A Cautionary Tale: Lack of Consistency in Allele Sizes between Two Laboratories for a Published Multilocus Microsatellite Typing System

Abstract

ABSTRACT For species with low genetic diversity, typing using the differences in PCR fragment length resulting from variations in numbers of short tandem repeats has been shown to provide a high level of discrimination. This technique has been called multilocus microsatellite typing (MLMT) or multiple-locus variable-number tandem repeat analysis, and studies usually employ genetic or sequence analyzers to size PCR fragments to a high degree of precision. We set out to validate one such system that has been developed for Aspergillus fumigatus (H. A. de Valk, J. F. G. M. Meis, I. M. Curfs, K. Muehlethaler, J. W. Mouton, and C. H. W. Klaassen, J. Clin. Microbiol. 43:4112-4120, 2005). The sizes of the alleles were compared both by sequencing and from two genotyping laboratories, where they used capillary electrophoresis (CE) for sizing. Size differences of up to 6 bases were found between the actual sizes reported by sequencing and the sizes reported by CE. In addition, because the two genotyping laboratories used different machines and running conditions, differences of up to 3 bases were identified between them. As the microsatellite markers used differ by repeat units of 3 or 4 bases, it was not possible to assign PCR fragments to the correct alleles without confirming the sizes of a range of alleles by direct sequencing. Lines of best fit were plotted for each CE machine against actual sizes and will therefore enable unsequenced PCR fragments to be assigned to the correct alleles. This study highlights the care required to ensure that an MLMT system undergoes a suitable correction procedure before data can be merged between different laboratories involved in the typing of individual species.