Sequences Capture on microarrays for targeted sequencing on regions of interest : development in test studies in Animal Genomics – CapSeqAn

Detecting polymorphisms, such as SNPs (Single Nucleotide Polymorphisms) or CNVs (Copy Number Variations) in candidate genomic regions associated with complex traits or diseases in relevant animals is a prerequisite for further genotyping and identification of causes that explain phenotypic variations. Moreover sequencing of common genomic regions of interest in related species is a powerful tool for phylogenetic or evolutive studies and characterisation of orthologous genes. A major difficulty to achieve sequencing of these target regions from several animals is to specifically isolate the DNA template from the billions of base pairs of DNA present in mammalian and other large genomes.
In CapSeqAn project, we propose to develop a method aiming at enriching genomic regions of interest by capture on microarray. It is an emergent and promising technology that is an alternative to conventional cloning and PCR methods. This technique, associated with high-throughput massively parallel sequencing, will speed up the recovery and characterisation of large candidate genomic regions from various individuals that are relevant for a studied trait or disease.
Throuch 3 pilot studies in different scientific areas, the CapSeqAn project will address two questions, related to the activities of CRB GADIE platform and users' request:
1) Is it relevant and feasible to propose the capture of genomic regions of interest as a routine service? For this purpose, we will transfer the technique in the CRB GADIE and optimise the methods from existing technologies. We will work on two candidate regions: MITF locus for melanoma-bearing pigs (MeLiM model) and POLL (hornless) locus in cattle.
2) Is it possible to develop this technology for complementary applications such as CNV screening before sequencing and capture of heterologous sequences from related species? For this purpose, we will carry out a pilot study on MHC locus in chicken (and Galloanserae) and in pig (and Suidae).
The pilots studies :
The porcine MITF gene is located in a genomic region predisposing to cutaneous melanoma in MeLiM swine, a biomedical model for skin cancer in human. Considering MITF function in melanocyte biology and melanoma development, it is of great interest to study its genetic variability and association with the pathology.
Over the past decades and thanks to the development of genetic markers in cattle, the existence of the POLL locus has been demonstrated and several authors reported genetic linkage between the POLL locus and microsatellite markers, mapping the POLL locus to the centromeric region of bovine chromosome 1 (BTA1). Comparative sequencing of the entire POLL region (2 Mb) for animals from different breeds and phenotypes should help resolving the mystery around the genetic determinism of the inheritance of the horns in cattle.
The MHC is a genetic locus comprising a large set of genes evolutionarily conserved in Vertebrates. It is one of the highest gene dense regions in vertebrate genomes. About 60% of these genes have functions related to immunity and inflammation, including genes coding for protein involved in antigen presentation to T-cells. Class I and class II genes display a very high rate of non-synonymous nucleotide polymorphism, particularly in the peptide binding region. Moreover, variation in copy number of these genes is also described between and within species. The study of MHC in pig and chicken aims here to integrate two levels of polymorphisms (SNPs and CNVs) in the characterisation of individual haplotypes.
MHC locus is a pilot region for evolutive analysis, due to its size and gene content with contrasted evolutionary rates. It is a key region to study host-pathogen interactions and the mechanism of pathogen-driven balance selection. The capture sequences from a phylogenetically consistent set of species related respectively to pig and chicken, would allow studying the pattern of evolution inside MHC locus, where unusually high genetic diversity is considered to be the consequence of pathogen driven balance selection.