| description |
The MHC region contains many genes that are key regulators of both innate and adaptive immunity including the polymorphic MHCI and MHCII genes. Consequently, the characterisation of the repertoire of MHC genes is critical to understanding the variation that determines the nature of immune responses. Currently our knowledge of the bovine MHCI repertoire is limited with only the Holstein-Friesian breed having been studied in any depth. Traditional methods of MHCI genotyping are of low resolution and laborious and this has been a major impediment to a more comprehensive analysis of the MHCI repertoire of other cattle breeds. Next-generation sequencing (NGS) technologies have been used to enable high throughput and much higher resolution MHCI typing in a number of species. In this study we have developed a MiSeq platform approach and requisite bioinformatics pipeline to facilitate typing of bovine MHCI repertoires. The method was validated initially on a cohort of Holstein-Friesian animals and then demonstrated to enable characterisation of MHCI repertoires in African cattle breeds, for which there was limited or no available data. During the course of these studies we identified >140 novel classical MHCI genes and defined 62 novel MHCI haplotypes, dramatically expanding the known bovine MHCI repertoire.Construction protocol: Total RNAs were extracted from white blood cell pellets prepared form blood obtained by venepuncture using Tri-reagent (Sigma-Aldrich, Dorset) and cDNA prepared using the Reverse Transcription Kit (Promega, WI); both according to the manufacturer’s protocols. PCR amplification was performed with the Phusion HF PCR kit (NEB, Herts.) using bovine MHCI primers that included Illumina sequencing primer annealing sites, multiple sample identifier tags (D501-508 and D701-712) and Illumina ligation adaptors. PCR products from ~96 animals were pooled to form a library which was then sequenced on the Illumina Miseq platform. |