair. This accession is in the context of the gene source used to identify the expected peak.

forward

Forward primer name.

reverse

Reverse primer name.

sample

Name of the sample in which the amplification was assayed. Usually this is the amplified RNA source name, but can be a more complex label naming other oligos, reagents or experimental conditions used.

bps

Amplicon length in base pairs.

nM

Molarity of the detected peak in nM.

total_nM

Sum of the molarites for all the peaks in this reaction (same primer pair and sample). This is the sum used to compute the ratio.

ratio

Molarity ratio for this peak, literaly column nM divided by column total_nM.

unexpected

1 if the peak is unexpected (based on unexpected peak alignment analysis), 0 otherwise.

share

Lists all peaks by their amplicon length that share the same detected peak as this one (space-separated). Here is sample data snippet for the 5 peaks of a reaction:

The first three peaks (at 239, 254, and 266 bps) all show two other peaks in the share column. This means that these three peaks were matched simultaneously to the same detected peak with 12.1 nM. Therefore, the 12.1 nM was counted only once to obtain the total molarity for this reaction: 24.3 nM.

All other columns appearing on the right side of share are not useful to view.

This data is made available for online viewing using Palace table view and for download as a CSV file (tab-separated).

Algorithm

Molarity ratios are calculated by surveying all detected peaks. The ratio applies to a single reaction and gives the molarity proportion for a peak with respect to the sum of the molarity of all peaks in the reaction.

The peak matching analysis is used to identify missing peaks, i.e. an expected peak not matched to any detected peak. Missing peaks are given 0 molarity ratio. Peak matching is also used to uncover dubious peak matching, which happens when a single detected peak is matched to many expected peaks. Molarity is counted once only in such a case.

The unexpected peak alignment analysis is used to flag unexpected peak in the data sheet (column unexpected).

Contacts

• Philippe Thibault

Credits

Philippe Thibault

Implementation of the analysis