Abstract
BackgroundCurrent techniques of screening bacterial artificial chromosome (BAC) libraries for molecular markers during the construction of physical maps are slow, laborious and often assign multiple BAC contigs to a single locus on a genetic map. These limitations are the principal impediment in the construction of physical maps of large eukaryotic genomes. It is hypothesized that this impediment can be overcome by screening multidimensional pools of BAC clones using the highly parallel Illumina GoldenGate™ assay.ResultsTo test the efficacy of the Golden Gate assay in BAC library screening, multidimensional pools involving 302976 Aegilops tauschii BAC clones were genotyped for the presence/absence of specific gene sequences with multiplexed Illumina GoldenGate oligonucleotide assays previously used to place single nucleotide polymorphisms on an Ae. tauschii genetic map. Of 1384 allele-informative oligonucleotide assays, 87.6% successfully clustered BAC pools into those positive for a BAC clone harboring a specific gene locus and those negative for it. The location of the positive BAC clones within contigs assembled from 199190 fingerprinted Ae. tauschii BAC clones was used to evaluate the precision of anchoring of BAC clones and contigs on the Ae. tauschii genetic map. For 41 (95%) assays, positive BAC clones were neighbors in single contigs. Those contigs could be unequivocally assigned to loci on the genetic map. For two (5%) assays, positive clones were in two different contigs and the relationships of these contigs to loci on the Ae. tauschii genetic map were equivocal. Screening of BAC libraries with a simple five-dimensional BAC pooling strategy was evaluated and shown to allow direct detection of positive BAC clones without the need for manual deconvolution of BAC clone pools.ConclusionThe highly parallel Illumina oligonucleotide assay is shown here to be an efficient tool for screening BAC libraries and a strategy for high-throughput anchoring of BAC contigs on genetic maps during the construction of physical maps of eukaryotic genomes. In most cases, screening of BAC libraries with Illumina oligonucleotide assays results in the unequivocal relationship of BAC clones with loci on the genetic map.
Highlights
Current techniques of screening bacterial artificial chromosome (BAC) libraries for molecular markers during the construction of physical maps are slow, laborious and often assign multiple BAC contigs to a single locus on a genetic map
We describe here a BAC library screening strategy that is largely devoid of these limitations and can be performed in a high-throughput mode
Genotyping of genomic DNA and BAC pool DNA with Illumina GoldenGate assays Genotyping of BAC super-pools consisting of pooled BAC plate-pools and containing DNA of either 10368 or 11520
Summary
Current techniques of screening bacterial artificial chromosome (BAC) libraries for molecular markers during the construction of physical maps are slow, laborious and often assign multiple BAC contigs to a single locus on a genetic map. These limitations are the principal impediment in the construction of physical maps of large eukaryotic genomes. Current screening techniques utilize either DNA-DNA hybridization or polymerase chain reaction (PCR) In some applications, such as the construction of a physical map, a BAC library must be screened for the presence of hundreds or thousands of different molecular markers. To maximize the efficiency of such screening, multidimensional pooling of clones or probes is employed [9]; probes are pooled if a library is screened by DNA-DNA hybridization [10] and clones are pooled if it is screened by PCR [11]
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