Tor Erik Jørgensen

Large-scale sequence analyses of Atlantic cod.

The Atlantic cod (Gadus morhua) is a key species in the North Atlantic ecosystem and commercial fisheries, with increasing aquacultural production in several countries. A Norwegian effort to sequence the complete 0.9Gbp genome by the 454 pyrosequencing technology has been initiated and is in progress. Here we review recent progress in large-scale sequence analyses of the nuclear genome, the mitochondrial genome and genome-wide microRNA identification in the Atlantic cod. The nuclear genome will be de novo sequenced with 25 times oversampling. A total of 120 mitochondrial genomes, sampled from several locations in the North Atlantic, are being completely sequenced by Sanger technology in a high-throughput pipeline. These sequences will be included in a new database for maternal marker reference of Atlantic cod diversity. High-throughput 454 sequencing, as well as Evolutionary Image Array (EvoArray) informatics, is used to investigate the complete set of expressed microRNAs and corresponding mRNA targets in various developmental stages and tissues. Information about microRNA profiles will be essential in the understanding of transcriptome complexity and regulation. Finally, developments and perspectives of Atlantic cod aquaculture are discussed in the light of next-generation high-throughput sequence technologies.

Halibut mitochondrial genomes contain extensive heteroplasmic tandem repeat arrays involved in DNA recombination

BackgroundHalibuts are commercially important flatfish species confined to the North Pacific and North Atlantic Oceans. We have determined the complete mitochondrial genome sequences of four specimens each of Atlantic halibut (Hippoglossus hippoglossus), Pacific halibut (Hippoglossus stenolepis) and Greenland halibut (Reinhardtius hippoglossoides), and assessed the nucleotide variability within and between species.ResultsAbout 100 variable positions were identified within the four specimens in each halibut species, with the control regions as the most variable parts of the genomes (10 times that of the mitochondrial ribosomal DNA). Due to tandem repeat arrays, the control regions have unusually large sizes compared to most vertebrate mtDNAs. The arrays are highly heteroplasmic in size and consist mainly of different variants of a 61-bp motif. Halibut mitochondrial genomes lacking arrays were also detected.ConclusionThe complexity, distribution, and biological role of the heteroplasmic tandem repeat arrays in halibut mitochondrial control regions are discussed. We conclude that the most plausible explanation for array maintenance includes both the slipped-strand mispairing and DNA recombination mechanisms.

Genomic divergence between the migratory and stationary ecotypes of Atlantic cod

Atlantic cod displays a range of phenotypic and genotypic variations, which includes the differentiation into coastal stationary and offshore migratory types of cod that co‐occur in several parts of its distribution range and are often sympatric on the spawning grounds. Differentiation of these ecotypes may involve both historical separation and adaptation to ecologically distinct environments, the genetic basis of which is now beginning to be unravelled. Genomic analyses based on recent sequencing advances are able to document genomic divergence in more detail and may facilitate the exploration of causes and consequences of genome‐wide patterns. We examined genomic divergence between the stationary and migratory types of cod in the Northeast Atlantic, using next‐generation sequencing of pooled DNA from each of two population samples. Sequence data was mapped to the published cod genome sequence, arranged in more than 6000 scaffolds (611 Mb). We identified 25 divergent scaffolds (26 Mb) with a higher than average gene density, against a backdrop of overall moderate genomic differentiation. Previous findings of localized genomic divergence in three linkage groups were confirmed, including a large (15 Mb) genomic region, which seems to be uniquely involved in the divergence of migratory and stationary cod. The results of the pooled sequencing approach support and extend recent findings based on single‐nucleotide polymorphism markers and suggest a high degree of reproductive isolation between stationary and migratory cod in the North‐east Atlantic.

RNA deep sequencing of the Atlantic cod transcriptome

The Atlantic cod (Gadus morhua) is an emerging aquaculture species. Efforts to develop and characterize its genomic recourses, including draft-grade genome sequencing, have been initiated by the research community. The transcriptome represents the whole complement of RNA transcripts in cells and tissues and reflects the expressed genes at various life stages, tissue types, physiological states, and environmental conditions. We are investigating the Atlantic cod transcriptome by Roche 454, Illumina GA, and ABI SOLiD deep sequencing platforms and corresponding bioinformatics. Both embryonic developmental stages and adult tissues are studied. Here we summarize our recent progress in the analyses of nuclear and mitochondrial polyA mRNAs, non-protein-coding intermediate RNAs, and regulatory microRNAs.

Characterization of mitochondrial mRNAs in codfish reveals unique features compared to mammals

Expression and processing of mitochondrial gene transcripts are fundamental to mitochondrial function, but information from early vertebrates like teleost fishes is essentially lacking. We have analyzed mitogenome sequences of ten codfishes (family Gadidae), and provide complete sequences from three new species (Saithe, Pollack and Blue whiting). Characterization of the mitochondrial mRNAs in Saithe and Atlantic cod identified a set of ten poly(A) transcripts, and six UAA stop codons are generated by posttranscriptional polyadenylation. Structural assessment of poly(A) sites is consistent with an RNaseP cleavage activity 5′ of tRNA acceptor-like stems. COI, ND5 and ND6 mRNAs were found to harbor 3′ UTRs with antisense potential extending into neighboring gene regions. While the 3′ UTR of COI mRNA is complementary to the tRNASer (UCN) and highly similar to that detected in human mitochondria, the ND5 and ND6 3′ UTRs appear more heterogenic. Deep sequencing confirms expression of all mitochondrial mRNAs and rRNAs, and provides information about the precise 5′ ends in mature transcripts. Our study supports an overall evolutionary conservation in mitochondrial RNA processing events among vertebrates, but reveals some unique 5′ and 3′ end characteristics in codfish mRNAs with implications to antisense regulation of gene expression.

Performance Comparison of Digital microRNA Profiling Technologies Applied on Human Breast Cancer Cell Lines

MicroRNA profiling represents an important first-step in deducting individual RNA-based regulatory function in a cell, tissue, or at a specific developmental stage. Currently there are several different platforms to choose from in order to make the initial miRNA profiles. In this study we investigate recently developed digital microRNA high-throughput technologies. Four different platforms were compared including next generation SOLiD ligation sequencing and Illumina HiSeq sequencing, hybridization-based NanoString nCounter, and miRCURY locked nucleic acid RT-qPCR. For all four technologies, full microRNA profiles were generated from human cell lines that represent noninvasive and invasive tumorigenic breast cancer. This study reports the correlation between platforms, as well as a more extensive analysis of the accuracy and sensitivity of data generated when using different platforms and important consideration when verifying results by the use of additional technologies. We found all the platforms to be highly capable for microRNA analysis. Furthermore, the two NGS platforms and RT-qPCR all have equally high sensitivity, and the fold change accuracy is independent of individual miRNA concentration for NGS and RT-qPCR. Based on these findings we propose new guidelines and considerations when performing microRNA profiling.

Distinct Small RNA Signatures in Extracellular Vesicles Derived from Breast Cancer Cell Lines

Breast cancer is a heterogeneous disease, and different subtypes of breast cancer show distinct cellular morphology, gene expression, metabolism, motility, proliferation, and metastatic potential. Understanding the molecular features responsible for this heterogeneity is important for correct diagnosis and better treatment strategies. Extracellular vesicles (EVs) and their associated molecules have gained much attention as players in intercellular communication, ability to precondition specific organs for metastatic invasion, and for their potential role as circulating cancer biomarkers. EVs are released from the cells and contain proteins, DNA, and long and small RNA species. Here we show by high-throughput small RNA-sequencing that EVs from nine different breast cancer cell lines share common characteristics in terms of small RNA content that are distinct from their originating cells. Most strikingly, a highly abundant small RNA molecule derived from the nuclear 28S rRNA is vastly enriched in EVs. The miRNA profiles in EVs correlate with the cellular miRNA expression pattern, but with a few exceptions that includes miR-21. This cancer-associated miRNA is retained in breast cancer cell lines. Finally, we report that EVs from breast cancer cell lines cluster together based on their small RNA signature when compared to EVs derived from other cancer cell lines. Altogether, our data demonstrate that breast cancer cell lines manifest a specific small RNA signature in their released EVs. This opens up for further evaluation of EVs as breast cancer biomarkers.

Mitogenome sequence variation in migratory and stationary ecotypes of North-east Atlantic cod.

Sequencing of mitochondrial gene fragments from specimens representing a wide range of geographical locations has indicated limited population structuring in Atlantic cod (Gadus morhua). We recently performed whole genome analysis based on next-generation sequencing of two pooled ecotype samples representing offshore migratory and inshore stationary cod from the North-east Atlantic Ocean. Here we report molecular features and variability of the 16.7kb mitogenome component that was collected from the datasets. These sequences represented more than 25 times coverage of each individual and more than 1100 times coverage of each ecotype sample. We estimated the mitogenome to have evolved 14 times more rapidly than the nuclear genome. Among the 365 single nucleotide polymorphism (SNP) sites identified, 121 were shared between ecotypes, and 151 and 93 were private within the migratory and stationary cod, respectively. We found 323 SNPs to be located in protein coding genes, of which 29 were non-synonymous. One synonymous site in ND2 was likely to be under positive selection. FST measurements indicated weak differentiation in ND1 and ND2 between ecotypes. We conclude that the Atlantic cod mitogenome and the nuclear genome apparently evolved by distinct evolutionary constraints, and that the reproductive isolation observed from whole genome analysis was not visible in the mtDNA sequences.

An evolutionary preserved intergenic spacer in gadiform mitogenomes generates a long noncoding RNA

BackgroundVertebrate mitogenomes are economically organized and usually lack intergenic sequences other than the control region. Intergenic spacers located between the tRNAThr and tRNAPro genes (“T-P spacers”) have been observed in several taxa, including gadiform species, but information about their biological roles and putative functions is still lacking.ResultsSequence characterization of the complete European hake Merluccius merluccius mitogenome identified a complex T-P spacer ranging in size from 223–532 bp. Further analyses of 32 gadiform species, representing 8 families and 28 genera, revealed the evolutionary preserved presence of T-P spacers across all taxa. Molecular complexity of the T-P spacers was found to be coherent with the phylogenetic relationships, supporting a common ancestral origin and gain of function during codfish evolution. Intraspecific variation of T-P spacer sequences was assessed in 225 Atlantic cod specimens and revealed 26 haplotypes. Pyrosequencing data representing the mito-transcriptome poly (A) fraction in Atlantic cod identified an abundant H-strand specific long noncoding RNA of about 375 nt. The T-P spacer corresponded to the 5′ part of this transcript, which terminated within the control region in a tail-to-tail configuration with the L-strand specific transcript (the 7S RNA).ConclusionsThe T-P spacer is inferred to be evolutionary preserved in gadiform mitogenomes due to gain of function through a long noncoding RNA. We suggest that the T-P spacer adds stability to the H-strand specific long noncoding RNA by forming stable hairpin structures and additional protein binding sites.

Elucidating the small regulatory RNA repertoire of the sea anemone Anemonia viridis based on whole genome and small RNA sequencing

Abstract Cnidarians harbor a variety of small regulatory RNAs that include microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs), but detailed information is limited. Here, we report the identification and expression of novel miRNAs and putative piRNAs, as well as their genomic loci, in the symbiotic sea anemone Anemonia viridis. We generated a draft assembly of the A. viridis genome with putative size of 313 Mb that appeared to be composed of about 36% repeats, including known transposable elements. We detected approximately equal fractions of DNA transposons and retrotransposons. Deep sequencing of small RNA libraries constructed from A. viridis adults sampled at a natural CO2 gradient off Vulcano Island, Italy, identified 70 distinct miRNAs. Eight were homologous to previously reported miRNAs in cnidarians, whereas 62 appeared novel. Nine miRNAs were recognized as differentially expressed along the natural seawater pH gradient. We found a highly abundant and diverse population of piRNAs, with a substantial fraction showing ping–pong signatures. We identified nearly 22% putative piRNAs potentially targeting transposable elements within the A. viridis genome. The A. viridis genome appeared similar in size to that of other hexacorals with a very high divergence of transposable elements resembling that of the sea anemone genus Exaiptasia. The genome encodes and expresses a high number of small regulatory RNAs, which include novel miRNAs and piRNAs. Differentially expressed small RNAs along the seawater pH gradient indicated regulatory gene responses to environmental stressors.