Pyrosequencing-based SNP allele frequency estimation in DNA pools.
Association screening involving numerous genetic markers is facilitated by the analysis of pooled DNA samples rather than individual samples. Several genotyping methods have shown high accuracy and precision of allele frequency estimation in pools. Here, we expand the validation of SNP allele frequency estimation in DNA pools using Pyrosequencing by analyzing 186 pools for three SNPs representing complex sequencing cases. The correlation coefficient between estimated and true allele frequencies ranged between 0.979 and 0.996 and tended to increase with pool size, whereas the difference between estimated and true allele frequencies was 2.37+/-0.11%, in post-PCR pools. The precision was 1.73%. Pool size had no significant effect on accuracy and precision. A comparison between post-PCR and pre-PCR pools showed that for pre-PCR pooling efforts to accurately quantify the genomic DNA samples to be pooled and subsequently amplified are critical. To conclude, Pyrosequencing can be used for allele frequency estimation in DNA pools of SNPs with complex sequencing scenarios with accuracy and precision values in ranges comparable with those of other SNP typing techniques. Considering the ease of use, short run and analysis times, and little instrument maintenance requirements, Pyrosequencing may even be a preferred option.
Lavebratt
Catharina
C
Department of Molecular Medicine, Karolinska Institutet, Karolinska Hospital, Stockholm, Sweden. catharina.lavebratt@cmm.ki.se
Sengul
Selim
S
Jansson
Marten
M
Schalling
Martin
M
D004247:DNA / Q000737:chemistry; D011756:Diphosphates / Q000032:analysis; D005787:Gene Frequency; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*
D011756:Diphosphates; D004247:DNA