Whole Genome Profiling provides a robust framework for physical mapping and sequencing in the highly complex and repetitive wheat genome

Abstract

BackgroundSequencing projects using a clone-by-clone approach require the availability of a robust physical map. The SNaPshot technology, based on pair-wise comparisons of restriction fragments sizes, has been used recently to build the first physical map of a wheat chromosome and to complete the maize physical map. However, restriction fragments sizes shared randomly between two non-overlapping BACs often lead to chimerical contigs and mis-assembled BACs in such large and repetitive genomes. Whole Genome Profiling (WGP™) was developed recently as a new sequence-based physical mapping technology and has the potential to limit this problem.ResultsA subset of the wheat 3B chromosome BAC library covering 230 Mb was used to establish a WGP physical map and to compare it to a map obtained with the SNaPshot technology. We first adapted the WGP-based assembly methodology to cope with the complexity of the wheat genome. Then, the results showed that the WGP map covers the same length than the SNaPshot map but with 30% less contigs and, more importantly with 3.5 times less mis-assembled BACs. Finally, we evaluated the benefit of integrating WGP tags in different sequence assemblies obtained after Roche/454 sequencing of BAC pools. We showed that while WGP tag integration improves assemblies performed with unpaired reads and with paired-end reads at low coverage, it does not significantly improve sequence assemblies performed at high coverage (25x) with paired-end reads.ConclusionsOur results demonstrate that, with a suitable assembly methodology, WGP builds more robust physical maps than the SNaPshot technology in wheat and that WGP can be adapted to any genome. Moreover, WGP tag integration in sequence assemblies improves low quality assembly. However, to achieve a high quality draft sequence assembly, a sequencing depth of 25x paired-end reads is required, at which point WGP tag integration does not provide additional scaffolding value. Finally, we suggest that WGP tags can support the efficient sequencing of BAC pools by enabling reliable assignment of sequence scaffolds to their BAC of origin, a feature that is of great interest when using BAC pooling strategies to reduce the cost of sequencing large genomes.

Integrating cereal genomics to support innovation in the Triticeae

“…KeyGene (www.keygene.com) recently developed a new approach called Whole Genome Profiling (WGP, van Oeveren et al 2011) that is based on the sequencing of the ends of restriction endonuclease fragments after digestion of BAC pools. The application of WGP to a subset of the wheat chromosome 3B physical map (Philippe et al 2012) demonstrated that this is a viable approach for a complex genome such as wheat and that it reduces the amount of chimeric and misassembled clones compared to the SNAPshot method while providing sequence tag information that can be used to support pooling strategies for sequencing. Thus, robust methodologies and protocols are now in place for assembling reliable physical maps in the Triticeae genomes.…”

Systems Biology for Smart Crops and Agricultural Innovation: Filling the Gaps between Genotype and Phenotype for Complex Traits Linked with Robust Agricultural Productivity and Sustainability

“…Substantial progress has been made in identifying more than 80% of the genes in the large genomes of Triticeae crops such as barley (Mayer et al, 2011) and wheat (Philippe et al, 2012). These resources allow us to investigate genome dynamics by using phylogenomics and comparative genomics analysis tools to facilitate the identification of key genes such as MONOPTEROS, ABI3, FUS3, LEC2, AFL B3 etc.…”

Development of the BAC Physical Maps of Wheat Chromosome 6B for Its Genomic Sequencing

“…Whole Genome Profi ling (WGP TM ) is an alternative method for the establishment of the chromosome physical maps developed based on a next-generation sequencingbased technology (van Oeveren et al 2011 ). The effi ciency of WGP on physical mappings of wheat chromosomes has been demonstrated by comparison with that of SNaPshot (Philippe et al 2012 ).…”

“…After fi ltering BACs, assembly of the fi ngerprints representing nine and ten times equivalent of 6BS and 6BL, respectively, was performed with the FingerPrinted Contigs (FPC) software (Soderlund et al 1997 ). A stepwise method was used for the assembly to further improve the quality of BAC contigs, which was defi ned by Paux et al ( 2008 ) during the construction of chromosome 3B physical map and fi nally modifi ed by Philippe et al ( 2012 ). The physical maps of chromosome 6B have been successfully established to have an estimated chromosomal coverage of more than 90 %.…”