Abstract
BackgroundA five-dimensional (5-D) clone pooling strategy for screening of bacterial artificial chromosome (BAC) clones with molecular markers utilizing highly-parallel Illumina GoldenGate assays and PCR facilitates high-throughput BAC clone and BAC contig anchoring on a genetic map. However, this strategy occasionally needs manual PCR to deconvolute pools and identify truly positive clones.ResultsA new implementation is reported here for our previously reported clone pooling strategy. Row and column pools of BAC clones are divided into sub-pools with 1~2× genome coverage. All BAC pools are screened with Illumina's GoldenGate assay and the BAC pools are deconvoluted to identify individual positive clones. Putative positive BAC clones are then further analyzed to find positive clones on the basis of them being neighbours in a contig. An exhaustive search or brute force algorithm was designed for this deconvolution and integrated into a newly developed software tool, FPCBrowser, for analyzing clone pooling data. This algorithm was used with empirical data for 55 Illumina GoldenGate SNP assays detecting SNP markers mapped on Aegilops tauschii chromosome 2D and Ae. tauschii contig maps. Clones in single contigs were successfully assigned to 48 (87%) specific SNP markers on the map with 91% precision.ConclusionA new implementation of 5-D BAC clone pooling strategy employing both GoldenGate assay screening and assembled BAC contigs is shown here to be a high-throughput, low cost, rapid, and feasible approach to screening BAC libraries and anchoring BAC clones and contigs on genetic maps. The software FPCBrowser with the integrated clone deconvolution algorithm has been developed and is downloadable at http://avena.pw.usda.gov/wheatD/fpcbrowser.shtml.
Highlights
A five-dimensional (5-D) clone pooling strategy for screening of bacterial artificial chromosome (BAC) clones with molecular markers utilizing highly-parallel Illumina GoldenGate assays and polymerase chain reaction (PCR) facilitates highthroughput BAC clone and BAC contig anchoring on a genetic map
A PCR-based six dimensional (6-D) BAC clone pooling strategy has been successfully used by other groups for BAC library screening in sorghum [6], maize [7]
Because the pooling procedure involves five different DNA pool sets, this design was called a five dimensional (5-D) clone pooling strategy [9]. (2) Super pools screened by Illumina’s GoldenGate assay and row and column pools screened by PCR. (3) Positive plate pools at 3-D intersections further verified by PCR to find positive plate pools among the putative positives
Summary
A five-dimensional (5-D) clone pooling strategy for screening of bacterial artificial chromosome (BAC) clones with molecular markers utilizing highly-parallel Illumina GoldenGate assays and PCR facilitates highthroughput BAC clone and BAC contig anchoring on a genetic map. In order to reduce pool number and relieve PCR workload, Luo et al (2009) [9] evaluated a high-throughput fivedimensional (5-D) clone pooling strategy based on both Illumina’s GoldenGate assay and PCR screening of Ae. tauschii BAC clones. (3) Positive plate pools at 3-D intersections further verified by PCR to find positive plate pools among the putative positives This strategy reduces pool number and adopts the highly parallel GoldenGate assay for clone screening, and makes high-throughput clone screening possible for large genomes.
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