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A high-throughput strategy for screening of bacterial artificial chromosome libraries and anchoring of clones on a genetic map constructed with single nucleotide polymorphisms
- Source :
- BMC Genomics, BMC Genomics, Vol 10, Iss 1, p 28 (2009)
- Publisher :
- Springer Nature
-
Abstract
- Background 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. It is hypothesized that this impediment can be overcome by screening multidimensional pools of BAC clones using the highly parallel Illumina GoldenGateā¢ assay. Results To 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. Conclusion The 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.
- Subjects :
- 0106 biological sciences
Genetic Markers
Chromosomes, Artificial, Bacterial
lcsh:QH426-470
DNA, Plant
Genotype
lcsh:Biotechnology
Biology
01 natural sciences
Genome
Polymorphism, Single Nucleotide
03 medical and health sciences
Gene mapping
lcsh:TP248.13-248.65
Genetics
Aegilops tauschii
Genomic library
Triticum
030304 developmental biology
Gene Library
0303 health sciences
Bacterial artificial chromosome
Contig
Methodology Article
Chromosome Mapping
food and beverages
biology.organism_classification
lcsh:Genetics
Genetic marker
DNA microarray
Genome, Plant
010606 plant biology & botany
Biotechnology
Subjects
Details
- Language :
- English
- ISSN :
- 14712164
- Volume :
- 10
- Issue :
- 1
- Database :
- OpenAIRE
- Journal :
- BMC Genomics
- Accession number :
- edsair.doi.dedup.....68f2d8980cbc1782782f600003bc4714
- Full Text :
- https://doi.org/10.1186/1471-2164-10-28