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A new implementation of high-throughput five-dimensional clone pooling strategy for BAC library screening.

Frank M You1, Ming-Cheng Luo, Kenong Xu

  • 1Department of Plant Sciences, University of California, Davis, CA 95516, USA.

BMC Genomics
|December 7, 2010
PubMed
Summary

A new 5-D bacterial artificial chromosome (BAC) clone pooling strategy improves high-throughput screening and genetic map anchoring. This enhanced method, using Illumina GoldenGate assays and FPCBrowser software, increases efficiency and accuracy in identifying positive clones.

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Area of Science:

  • Genomics
  • Molecular Biology
  • Bioinformatics

Background:

  • Bacterial artificial chromosome (BAC) clone pooling strategies are crucial for high-throughput screening and genetic map anchoring.
  • Existing 5-dimensional (5-D) pooling methods using Illumina GoldenGate assays and PCR can require manual intervention for clone identification.

Purpose of the Study:

  • To report a new implementation of a 5-D BAC clone pooling strategy.
  • To enhance the efficiency and accuracy of BAC clone screening and genetic map anchoring.

Main Methods:

  • Dividing BAC clone pools into sub-pools (1-2x genome coverage).
  • Screening all pools using Illumina's GoldenGate assay for deconvolution.
  • Developing and integrating a brute force algorithm into FPCBrowser software for clone deconvolution.

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  • Analyzing empirical data from 55 SNP assays on Aegilops tauschii chromosome 2D.

    • Main Results:

    • Successfully assigned 48 out of 55 (87%) specific SNP markers to clones within single contigs.
    • Achieved 91% precision in assigning clones to SNP markers.
    • Demonstrated the effectiveness of the FPCBrowser software and integrated algorithm.

    Conclusions:

    • The new 5-D BAC clone pooling strategy is high-throughput, low-cost, rapid, and feasible for screening BAC libraries.
    • This approach effectively anchors BAC clones and contigs to genetic maps.
    • The FPCBrowser software with its deconvolution algorithm is available for public use.