Bioinformatics' approaches to detect genetic variation in whole genome sequencing data

Current genetic marker repositories are not sufficient or even are completely lacking for most farm animals. However, genetic markers are essential for the development of a research tool facilitating discovery of genetic factors that contribute to resistance to disease and the overall welfare and performance in farm animals. By large scale identification of Single Nucleotide Polymorphisms (SNPs) and Structural Variants (SVs) we aimed to contribute to the development of a repository of genetic variants for farm animals. For this purpose bioinformatics data pipelines were designed and validated to address the challenge of the cost effective identification of genetic markers in DNA sequencing data even in absence of a fully sequenced reference genome. To find SNPs in pig, we analysed publicly available whole genome shotgun sequencing datasets by sequence alignment and clustering. Sequence clusters were assigned to genomic locations using publicly available BAC sequencing and BAC mapping data. Within the sequence clusters thousands of SNPs were detected of which the genomic location is roughly known. For turkey and duck, species that both were lacking a sufficient sequence data repository for variant discovery, we applied next-generation sequencing (NGS) on a reduced genome representation of a pooled DNA sample. For turkey a genome reference was reconstructed from our sequencing data and available public sequencing data whereas in duck the reference genome constructed by a (NGS) project was used. SNPs obtained by our cost-effective SNP detection procedure still turned out to cover, at intervals, the whole turkey and duck genomes and are of sufficient quality to be used in genotyping studies. Allele frequencies, obtained by genotyping animal panels with a subset our SNPs, correlated well with those observed during SNP detection. The availability of two external duck SNP datasets allowed for the construction of a subset of SNPs which we had in common with these sets. Genotyping turned out that this subset was of outstanding quality and can be used for benchmarking other SNPs that we identified within duck. Ongoing developments in (NGS) allowed for paired end sequencing which is an extension on sequencing analysis that provides information about which pair of reads are coming from the outer ends of one sequenced DNA fragment. We applied this technique on a reduced genome representation of four chicken breeds to detect SVs. Paired end reads were mapped to the chicken reference genome and SVs were identified as abnormally aligned read pairs that have orientation or span sizes discordant from the reference genome. SV detection parameters, to distinguish true structural variants from false positives, were designed and optimized by validation of a small representative sample of SVs using PCR and traditional capillary sequencing. To conclude: we developed SNP repositories which fulfils a requirement for SNPs to perform linkage analysis, comparative genomics QTL studies and ultimately GWA studies in a range of farm animals. We also set the first step in developing a repository for SVs in chicken, a relatively new genetic marker in animal sciences. <br/

Dynamics of gene silencing during X inactivation using allele-specific RNA-seq

Background: During early embryonic development, one of the two X chromosomes in mammalian female cells is inactivated to compensate for a potential imbalance in transcript levels with male cells, which contain a single X chromosome. Here, we use mouse female embryonic stem cells (ESCs) with non-random X chromosome inactivation (XCI) and polymorphic X chromosomes to study the dynamics of gene silencing over the inactive X chromosome by high-resolution allele-specific RNA-seq. Results: Induction of XCI by differentiation of female ESCs shows that genes proximal to the X-inactivation center are silenced earlier than distal genes, while lowly expressed genes show faster XCI dynamics than highly expressed genes. The active X chromosome shows a minor but significant increase in gene activity during differentiation, resulting in complete dosage compensation in differentiated cell types. Genes escaping XCI show little or no silencing during early propagation of XCI. Allele-specific RNA-seq of neural progenitor cells generated from the female ESCs identifies three regions distal to the X-inactivation center that escape XCI. These regions, which stably escape during propagation and maintenance of XCI, coincide with topologically associating domains (TADs) as present in the female ESCs. Also, the previously characterized gene clusters escaping XCI in human fibroblasts correlate with TADs. Conclusions: The gene silencing observed during XCI provides further insight in the establishment of the repressive complex formed by the inactive X chromosome. The association of e

Transcriptomics of enterotoxigenic Escherichia coli infection. Individual variation in intestinal gene expression correlates with intestinal function

Acute secretory diarrhea is a major cause of morbidity and mortality in young animals and humans. Deaths result from excessive fluid and electrolyte losses. The disease is caused by non-invasive bacteria such as Vibrio cholerae and Escherichia coli which produce enterotoxins, however, much less is known about the role of individual host responses. Here we report the response of intact porcine small intestinal mucosa to infection with enterotoxigenic E. coli (ETEC). Jejunal segments in four piglets were infused with or without ETEC, and perfused for 8 h, and net absorption measured. Microarray analysis at 8 h post-infection showed significant differential regulation of on average fifteen transcripts in mucosa, with considerable individual variation. Differential net absorption varied between animals, and correlated negatively with the number of up regulated genes, and with one individual gene (THO complex 4). This shows that quantitative differences in gene regulation can be functionally linked to the physiological response in these four animals

Development of a porcine small intestinal cDNA micro-array: characterization and functional analysis of the response to enterotoxigenic E-coli

The intestine is a complex and dynamic ecosystem, in which nutrients, exogenous compounds and micro-flora interact, and its condition is influenced by the complex interaction between these factors and host genetic elements. Furthermore, interactions of immune cells with the other components of the intestinal mucosa are essential in the defense against pathogens. The outcomes of these complex interactions determine resistance to infectious diseases. The development of genomic tools and techniques allows for analysis of multiple and complex host responses. We have constructed a porcine small intestinal micro-array, based on cDNA from jejunal mucosal scrapings. Material from two developmental distinct stages (4- and 12-week-old pigs) was used in order to assure a reasonably broad representation of mucosal transcripts. The micro-array consists of 3468 cDNAs spotted in quadruplicate. Comparison of the 4-week-old versus 12-week-old pigs revealed a differential expression in at least 300 spots. Furthermore, we report the early gene expression response of pig small intestine jejunal mucosa to infection with enterotoxigenic E. coli (ETEC) using the small intestinal segment perfusion (SISP) technique. A response pattern was found in which a marker for innate defense dominated, demonstrating the strength of this applied technology. Further analysis of these response patterns will contribute to a better understanding of enteric health and disease in pigs. The great similarity between pig and human suggest results from these continuing studies should be applicable for both agricultural and human biomedical purpose

Development of a porcine small intestinal cDNA micro-array: characterization and functional analysis of the response to enterotoxigenic E-coli

The intestine is a complex and dynamic ecosystem, in which nutrients, exogenous compounds and micro-flora interact, and its condition is influenced by the complex interaction between these factors and host genetic elements. Furthermore, interactions of immune cells with the other components of the intestinal mucosa are essential in the defense against pathogens. The outcomes of these complex interactions determine resistance to infectious diseases. The development of genomic tools and techniques allows for analysis of multiple and complex host responses. We have constructed a porcine small intestinal micro-array, based on cDNA from jejunal mucosal scrapings. Material from two developmental distinct stages (4- and 12-week-old pigs) was used in order to assure a reasonably broad representation of mucosal transcripts. The micro-array consists of 3468 cDNAs spotted in quadruplicate. Comparison of the 4-week-old versus 12-week-old pigs revealed a differential expression in at least 300 spots. Furthermore, we report the early gene expression response of pig small intestine jejunal mucosa to infection with enterotoxigenic E. coli (ETEC) using the small intestinal segment perfusion (SISP) technique. A response pattern was found in which a marker for innate defense dominated, demonstrating the strength of this applied technology. Further analysis of these response patterns will contribute to a better understanding of enteric health and disease in pigs. The great similarity between pig and human suggest results from these continuing studies should be applicable for both agricultural and human biomedical purpose

Genome-wide SNP detection in the great tit Parus major using high throughput sequencing

Identifying genes that underlie ecological traits will open exiting possibilities to study gene–environment interactions in shaping phenotypes and in measuring natural selection on genes. Evolutionary ecology has been pursuing these objectives for decades, but they come into reach now that next generation sequencing technologies have dramatically lowered the costs to obtain the genomic sequence information that is currently lacking for most ecologically important species. Here we describe how we generated over 2 billion basepairs of novel sequence information for an ecological model species, the great tit Parus major. We used over 16 million short sequence reads for the de novo assembly of a reference sequence consisting of 550 000 contigs, covering 2.5%of the genome of the great tit. This reference sequence was used as the scaffold for mapping of the sequence reads, which allowed for the detection of over 20 000 novel single nucleotide polymorphisms. Contigs harbouring 4272 of the single nucleotide polymorphisms could be mapped to a unique location on the recently sequenced zebra finch genome. Of all the great tit contigs, significantly more were mapped to the microchromosomes than to the intermediate and the macrochromosomes of the zebra finch, indicating a higher overall level of sequence conservation on the microchromosomes than on the other types of chromosomes. The large number of great tit contigs that can be aligned to the zebra finch genome shows that this genome provides a valuable framework for large scale genetics, e.g. QTL mapping or whole genome association studies, in passerines.

Allele-specific RNA-seq expression profiling of imprinted genes in mouse isogenic pluripotent states

Contains fulltext : 201538.pdf (publisher's version ) (Open Access)21 p

Dynamics of gene silencing during X inactivation using allele-specific RNA-seq (vol 16, 149, 2015)

Contains fulltext : 157244.pdf (publisher's version ) (Open Access

Erratum to: Dynamics of gene silencing during X inactivation using allele-specific RNA-seq

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Dynamics of gene silencing during X inactivation using allele-specific RNA-seq (vol 16, 149, 2015)

Contains fulltext : 157244.pdf (publisher's version ) (Open Access