A new implementation of high-throughput five-dimensional clone pooling strategy for BAC library screening
<p>Abstract</p> <p>Background</p> <p>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 high-throughput BAC clone a...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2010-12-01
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| Series: | BMC Genomics |
| Online Access: | http://www.biomedcentral.com/1471-2164/11/692 |
| _version_ | 1811315185247322112 |
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| author | Deal Karin R Xu Kenong Luo Ming-Cheng You Frank M Anderson Olin D Dvorak Jan |
| author_facet | Deal Karin R Xu Kenong Luo Ming-Cheng You Frank M Anderson Olin D Dvorak Jan |
| author_sort | Deal Karin R |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>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 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.</p> <p>Results</p> <p>A 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 <it>Aegilops tauschii </it>chromosome 2D and <it>Ae. tauschii </it>contig maps. Clones in single contigs were successfully assigned to 48 (87%) specific SNP markers on the map with 91% precision.</p> <p>Conclusion</p> <p>A 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 <url>http://avena.pw.usda.gov/wheatD/fpcbrowser.shtml</url>.</p> |
| first_indexed | 2024-04-13T11:25:40Z |
| format | Article |
| id | doaj.art-b0abeec823344871b764145f20f67d3f |
| institution | Directory Open Access Journal |
| issn | 1471-2164 |
| language | English |
| last_indexed | 2024-04-13T11:25:40Z |
| publishDate | 2010-12-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Genomics |
| spelling | doaj.art-b0abeec823344871b764145f20f67d3f2022-12-22T02:48:43ZengBMCBMC Genomics1471-21642010-12-0111169210.1186/1471-2164-11-692A new implementation of high-throughput five-dimensional clone pooling strategy for BAC library screeningDeal Karin RXu KenongLuo Ming-ChengYou Frank MAnderson Olin DDvorak Jan<p>Abstract</p> <p>Background</p> <p>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 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.</p> <p>Results</p> <p>A 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 <it>Aegilops tauschii </it>chromosome 2D and <it>Ae. tauschii </it>contig maps. Clones in single contigs were successfully assigned to 48 (87%) specific SNP markers on the map with 91% precision.</p> <p>Conclusion</p> <p>A 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 <url>http://avena.pw.usda.gov/wheatD/fpcbrowser.shtml</url>.</p>http://www.biomedcentral.com/1471-2164/11/692 |
| spellingShingle | Deal Karin R Xu Kenong Luo Ming-Cheng You Frank M Anderson Olin D Dvorak Jan A new implementation of high-throughput five-dimensional clone pooling strategy for BAC library screening BMC Genomics |
| title | A new implementation of high-throughput five-dimensional clone pooling strategy for BAC library screening |
| title_full | A new implementation of high-throughput five-dimensional clone pooling strategy for BAC library screening |
| title_fullStr | A new implementation of high-throughput five-dimensional clone pooling strategy for BAC library screening |
| title_full_unstemmed | A new implementation of high-throughput five-dimensional clone pooling strategy for BAC library screening |
| title_short | A new implementation of high-throughput five-dimensional clone pooling strategy for BAC library screening |
| title_sort | new implementation of high throughput five dimensional clone pooling strategy for bac library screening |
| url | http://www.biomedcentral.com/1471-2164/11/692 |
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