A cautionary tale: Lack of consistency in allele sizes between two laboratories for a published multilocus microsatellite typing system
A cautionary tale: Lack of consistency in allele sizes between two laboratories for a published multilocus microsatellite typing system
复制标题
DOI：
10.1128/jcm.02136-06
复制
发表时间：
2007-02-01
影响因子：
9.4
通讯作者：
Anderson, Michael J.
中科院分区：
文献类型：
--
作者：
Pasqualotto, Alessandro C.;Denning, David W.;Anderson, Michael J.
对于具有低遗传多样性的物种，使用短串联重复序列数量变化导致的PCR片段长度差异进行分型已被证明提供了高水平的区分。这种技术被称为多位点微卫星分型（MLMT）或多位点可变数目串联重复序列分析，研究通常使用遗传或序列分析仪来高度精确地确定PCR片段的大小。我们着手验证一个这样的系统，已开发的烟曲霉（H。A. de Valk，J. F. G. M.梅斯岛M. Curfs，K. Muehlethaler，J. W. Mouton，和C. H. W. Klaassen，J. Clin. Microbiol. 43：4112-4120，2005）。通过测序和两个基因分型实验室比较等位基因的大小，在那里他们使用毛细管电泳（CE）进行大小测定。测序报告的实际大小与CE报告的大小之间存在多达6个碱基的大小差异。此外，由于两个基因分型实验室使用不同的机器和运行条件，因此在它们之间鉴定出多达3个碱基的差异。由于所用的微卫星标记的差异在于3或4个碱基的重复单位，因此在不通过直接测序确认一系列等位基因的大小的情况下，不可能将PCR片段分配给正确的等位基因。针对每个CE机器的实际大小绘制最佳拟合线，因此将使未测序的PCR片段能够分配给正确的等位基因。这项研究强调了需要小心，以确保MLMT系统进行适当的校正程序之前，数据可以合并在不同的实验室参与分型的个别物种。
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.