A simple method for automated allele binning in microsatellite markers

Abstract

High-throughput fluorescent genotyping requires a considerable amount of automation for accurate and efficient processing of genetic markers. Automated DNA sequencers and corresponding software products are commercially available that contribute substantially to increased throughput rates for large-scale genotyping projects. However, some conceptually simple tasks still require time-consuming manual intervention that imposes bottlenecks on throughput capacity. One of these tasks is the conversion of imprecise DNA fragment sizes determined by commercial software programs to the underlying discrete alleles that the sizes represent. Here we describe a simple method for assigning allele sizes into their appropriate allele "bins" using least-squares minimization procedures. The method requires no special treatment of family data on plates, internal/external size standards, or electropherogram data manipulation. Tests of the method using the ABI 373A automated DNA sequencer and accompanying Genescan/Genotyper software resulted in accurate automatic classification of all alleles in >80% of 208 markers analyzed, with the remaining 20% being appropriately identified as requiring additional attention to laboratory conditions. Specific characteristics of different markers, including differences in PCR product size and inexact repeat lengths (e.g., 1. 9 bp for a dinucleotide repeat), are accommodated by the method and their properties discussed.

Figure 1

Frequency histograms of two markers having different quality of allele sizes. Both markers were typed on 642 individuals using the ABI 373A and accompanying Genescan/Genotyper software. Marker D10S535 has a very clear separation of alleles, while marker D13S1325 has poor separation between most alleles.

Figure 2

Histogram representing standard deviations, S_w for all di- and tetranucleotide markers tested. Light shaded bars represent dinucleotide repeats and dark shaded bars represent tetranucleotide repeat markers.

Figure 3

Frequency histogram of illustrative marker which has nonstandard repeat lengths between adjacent alleles. In this dinucleotide marker, the expected difference between alleles is 2 bp, yet the average spacing is 1.90 bp. The automated binning procedure accurately assigns all bins only when allowing for this discrepancy of repeat length, referred to as allelic drift.