Software for Automated Analysis of DNA Fingerprinting Gels

Department of Electrical Engineering, Washington University, St. Louis, Missouri 63130, USA.
Genome Research (Impact Factor: 14.63). 06/2003; 13(5):940-53. DOI: 10.1101/gr.904303
Source: PubMed


Here we describe software tools for the automated detection of DNA restriction fragments resolved on agarose fingerprinting gels. We present a mathematical model for the location and shape of the restriction fragments as a function of fragment size, with model parameters determined empirically from "marker" lanes containing molecular size standards. Automated identification of restriction fragments involves several steps, including: image preprocessing, to put the data in a form consistent with a linear model; marker lane analysis, for determination of the model parameters; and data lane analysis, a procedure for detecting restriction fragment multiplets while simultaneously determining the amplitude curve that describes restriction fragment amplitude as a function of mobility. In validation experiments conducted on fingerprinted and sequenced Bacterial Artificial Chromosome (BAC) clones, sensitivity and specificity of restriction fragment identification exceeded 96% on restriction fragments ranging in size from 600 base pairs (bp) to 30,000 bp. The integrated suite of software tools, written in MATLAB and collectively called BandLeader, is in use at the BC Cancer Agency Genome Sciences Centre (GSC) and the Washington University Genome Sequencing Center, and has been provided to the Wellcome Trust Sanger Institute and the Whitehead Institute. Employed in a production mode at the GSC, BandLeader has been used to perform automated restriction fragment identification for more than 850,000 BAC clones for mouse, rat, bovine, and poplar fingerprint mapping projects.

Download full-text


Available from: John Mcpherson, Apr 16, 2014
29 Reads
  • Source
    • "Gel images were acquired using a Molecular Dynamics Fluorimager 595. Lane tracking of the digitized gel images was manually adjusted using Image software (, and restriction fragments were identified and sized automatically using BandLeader software (Fuhrmann et al. 2003). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Drosophila melanogaster plays an important role in molecular, genetic and genomic studies of heredity, development, metabolism, behavior and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117 Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy and middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence, and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9 Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres are largely inaccessible to current sequencing and assembly methods and remain poorly represented. Further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads. Published by Cold Spring Harbor Laboratory Press.
    Genome Research 01/2015; 25(3). DOI:10.1101/gr.185579.114 · 14.63 Impact Factor
  • Source
    • "Another avenue, perhaps more pragmatic, would be to assess corrections on a simulated project. For example, digesting finished sequences in silico [27] enables one to use the resulting simulated fingerprints to see how well a map could be reconstructed. Several variations on this method are possible [28,29]. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The Sulston score is a well-established, though approximate metric for probabilistically evaluating postulated clone overlaps in DNA fingerprint mapping. It is known to systematically over-predict match probabilities by various orders of magnitude, depending upon project-specific parameters. Although the exact probability distribution is also available for the comparison problem, it is rather difficult to compute and cannot be used directly in most cases. A methodology providing both improved accuracy and computational economy is required. We propose a straightforward algebraic correction procedure, which takes the Sulston score as a provisional value and applies a power-law equation to obtain an improved result. Numerical comparisons indicate dramatically increased accuracy over the range of parameters typical of traditional agarose fingerprint mapping. Issues with extrapolating the method into parameter ranges characteristic of newer capillary electrophoresis-based projects are also discussed. Although only marginally more expensive to compute than the raw Sulston score, the correction provides a vastly improved probabilistic description of hypothesized clone overlaps. This will clearly be important in overlap assessment and perhaps for other tasks as well, for example in using the ranking of overlap probabilities to assist in clone ordering.
    BMC Bioinformatics 02/2007; 8:127. DOI:10.1186/1471-2105-8-127 · 2.58 Impact Factor
  • Source
    • "Wngerprinting, Wngerprint map assembly and manual editing were performed as previously described (Marra et al., 1997; Marra et al., 1999; McPherson et al., 2001; Schein et al., 2002) except that restriction fragment identiWcation, fragment mobility and size determination were performed using automated analysis software (Fuhrmann et al., 2003). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Sex in basidiomycete fungi is controlled by tetrapolar mating systems in which two unlinked gene complexes determine up to thousands of mating specificities, or by bipolar systems in which a single locus (MAT) specifies different sexes. The genus Ustilago contains bipolar (Ustilago hordei) and tetrapolar (Ustilago maydis) species and sexual development is associated with infection of cereal hosts. The U. hordei MAT-1 locus is unusually large (approximately 500 kb) and recombination is suppressed in this region. We mapped the genome of U. hordei and sequenced the MAT-1 region to allow a comparison with mating-type regions in U. maydis. Additionally the rDNA cluster in the U. hordei genome was identified and characterized. At MAT-1, we found 47 genes along with a striking accumulation of retrotransposons and repetitive DNA; the latter features were notably absent from the corresponding U. maydis regions. The tetrapolar mating system may be ancestral and differences in pathogenic life style and potential for inbreeding may have contributed to genome evolution.
    Fungal Genetics and Biology 10/2006; 43(9):655-66. DOI:10.1016/j.fgb.2006.04.002 · 2.59 Impact Factor
Show more