RH mapping by sequencing: chromosome-scale assembly of the duck genome

Like many other species, the duck genome has been sequenced thanks to the technological breakthrough provided by the emergence of Next Generation Sequencing (NGS). The resulting de novo assemblies are however made of thousands of scattered scaffolds. To achieve chromosome-scale contiguity, long-range intermediate genome maps remain indispensable. Radiation Hybrid (RH) maps have been used to assist the generation of chromosome-scale genome assemblies by taking advantage of the high density SNP chips that provide a large number of markers that can be efficiently genotyped on the panel.In the absence of such a resource in duck, we sequenced 100 hybrid clones of a duck RH panel enabling direct genotyping of the assembly scaffolds on the panel. The rationale is to use scaffolds as markers and to genotype the scaffolds by sequencing the clones: the presence/absence of a scaffold in a particular sequenced hybrid is attested by the presence/absence of reads mapping specifically to this scaffold. The detection of scaffolds exhibiting a chromosomal breakage resulting from the irradiation process revealed itself to be a critical issue of this genotyping by sequencing process. This process resulted in the construction of RH vectors for 2,027 scaffolds, representing a total of about 1 Gb of sequences (95% of the current Duck genome assembly). The subsequent linkage analysis enabled the construction of RH maps and therefore to organize, i.e. order and orient, the scaffolds into pseudomolecules associated to the corresponding duck chromosomes. We describe here the whole mapping process, from sequence-based genotyping to the construction of comparative maps, as well as few examples of intra-chromosomal rearrangements that have been identified by the comparison with the chicken, turkey and zebra finch genomes and subsequently confirmed by FISH.We describe a method to order and orient sequence scaffolds into super-scaffolds spanning entire chromosomes. The method, which requires a pre-existing RH panel and sequence scaffolds from an NGS assembly, relies on a shallow sequencing of the RH clones. This approach was applied to the duck genome and produced chromosome-scale scaffolds for 29 out of the 41 duck chromosomes.

Linking porcine microsatellite markers to known genome regions by identifying their human orthologs

Microsatellites, or tandem simple sequence repeats (SSRs), have become one of the most popular molecular markers in genome mapping because of their abundance across genomes and because of their high levels of polymorphism. However, information on which genes surround or flank them has remained very limited for most SSRs, especially in livestock species. In this study, an in silico comparative mapping approach was developed to link porcine SSRs to known genome regions by identifying their human orthologs. From a total of 1321 porcine microsatellites used in this study, 228 were found to have blocks in alignment with human genomic sequences. These 228 SSRs span about 1459 cM of the porcine genome, but with uneven distributions, ranging from 2 on SSC12 to 24 on SSC14. Linking these porcine SSRs to the known genome regions in the human genome also revealed 16 new putative synteny groups between these two species. Fifteen SSRs on SSC3 with identified human orthologs were typed on a pig-hamster radiation hybrid (RH) panel and used in a joint analysis with 80 known gene markers previously mapped on SSC3 using the same panel. The analysis revealed that they were all highly linked to either one or both adjacent markers. These results indicated that assigning the porcine SSRs to known genome regions by identifying their human orthologs is a reliable approach. The process will provide a foundation for positional cloning of causative genes for economically important traits.Key words: pig, microsatellite markers, human orthologs, RH mapping.

A Cattle–Human Comparative Map Built with Cattle BAC-Ends and Human Genome Sequence

As a step toward the goal of adding the cattle genome to those available for multispecies comparative genome analysis, 40,224 cattle BAC clones were end-sequenced, yielding 60,547 sequences (BAC end sequences, BESs) after trimming with an average read length of 515 bp. Cattle BACs were anchored to the human and mouse genome sequences by BLASTN search, revealing 29.4% and 10.1% significant hits (E < e-5), respectively. More than 60% of all cattle BES hits in both the human and mouse genomes are located within known genes. In order to confirm in silico predictions of orthology and their relative position on cattle chromosomes, 84 cattle BESs with similarity to sequences on HSA11 were mapped using a cattle–hamster radiation hybrid (RH) panel. Resulting RH maps of BTA15 and BTA29 cover ∼85% of HSA11 sequence, revealing a complex patchwork shuffling of segments not explained by a simple translocation followed by internal rearrangements. Overlay of the mouse conserved syntenies onto HSA11 revealed that segmental boundaries appear to be conserved in all three species. The BAC clone-based comparative map provides a foundation for the evolutionary analysis of mammalian karyotypes and for sequencing of the cattle genome.