Genome-wide association studies in livestock based on high-resolution genotyping and sequencing have revealed that the majority of signals associated with complex phenotypic traits are located outside of annotated protein-coding regions in the genome. The approaches of next-generation sequencing applied to whole transcriptome and chromatin profiles have provided information about existing genome-wide transcriptional activity and have revealed that the genomes are templates for thousands of long noncoding transcripts (lncRNAs). Despite their lack of coding capacity, many lncRNAs have been found to play functional roles in a variety of biological processes, which is adding a novel regulatory network to the complex structural organization and function of the genome. Here, we summarize main features of lncRNAs, provide an overview about computational tools and pipelines used for identification of lncRNAs from whole transcriptome datasets and review the current state of knowledge about lncRNAs in livestock species. Although lncRNAs are increasingly emerging as an integral component of the regulatory information encoded in the genome, the complexity of the transcriptomes in domesticated animals is inadequately characterized in comparison to human and mouse. Progress in elucidating whole transcriptomes of livestock species, including identification, functional annotation and characterization of lncRNAs, will be essential for a better understanding of basic biological processes associated with developmental, metabolic and immunological regulation and adaptation and phenotypic variation of complex traits in domesticated animals.
Bovine neonatal pancytopenia (BNP) is a new fatal, alloimmune/alloantibody mediated disease of new-born calves induced by ingestion of colostrum from cows, which had been vaccinated with a specific vaccine against the Bovine Virus Diarrhoea Virus (BVDV). The hypothesis of pathogenic MHC class I molecules in the vaccine had been put up, but no formal proof of specific causal MHC class I alleles has been provided yet. However, the unique features of the vaccine obviously result in extremely high specific antibody titres in the vaccinated animals, but apparently also in further molecules inducing BNP. Thus, a comprehensive picture of the immune response to the vaccine is essential. Applying the novel approach of next generation RNA sequencing (RNAseq), our study provides a new holistic, comprehensive analysis of the blood transcriptome regulation after vaccination with the specific BVDV vaccine. Our RNAseq approach identified a novel cytokine-like gene in the bovine genome that is highly upregulated after vaccination. This gene has never been described before in any other species and might be specific to ruminant immune response. Furthermore, our data revealed a very coordinated immune response to double-stranded (ds) RNA or a dsRNA analogue after vaccination with the inactivated single-stranded (ss) RNA vaccine. This would suggest either a substantial contamination of the vaccine with dsRNA from host cells after virus culture or a dsRNA analogue applied to the vaccine. The first option would highlight the potential risks associated with virus culture on homologous cells during vaccine production; the latter option would emphasise the potential risks associated with immune stimulating adjuvants used in vaccine production.
Usually, reads from transcriptome sequencing data unmapped to the target species' reference genome are disregarded. A recent RNAseq project on the new fatal disease Bovine Neonatal Pancytopenia had indicated an unexplained immune response signature to a double-stranded RNA virus. To unravel its background, contigs were de novo assembled from unmapped RNAseq reads and aligned against the bovine genome assemblies and multispecies NCBI databases. Lack of genuine virus sequence contigs rejected the hypothesis of a live virus being causal for the unexplained immune response. Alignment data also demonstrated incomplete bovine reference genome assemblies. In addition, we found that several parasite and virus genome reference assemblies in NCBI were contaminated with bovine DNA and confirmed recombination of bovine DNA into BVD virus strains. Exploring unmapped reads can extract useful biological information regarding the presence of microorganisms and can highlight issues with reference genome assemblies of host and pathogen species.
BackgroundMHC class I genotyping is essential for a wide range of biomedical, immunological and biodiversity applications. Whereas in human a comprehensive MHC class I allele catalogue is available, respective data in non-model species is scarce in spite of decades of research.ResultsTaking advantage of the new high-throughput RNA sequencing technology (RNAseq), we developed a novel RNAseq-assisted method (RAMHCIT) for MHC class I typing at nucleotide level. RAMHCIT is performed on white blood cells, which highly express MHC class I molecules enabling reliable discovery of new alleles and discrimination of closely related alleles due to the high coverage of alleles with reads. RAMHCIT is more comprehensive than previous methods, because no targeted PCR pre-amplification of MHC loci is necessary, which avoids preselection of alleles as usually encountered, when amplification with MHC class I primers is performed prior to sequencing. In addition to allele identification, RAMHCIT also enables quantification of MHC class I expression at allele level, which was remarkably consistent across individuals.ConclusionsSuccessful application of RAMHCIT is demonstrated on a data set from cattle with different phenotype regarding a lethal, vaccination-induced alloimmune disease (bovine neonatal pancytopenia), for which MHC class I alleles had been postulated as causal agents.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2688-0) contains supplementary material, which is available to authorized users.
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