Accurate determination of microsatellite allele frequencies in pooled DNA samples

Abstract

Pooling of DNA samples instead of individual genotyping can speed up genetic association studies. However, for microsatellite markers, the electrophoretic pattern of DNA pools can be complex, and procedures for deriving allele frequencies are often confounded by PCR-induced stutter artefacts. We have developed a mathematical procedure to remove stutter noise and accurately determine allele frequencies in pools. A stutter correction model can be reliably derived from one standard 'training set' of the same 10 individual DNA samples for each marker, which can also include heterozygous patterns with partially overlapping peaks. Compared with earlier methods, this reduces the number of genotypes needed in the training set considerably, and allows standardization of analyses for different markers. Moreover, the use of a procedure that fits all data simultaneously makes the method less sensitive to aberrant data. The model was tested with 34 markers, 18 of which were newly defined from human sequence data. Allele frequencies derived from stutter-corrected DNA pool patterns were compared with the summed individual genotyping results of all the individuals in the pools (n ¼ 109 and n ¼ 64). We show that the model is robust and accurately extracts allele frequencies from pooled DNA samples for 32 of the 34 microsatellite markers tested. Finally, we performed a case -control study in celiac disease and found that weakly associated disease alleles, identified by individual genotyping, were only detectable in pools after stutter correction. This efficient method for correcting stutter artefacts in microsatellite markers enables large-scale genetic association studies using DNA pools to be performed.

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

“…The procedures implemented in previous studies (Perlin et al 1995;Barcellos et al 1997;Band and Ron 1998;Lipkin et al 1998Lipkin et al , 2002Kirov et al 2000;Mosig et al 2001;Schnack et al 2004) have used some version of this estimator, but a probabilistic basis for the estimator has not been presented. Our results show explicitly that the estimator depends only on the relative fluorescence intensities associated with each allele, explaining why allele frequency estimation from DNA pools is tractable empirically, even if absolute fluorescence intensities are quite variable.…”

“…In our present study, we found that unbiasedness vanishes between pool sizes of $6 and 12, depending on the locus. Additional research on the best methods for estimating R is needed (Schnack et al 2004).…”

“…Small pools offer the possibility of unbiased and even exact estimates. Larger pools can yield biased estimates, but may remain useful for assessing relative differences in allele frequencies among populations, the main approach of previous pooling studies (Barcellos et al 1997;Band and Ron 1998;Lipkin et al 1998Lipkin et al , 2002Kirov et al 2000;Mosig et al 2001;Schnack et al 2004). We used our estimates of technical variance to assess the cost-effectiveness of DNA pooling relative to individual genotyping.…”

“…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.…”

“…DNA pooling incurs the cost of DNA quantitation and pooling, but saves on PCR amplification and electrophoresis since a pool can be used repeatedly for different multiplexes (e.g., Kirov et al 2000;Schnack et al 2004). If the same number of individuals is used in a single pool so that m ¼ n, then the percentage of savings from DNA pooling is 100 1 À C P C I % ¼ 100 1 À nðc I 1 c Q Þ 1 lðc A 1 c E Þ nc I 1 lnðc A 1 c E Þ…”