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Identification of novel transcripts and noncoding RNAs in bovine skin by deep next generation sequencing.

ABSTRACT: BACKGROUND: Deep RNA sequencing (RNAseq) has opened a new horizon for understanding global gene expression. The functional annotation of non-model mammalian genomes including bovines is still poor compared to that of human and mouse. This particularly applies to tissues without direct significance for milk and meat production, like skin, in spite of its multifunctional relevance for the individual. Thus, applying an RNAseq approach, we performed a whole transcriptome analysis of pigmented and nonpigmented bovine skin to describe the comprehensive transcript catalogue of this tissue. RESULTS: A total of 39,577 unique primary skin transcripts were mapped to the bovine reference genome assembly. The majority of the transcripts were mapped to known transcriptional units (65%). In addition to the reannotation of known genes, a substantial number (10,884) of unknown transcripts (UTs) were discovered, which had not previously been annotated. The classification of UTs was based on the prediction of their coding potential and comparative sequence analysis, subsequently followed by meticulous manual curation. The classification analysis and experimental validation of selected UTs confirmed that RNAseq data can be used to amend the annotation of known genes by providing evidence for additional exons, untranslated regions or splice variants, by approving genes predicted in silico and by identifying novel bovine loci. A large group of UTs (4,848) was predicted to potentially represent long noncoding RNA (lncRNA). Predominantly, potential lncRNAs mapped in intergenic chromosome regions (4,365) and therefore, were classified as potential intergenic lncRNA. Our analysis revealed that only about 6% of all UTs displayed interspecies conservation and discovered a variety of unknown transcripts without interspecies homology but specific expression in bovine skin. CONCLUSIONS: The results of our study demonstrate a complex transcript pattern for bovine skin and suggest a possible functional relevance of novel transcripts, including lncRNA, in the modulation of pigmentation processes. The results also indicate that the comprehensive identification and annotation of unknown transcripts from whole transcriptome analysis using RNAseq data remains a tremendous future challenge.

SUBMITTER: Weikard R

PROVIDER: S-EPMC3833843 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: The advent of large-scale gene expression technologies has helped to reveal in eukaryotic cells, the existence of thousands of non-coding transcripts, whose function and significance remain mostly poorly understood. Among these non-coding transcripts, long non-coding RNAs (lncRNAs) are the least well-studied but are emerging as key regulators of diverse cellular processes. In the present study, we performed a survey in bovine Longissimus thoraci of lincRNAs (long intergenic non-coding RNAs not overlapping protein-coding transcripts). To our knowledge, this represents the first such study in bovine muscle. RESULTS: To identify lincRNAs, we used paired-end RNA sequencing (RNA-Seq) to explore the transcriptomes of Longissimus thoraci from nine Limousin bull calves. Approximately 14-45 million paired-end reads were obtained per library. A total of 30,548 different transcripts were identified. Using a computational pipeline, we defined a stringent set of 584 different lincRNAs with 418 lincRNAs found in all nine muscle samples. Bovine lincRNAs share characteristics seen in their mammalian counterparts: relatively short transcript and gene lengths, low exon number and significantly lower expression, compared to protein-encoding genes. As for the first time, our study identified lincRNAs from nine different samples from the same tissue, it is possible to analyse the inter-individual variability of the gene expression level of the identified lincRNAs. Interestingly, there was a significant difference when we compared the expression variation of the 418 lincRNAs with the 10,775 known selected protein-encoding genes found in all muscle samples. In addition, we found 2,083 pairs of lincRNA/protein-encoding genes showing a highly significant correlated expression. Fourteen lincRNAs were selected and 13 were validated by RT-PCR. Some of the lincRNAs expressed in muscle are located within quantitative trait loci for meat quality traits. CONCLUSIONS: Our study provides a glimpse into the lincRNA content of bovine muscle and will facilitate future experimental studies to unravel the function of these molecules. It may prove useful to elucidate their effect on mechanisms underlying the genetic variability of meat quality traits. This catalog will complement the list of lincRNAs already discovered in cattle and therefore will help to better annotate the bovine genome.

Project description:We conducted an unbiased metagenomics survey using plasma from patients with chronic hepatitis B, chronic hepatitis C, autoimmune hepatitis (AIH), non-alcoholic steatohepatitis (NASH), and patients without liver disease (control). RNA and DNA libraries were sequenced from plasma filtrates enriched in viral particles to catalog virus populations. Hepatitis viruses were readily detected at high coverage in patients with chronic viral hepatitis B and C, but only a limited number of sequences resembling other viruses were found. The exception was a library from a patient diagnosed with hepatitis C virus (HCV) infection that contained multiple sequences matching GB virus C (GBV-C). Abundant GBV-C reads were also found in plasma from patients with AIH, whereas Torque teno virus (TTV) was found at high frequency in samples from patients with AIH and NASH. After taxonomic classification of sequences by BLASTn, a substantial fraction in each library, ranging from 35% to 76%, remained unclassified. These unknown sequences were assembled into scaffolds along with virus, phage and endogenous retrovirus sequences and then analyzed by BLASTx against the non-redundant protein database. Nearly the full genome of a heretofore-unknown circovirus was assembled and many scaffolds that encoded proteins with similarity to plant, insect and mammalian viruses. The presence of this novel circovirus was confirmed by PCR. BLASTx also identified many polypeptides resembling nucleo-cytoplasmic large DNA viruses (NCLDV) proteins. We re-evaluated these alignments with a profile hidden Markov method, HHblits, and observed inconsistencies in the target proteins reported by the different algorithms. This suggests that sequence alignments are insufficient to identify NCLDV proteins, especially when these alignments are only to small portions of the target protein. Nevertheless, we have now established a reliable protocol for the identification of viruses in plasma that can also be adapted to other patient samples such as urine, bile, saliva and other body fluids.

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Identification of novel transcripts and noncoding RNAs in bovine skin by deep next generation sequencing.

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