Evaluation of DNA pooling for the estimation of microsatellite allele frequencies: a case study using striped bass (Morone saxatilis).

Using striped bass (Morone saxatilis) and six multiplexed microsatellite markers, we evaluated procedures for estimating allele frequencies by pooling DNA from multiple individuals, a method suggested as cost-effective relative to individual genotyping. Using moment-based estimators, we estimated allele frequencies in experimental DNA pools and found that the three primary laboratory steps, DNA quantitation and pooling, PCR amplification, and electrophoresis, accounted for 23, 48, and 29%, respectively, of the technical variance of estimates in pools containing DNA from 2-24 individuals. Exact allele-frequency estimates could be made for pools of sizes 2-8, depending on the locus, by using an integer-valued estimator. Larger pools of size 12 and 24 tended to yield biased estimates; however, replicates of these estimates detected allele frequency differences among pools with different allelic compositions. We also derive an unbiased estimator of Hardy-Weinberg disequilibrium coefficients that uses multiple DNA pools and analyze the cost-efficiency of DNA pooling. DNA pooling yields the most potential cost savings when a large number of loci are employed using a large number of individuals, a situation becoming increasingly common as microsatellite loci are developed in increasing numbers of taxa.