Whole-genome validation of high-information-content fingerprinting - PubMed (original) (raw)

Whole-genome validation of high-information-content fingerprinting

William M Nelson et al. Plant Physiol. 2005 Sep.

Abstract

Fluorescent-based high-information-content fingerprinting (HICF) techniques have recently been developed for physical mapping. These techniques make use of automated capillary DNA sequencing instruments to enable both high-resolution and high-throughput fingerprinting. In this article, we report the construction of a whole-genome HICF FPC map for maize (Zea mays subsp. mays cv B73), using a variant of HICF in which a type IIS restriction enzyme is used to generate the fluorescently labeled fragments. The HICF maize map was constructed from the same three maize bacterial artificial chromosome libraries as previously used for the whole-genome agarose FPC map, providing a unique opportunity for direct comparison of the agarose and HICF methods; as a result, it was found that HICF has substantially greater sensitivity in forming contigs. An improved assembly procedure is also described that uses automatic end-merging of contigs to reduce the effects of contamination and repetitive bands. Several new features in FPC v7.2 are presented, including shared-memory multiprocessing, which allows dramatically faster assemblies, and automatic end-merging, which permits more accurate assemblies. It is further shown that sequenced clones may be digested in silico and located accurately on the HICF assembly, despite size deviations that prevent the precise prediction of experimental fingerprints. Finally, repetitive bands are isolated, and their effect on the assembly is studied.

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Figures

Figure 1.

Figure 1.

Three-color fluorescent fingerprinting based on the type IIS restriction enzyme _Ear_I and 4-cutter _Taq_I. Shown are the (A) restriction enzyme cutting patterns, (B) the assignments of dye label to ddNTP and the ROX (−250 bp) standard that was used, (C) an example of enzyme cutting and labeling, and (D) a sample HICF trace (ZMMBBc0519B22) displayed in Genescan.

Figure 2.

Figure 2.

Distribution of fragment sizing error from ABI 3700 sequencers, as determined by comparing in silico size predictions with observed fragment sizes for 22 sequenced maize clones (see text). The data are plotted as a histogram with a bin size of 0.25 bp.

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