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=109 and n=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.
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Acknowledgements
We acknowledge the special contribution of Lodewijk Sandkuijl in formulating some of the basic concepts embodied in this work; he died shortly before completion of the manuscript. We also thank Martine van Belzen for providing the celiac disease samples and individual genotypes, and to Jackie Senior for critically reading the manuscript.
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Electronic Database Information The PoolFitter program and more illustrating figures are available at our website: http://www.smri.nl/microsatellitesCLUMP (DOS version): http://www.mds.qmw.ac.uk/statgen/dcurtis/software.htmlPrimer3: http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgiTandem Repeat Finder: http://c3.biomath.mssm.edu/trf.htmlGenome Database (mirror site): http://gdbwww.dkfz-heidelberg.de/
Marshfield Center for Medical Genetics: http://research.marshfieldclinic.org/genetics/
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Schnack, H., Bakker, S., van't Slot, R. et al. Accurate determination of microsatellite allele frequencies in pooled DNA samples. Eur J Hum Genet 12, 925–934 (2004). https://doi.org/10.1038/sj.ejhg.5201234
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DOI: https://doi.org/10.1038/sj.ejhg.5201234
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