Dataset Information

High-throughput physical map anchoring via BAC-pool sequencing.

ABSTRACT: Physical maps created from large insert DNA libraries, typically cloned in BAC vector, are valuable resources for map-based cloning and de novo genome sequencing. The maps are most useful if contigs of overlapping DNA clones are anchored to chromosome(s), and ordered along them using molecular markers. Here we present a novel approach for anchoring physical maps, based on sequencing three-dimensional pools of BAC clones from minimum tilling path.We used physical map of wheat chromosome arm 3DS to validate the method with two different DNA sequence datasets. The first comprised 567 genes ordered along the chromosome arm based on syntenic relationship of wheat with the sequenced genomes of Brachypodium, rice and sorghum. The second dataset consisted of 7,136 SNP-containing sequences, which were mapped genetically in Aegilops tauschii, the donor of the wheat D genome. Mapping of sequence reads from individual BAC pools to the first and the second datasets enabled unambiguous anchoring 447 and 311 3DS-specific sequences, respectively, or 758 in total.We demonstrate the utility of the novel approach for BAC contig anchoring based on mass parallel sequencing of three-dimensional pools prepared from minimum tilling path of physical map. The existing genetic markers as well as any other DNA sequence could be mapped to BAC clones in a single in silico experiment. The approach reduces significantly the cost and time needed for anchoring and is applicable to any genomic project involving the construction of anchored physical map.

SUBMITTER: Cvikova K

PROVIDER: S-EPMC4407875 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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High-throughput physical map anchoring via BAC-pool sequencing.

Cviková Kateřina K Cattonaro Federica F Alaux Michael M Stein Nils N Mayer Klaus Fx KF Doležel Jaroslav J Bartoš Jan J

BMC plant biology 20150411

<h4>Background</h4>Physical maps created from large insert DNA libraries, typically cloned in BAC vector, are valuable resources for map-based cloning and de novo genome sequencing. The maps are most useful if contigs of overlapping DNA clones are anchored to chromosome(s), and ordered along them using molecular markers. Here we present a novel approach for anchoring physical maps, based on sequencing three-dimensional pools of BAC clones from minimum tilling path.<h4>Results</h4>We used physica ...[more]

PMID: 25887276

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Project description:BackgroundPotato is the world's third most important food crop, yet cultivar improvement and genomic research in general remain difficult because of the heterozygous and tetraploid nature of its genome. The development of physical map resources that can facilitate genomic analyses in potato has so far been very limited. Here we present the methods of construction and the general statistics of the first two genome-wide BAC physical maps of potato, which were made from the heterozygous diploid clone RH89-039-16 (RH).ResultsFirst, a gel electrophoresis-based physical map was made by AFLP fingerprinting of 64478 BAC clones, which were aligned into 4150 contigs with an estimated total length of 1361 Mb. Screening of BAC pools, followed by the KeyMaps in silico anchoring procedure, identified 1725 AFLP markers in the physical map, and 1252 BAC contigs were anchored the ultradense potato genetic map. A second, sequence-tag-based physical map was constructed from 65919 whole genome profiling (WGP) BAC fingerprints and these were aligned into 3601 BAC contigs spanning 1396 Mb. The 39733 BAC clones that overlap between both physical maps provided anchors to 1127 contigs in the WGP physical map, and reduced the number of contigs to around 2800 in each map separately. Both physical maps were 1.64 times longer than the 850 Mb potato genome. Genome heterozygosity and incomplete merging of BAC contigs are two factors that can explain this map inflation. The contig information of both physical maps was united in a single table that describes hybrid potato physical map.ConclusionsThe AFLP physical map has already been used by the Potato Genome Sequencing Consortium for sequencing 10% of the heterozygous genome of clone RH on a BAC-by-BAC basis. By layering a new WGP physical map on top of the AFLP physical map, a genetically anchored genome-wide framework of 322434 sequence tags has been created. This reference framework can be used for anchoring and ordering of genomic sequences of clone RH (and other potato genotypes), and opens the possibility to finish sequencing of the RH genome in a more efficient way via high throughput next generation approaches.

Project description:BackgroundCucumis melo (melon) belongs to the Cucurbitaceae family, whose economic importance among horticulture crops is second only to Solanaceae. Melon has high intra-specific genetic variation, morphologic diversity and a small genome size (450 Mb), which make this species suitable for a great variety of molecular and genetic studies that can lead to the development of tools for breeding varieties of the species. A number of genetic and genomic resources have already been developed, such as several genetic maps and BAC genomic libraries. These tools are essential for the construction of a physical map, a valuable resource for map-based cloning, comparative genomics and assembly of whole genome sequencing data. However, no physical map of any Cucurbitaceae has yet been developed. A project has recently been started to sequence the complete melon genome following a whole-genome shotgun strategy, which makes use of massive sequencing data. A BAC-based melon physical map will be a useful tool to help assemble and refine the draft genome data that is being produced.ResultsA melon physical map was constructed using a 5.7 x BAC library and a genetic map previously developed in our laboratories. High-information-content fingerprinting (HICF) was carried out on 23,040 BAC clones, digesting with five restriction enzymes and SNaPshot labeling, followed by contig assembly with FPC software. The physical map has 1,355 contigs and 441 singletons, with an estimated physical length of 407 Mb (0.9 x coverage of the genome) and the longest contig being 3.2 Mb. The anchoring of 845 BAC clones to 178 genetic markers (100 RFLPs, 76 SNPs and 2 SSRs) also allowed the genetic positioning of 183 physical map contigs/singletons, representing 55 Mb (12%) of the melon genome, to individual chromosomal loci. The melon FPC database is available for download at http://melonomics.upv.es/static/files/public/physical_map/.ConclusionsHere we report the construction of the first physical map of a Cucurbitaceae species described so far. The physical map was integrated with the genetic map so that a number of physical contigs, representing 12% of the melon genome, could be anchored to known genetic positions. The data presented is already helping to improve the quality of the melon genomic sequence available as a result of a project currently being carried out in Spain, adopting a whole genome shotgun approach based on 454 sequencing data.

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High-throughput physical map anchoring via BAC-pool sequencing.

Publications

High-throughput physical map anchoring via BAC-pool sequencing.

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