Heterozygous Balance

What are heterozygote peaks?

two distinct peaks in a chromatogram that indicate an individual has two different alleles at a particular locus

What is heterozygous balance?

The ratio of peak heights between the two alleles of a heterozygote. The peak heights of both alleles should be the same or very similar because the amplification efficiency during PCR should be the same.

How to work out heterozygous balance

(area of smaller allele/area of larger allele) x 100

Between 0-1

Closer to 1 = more balanced

Analytical threshold for heterozygous balance

Laboratories set a threshold by taking multiple measurements in validation studies.

It is usually 85%. Peaks above this are accepted as heterozygote peaks, signals below have to be questioned as it may indicate a mixture and/or stochastic amplification.

Why may heterozygous imbalance occur

Stochastic effects - alleles may amplify differently because of minimal/degraded DNA

Preferential amplification: one allele is amplified more efficiently than the another.

Primer-binding mutations can cause uneven amplification

What can complicate interpretation

High stutter peaks above stutter threshold - most commonly occurs with long fragments

Mixture - multiple alleles displayed at each locus, 2 different homozygote peaks wrongfully classified as heterozygote

Allelic drop in - additional alleles in a DNA profile, random DNA fragments from other sources in a PCR - homozygote mistaken for a heterozygote?

Allelic drop out - missing alleles at a genetic locus - alleles wrongfully classified as homozygous

null alleles - alleles in samples that do not amplify due to primer binding site mutations - wrongfully classified as homozygote.

Stochastic effects - variations in amplifications