A Simple Method for Analyzing Microsatellite Allele Image Patterns Generated from DNA Pools and Its Application to Allelic Association Studies

Abstract

Allelic association studies provide the most powerful method for locating genes of small effect contributing to complex diseases and traits. However, in outbred populations, allelic association is usually maintained only over distances of <=1 cM. Therefore, systematic searches over large regions are costly. Here we present a method involving DNA pooling that can be used as a rapid preliminary screen for allelic association with the most common class of polymorphic markers, single-sequence repeats. Patient and control samples are pooled separately, and markers are typed in the two pools. By use of primers with fluorescent 5' ends, PCR products can be analyzed on an automated sequencing apparatus. Allele image patterns (AIPs) produced for the two groups are overlaid and differences in pattern area between pools computed. From this, a DeltaAIP statistic is calculated from the difference in areas between the two AIPs expressed as a fraction of the total shared and nonshared area. AIPs of a range of different-sized pools were generated by computer simulation for markers with a range of allele sizes and frequencies. DeltaAIPs from pools and chi2 values for individual genotypings were compared, with both simulated and real data from microsatellite markers. The results demonstrated a high correlation between DeltaAIP and chi2 values. DeltaAIP analysis of real microsatellite data indicated the feasibility of using this method in systematic searches for allelic association and generated a small number of false positives but few false negatives. We conclude that DeltaAIP analysis of DNA pools can be used effectively and efficiently as a rapid screen for allelic association in case-control studies.

Evaluation of DNA Pooling for the Estimation of Microsatellite Allele Frequencies: A Case Study Using Striped Bass (Morone saxatilis)

“…In many actual applications (e.g., Daniels et al 1998;Moritz et al 2003;Lee et al 2004), however, the actual value of population allele frequencies is not relevant; instead the main concern is the relative frequencies of alleles between two or more populations. In this situation bias may not be a concern, and we can ask how well larger DNA pools detect differences in allelic composition between populations.…”

“…Quantitative genetic studies in human and agricultural systems have sought to find an association between a trait and a marker by pooling DNA from individuals that share a common phenotype and/or ancestry, a technique sometimes called ''bulked segregant analysis.'' Methods for comparing microsatellite DNA pools among experimental populations vary from more qualitative approaches such as counting alleles (Hillel et al 2003) or comparing measures of relative fluorescence intensity (Daniels et al 1998;Moritz et al 2003;Lee et al 2004) to more quantitative approaches such as attempting to estimate allele frequencies within a pool (Perlin et al 1995;Barcellos et al 1997;Band and Ron 1998;Lipkin et al 1998Lipkin et al , 2002Rutyer-Spira et al 1998;Kirov et al 2000;Mosig et al 2001;Schnack et al 2004). More generally, DNA pooling might be applied in any genetic study relying on allele frequency estimation, including marker screening and association mapping, analyses of population differentiation, and analyses of effective population size.…”

Replication in genetic studies of complex traits

“…Alternatively, ascertainment can favour certain alleles by increasing their frequencies in the sample. Apart from the recruitment of individuals, ascertainment may also include selection (preliminary screening) of markers based on their estimated allele frequencies from pooled DNA samples (Daniels et al 1998;Shaw et al 1998;Collins et al 2000;Sham et al 2002). Even if the allele frequencies in two different studies are equal, use of dissimilar designs and ascertainment schemes may lead to difficulties in obtaining consistent results (Bruun & Ewald, 1999;McCarthy et al 1998).…”

Accurate determination of microsatellite allele frequencies in pooled DNA samples

“…The recently proposed DAIP and DTAC methods compare overall differences in peak area or peak height between pool patterns. 8,9 However, both methods assume a single fixed stutter profile for all markers and simulate large numbers of pool patterns to determine what proportion by chance will deviate significantly. Since the heights as well as the number of stutter peaks can differ greatly between markers, these methods raise the question whether realistic significance levels can be calculated in this way.…”