Tiefencharakterisierung des intestinalen Transkriptoms der Maus mittels RNA-Seq

Die Schleimhaut des Darmtraktes ist charakterisiert durch komplexe metabolische und immunologische Prozesse und wird gesteuert durch hochdynamische Genexpressions-Programme. Mit der Verfügbarkeit von next generation sequencing und ihrer Nutzbarkeit für die Analyse von RNA-Sequenzen wurde die Genauigkeit über die globale Architektur des Transkriptoms gegenüber anderen Methoden wie microarrays auf eine neue Ebene befördert. Basierend auf dem 3‘ Oligo-dT priming von polyadenylierten Boten-RNA gefolgt von reverser Transkription und einem sogenannten template switch wurde eine Methode für RNA-Seq auf der Life Technologies SOLiD Plattform etabliert. Dieser Ansatz zeigte zuverlässige Informationen über das untersuchte Gewebe, z. B. in Bezug auf Genexpression und der Beobachtung von nicht-annotierten, transkriptionell aktiven Bereichen im Genom. In dieser Arbeit wird über die Tiefencharakterisierung des polyadenylierten Transkriptoms in zwei nah verwandten, dennoch unterschiedlichen Geweben des murinen Intestinaltrakts (Dünn- und Dickdarm) berichtet. Eine gewebespezifische Architektur des Transkriptoms und die Präsenz von zuvor nicht bekannten Bereichen transkriptioneller Aktivität wurden gefunden. Im ersten Schritt wurden Signaturen von 20.541 NCBI-RefSeq-Transkripten im Darm identifiziert (74,1 % der annotierten Gene), davon fanden sich 16.742 in beiden untersuchten Geweben. Obwohl die Mehrheit der Sequenzen annotierten Genen zugeordnet werden konnten, fanden sich 27.543 nicht-annotierte, transkriptionell aktive Regionen im Genom im Widerspruch zur aktuellen Genannotationen von RefSeq oder Ensembl. Unter Nutzung eines zweiten, unabhängigen, strangspezifischen Protokolls konnten 20.966 dieser Befunde bestätigt werden, die Mehrheit davon in direkter Nähe zu bekannten Genen. In der Folge wurden die Befunde bezüglich ihrer Nähe zu beschriebenen Exon-Elementen kategorisiert. Regionale Unterschiede zwischen Dünn- und Dickdarm dieser transkriptionell-aktiven, aber nicht annotierten Elemente wurden untersucht. Die vorliegende Arbeit demonstriert die Komplexität eines typischen intestinalen mRNA-Transkriptoms von Säugetieren anhand einer strangspezifischen Auflösung bis hin zur Einzelbase. Die Analysen zeigten zum ersten Mal ein strangspezifisches Bild von nicht-annotierten, transkriptionell aktiven Bereichen in zwei Geweben und repräsentieren eine Ressource für weitere Untersuchungen transkriptioneller Prozesse, welche zur molekularen Gewebeidentität beitragen. Die mittels RNA-Seq in dieser Arbeit erhobenen Daten waren von hoher, zuvor nicht zugänglicher Qualität, so dass RNA-Seq in der Zukunft vermutlich die neue Standardmethode für die genomweite Untersuchung des Transkriptoms darstellen wird.The intestinal mucosa is characterized by complex metabolic and immunological processes driven highly dynamic gene expression programs. With the advent of next generation sequencing and its utilization for the analysis of the RNA sequence space, the level of detail on the global architecture of the transcriptome reached a new order of magnitude compared to other methods like microarrays. A method for RNA-Seq based on 3’ oligo-dT priming of polyadenylated messenger RNA followed by reverse transcription and a template switch was established on the Life Technologies SOLiD platform. This approach showed robust information about the characterized tissue, for example in terms of gene expression and the observation of non-annotated transcriptionally active regions of the genome. This thesis reports the ultra-deep characterization of the polyadenylated transcriptome in two closely related, yet distinct regions of the mouse intestinal tract (small intestine and colon). The tissue-specific transcriptomal architecture and the presence of novel transcriptionally active regions were assessed. In the first step, signatures of 20,541 NCBI RefSeq transcripts could be identified in the intestine (74.1% of annotated genes), thereof 16,742 are common in both tissues. Although the majority of reads could be linked to annotated genes, 27,543 non-annotated transcriptionally active regions not consistent with current gene annotations in RefSeq or Ensembl were identified. By use of a second independent strand-specific RNA-Seq protocol, 20,966 of these nTARs were confirmed, most of them in vicinity of known genes. This thesis further categorized the findings by their relative adjacency to described exonic elements and investigated regional differences of novel transcribed elements in small intestine and colon. The current study demonstrates the complexity of an archetypal mammalian intestinal mRNA transcriptome in high resolution and identifies novel transcriptionally active regions at strand-specific, single base resolution. The analysis for the first time shows a strand-specific comparative picture of non-annotated transcriptionally active regions in two tissues and represents a resource for further investigation of the transcriptional processes that contribute to molecular tissue identity. RNA-Seq generated data showed high, to date unseen quality. This suggests that RNA-Seq will be the new standard method for genome-wide analysis of the transcriptome

Janus—a comprehensive tool investigating the two faces of transcription

Motivation: Protocols to generate strand-specific transcriptomes with next-generation sequencing platforms have been used by the scientific community roughly since 2008. Strand-specific reads allow for detection of antisense events and a higher resolution of expression profiles enabling extension of current transcript annotations. However, applications making use of this strandedness information are still scarce. Results: Here we present a tool (Janus), which focuses on the identification of transcriptional active regions in antisense orientation to known and novel transcribed elements of the genome. Janus can compare the antisense events of multiple samples and assigns scores to identify mutual expression of either transcript in a sense/antisense pair, which could hint to regulatory mechanisms. Janus is able to make use of single-nucleotide variant (SNV) and methylation data, if available, and reports the sense to antisense ratio of regions in the vicinity of the identified genetic and epigenetic variation. Janus interrogates positions of heterozygous SNVs to identify strand-specific allelic imbalance. Availability: Janus is written in C/C++ and freely available at http://www.ikmb.uni-kiel.de/janus/janus.html under terms of GNU General Public License, for both, Linux and Windows 64×. Although the binaries will work without additional downloads, the software depends on bamtools (https://github.com/pezmaster31/bamtools) for compilation. A detailed tutorial section is included in the first section of the supplemental material and included as brief readme.txt in the tutorial archive. Contact: [email protected] or [email protected] Supplementary information: Supplementary data are available at Bioinformatics onlin

Genome and low-iron response of an oceanic diatom adapted to chronic iron limitation

ABSTRACT: BACKGROUND: Biogeochemical elemental cycling is driven by primary production of biomass via phototrophic phytoplankton growth, with 40% of marine productivity being assigned to diatoms. Phytoplankton growth is widely limited by the availability of iron, an essential component of the photosynthetic apparatus. The oceanic diatom Thalassiosira oceanica shows a remarkable tolerance to low-iron conditions and was chosen as a model for deciphering the cellular response upon shortage of this essential micronutrient. RESULTS: The combined efforts in genomics, transcriptomics and proteomics reveal an unexpected metabolic flexibility in response to iron availability for T. oceanica CCMP1005. The complex response comprises cellular retrenchment as well as remodeling of bioenergetic pathways, where the abundance of iron-rich photosynthetic proteins is lowered, whereas iron-rich mitochondrial proteins are preserved. As a consequence of iron deprivation, the photosynthetic machinery undergoes a remodeling to adjust the light energy utilization with the overall decrease in photosynthetic electron transfer complexes. CONCLUSIONS: Beneficial adaptations to low-iron environments include strategies to lower the cellular iron requirements and to enhance iron uptake. A novel contribution enhancing iron economy of phototrophic growth is observed with the iron-regulated substitution of three metal-containing fructose-bisphosphate aldolases involved in metabolic conversion of carbohydrates for enzymes that do not contain metals. Further, our data identify candidate components of a high-affinity iron-uptake system, with several of the involved genes and domains originating from duplication events. A high genomic plasticity, as seen from the fraction of genes acquired through horizontal gene transfer, provides the platform for these complex adaptations to a low-iron world

A Powerful Method for Transcriptional Profiling of Specific Cell Types in Eukaryotes: Laser-Assisted Microdissection and RNA Sequencing

The acquisition of distinct cell fates is central to the development of multicellular organisms and is largely mediated by gene expression patterns specific to individual cells and tissues. A spatially and temporally resolved analysis of gene expression facilitates the elucidation of transcriptional networks linked to cellular identity and function. We present an approach that allows cell type-specific transcriptional profiling of distinct target cells, which are rare and difficult to access, with unprecedented sensitivity and resolution. We combined laser-assisted microdissection (LAM), linear amplification starting from <1 ng of total RNA, and RNA-sequencing (RNA-Seq). As a model we used the central cell of the Arabidopsis thaliana female gametophyte, one of the female gametes harbored in the reproductive organs of the flower. We estimated the number of expressed genes to be more than twice the number reported previously in a study using LAM and ATH1 microarrays, and identified several classes of genes that were systematically underrepresented in the transcriptome measured with the ATH1 microarray. Among them are many genes that are likely to be important for developmental processes and specific cellular functions. In addition, we identified several intergenic regions, which are likely to be transcribed, and describe a considerable fraction of reads mapping to introns and regions flanking annotated loci, which may represent alternative transcript isoforms. Finally, we performed a de novo assembly of the transcriptome and show that the method is suitable for studying individual cell types of organisms lacking reference sequence information, demonstrating that this approach can be applied to most eukaryotic organisms

A tissue-specific landscape of sense/antisense transcription in the mouse intestine

<p>Abstract</p> <p>Background</p> <p>The intestinal mucosa is characterized by complex metabolic and immunological processes driven highly dynamic gene expression programs. With the advent of next generation sequencing and its utilization for the analysis of the RNA sequence space, the level of detail on the global architecture of the transcriptome reached a new order of magnitude compared to microarrays.</p> <p>Results</p> <p>We report the ultra-deep characterization of the polyadenylated transcriptome in two closely related, yet distinct regions of the mouse intestinal tract (small intestine and colon). We assessed tissue-specific transcriptomal architecture and the presence of novel transcriptionally active regions (nTARs). In the first step, signatures of 20,541 NCBI RefSeq transcripts could be identified in the intestine (74.1% of annotated genes), thereof 16,742 are common in both tissues. Although the majority of reads could be linked to annotated genes, 27,543 nTARs not consistent with current gene annotations in RefSeq or ENSEMBL were identified. By use of a second independent strand-specific RNA-Seq protocol, 20,966 of these nTARs were confirmed, most of them in vicinity of known genes. We further categorized our findings by their relative adjacency to described exonic elements and investigated regional differences of novel transcribed elements in small intestine and colon.</p> <p>Conclusions</p> <p>The current study demonstrates the complexity of an archetypal mammalian intestinal mRNA transcriptome in high resolution and identifies novel transcriptionally active regions at strand-specific, single base resolution. Our analysis for the first time shows a strand-specific comparative picture of nTARs in two tissues and represents a resource for further investigating the transcriptional processes that contribute to tissue identity.</p

Apomictic and sexual germline development differ with respect to cell cycle, transcriptional, hormonal and epigenetic regulation

Seeds of flowering plants can be formed sexually or asexually through apomixis. Apomixis occurs in about 400 species and is of great interest for agriculture as it produces clonal offspring. It differs from sexual reproduction in three major aspects: (1) While the sexual megaspore mother cell (MMC) undergoes meiosis, the apomictic initial cell (AIC) omits or aborts meiosis (apomeiosis); (2) the unreduced egg cell of apomicts forms an embryo without fertilization (parthenogenesis); and (3) the formation of functional endosperm requires specific developmental adaptations. Currently, our knowledge about the gene regulatory programs underlying apomixis is scarce. We used the apomict Boechera gunnisoniana, a close relative of Arabidopsis thaliana, to investigate the transcriptional basis underlying apomeiosis and parthenogenesis. Here, we present the first comprehensive reference transcriptome for reproductive development in an apomict. To compare sexual and apomictic development at the cellular level, we used laser-assisted microdissection combined with microarray and RNA-Seq analyses. Conservation of enriched gene ontologies between the AIC and the MMC likely reflects functions of importance to germline initiation, illustrating the close developmental relationship of sexuality and apomixis. However, several regulatory pathways differ between sexual and apomictic germlines, including cell cycle control, hormonal pathways, epigenetic and transcriptional regulation. Enrichment of specific signal transduction pathways are a feature of the apomictic germline, as is spermidine metabolism, which is associated with somatic embryogenesis in various plants. Our study provides a comprehensive reference dataset for apomictic development and yields important new insights into the transcriptional basis underlying apomixis in relation to sexual reproduction

Comparisons between microarray and RNA-Seq data.

<p>(A) The average number of hits (log2(x+1)) for each gene are plotted on the y-axis and the corresponding normalized expression values from the array data are shown on the x-axis. Expression values of the genes having a probeset on the array are well correlated between the technologies (Spearman correlation: 0.63). (B) A Venn diagram summarizing the overlap between genes detected to be expressed in the RNA-Seq data sets and the array data.</p

Examples of sequence coverage in annotated (A) and unannotated (B) regions.

<p>Graphs in the upper parts of the panels represent the number of hits per base within the two replicates (CC1: cyan, CC2: yellow). Transcripts are drawn in the lower parts of the panels: dark boxes represent exons, bright lines mark introns and the arrowhead depicts the direction of transcription. (A) Sequence coverage at the region around the locus <i>AT4G27960</i> (<i>UBC9</i>) on chromosome 4. The two transcripts represent two isoforms of <i>AT4G27960</i>. Clearly visible is the lack of coverage at the introns and the non-uniformity of sequence coverage with the maxima close to the 3′ end of the transcripts. (B) Sequence coverage at a region on chromosome 5, which is not annotated as being transcribed. Hits in this region were assembled into transcripts using cufflinks <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029685#pone.0029685-Trapnell1" target="_blank">[11]</a>. For each replicate, two transcripts with overlapping 3′ ends could be assembled (CC1: cyan, CC2: yellow). Notably, the sequence coverage along these transcripts resembles the coverage observed at annotated transcripts (A). Also visible are the unsharp transcript boundaries which vary between the replicates.</p

Comparisons of expression values between the two RNA-Seq replicates.

<p>In each panel, the expression values (log2 of the number of hits plus one) for each feature are plotted on the x-axis (CC2) and the y-axis (CC1). Colors indicate the point density: red and blue indicate the highest, respectively lowest, densities. (A) refers to the approach that was based on the alignment of reads to the reference genome: given are the expression values of the “expressed” genes (Pearson correlation: 0.99, Spearman correlation: 0.83). (B) refers to the approach that was based on <i>de novo</i> assembly of the short reads. Reads from both replicates were pooled and assembled together. To calculate expression values, reads from both replicates were aligned to the assembled transcriptome (Spearman correlation: 0.87).</p

Schematic representation of the flower and the embryo sac of <i>Arabidopsis thaliana</i>.

<p>The flower of <i>Arabidopsis thaliana</i> consists of four whorls of organs: sepals, petals, anthers (male reproductive organs) and carpels (female reproductive organs). The carpels are fused and form the ovary, which harbors around fifty ovules. During ovule development, one embryo sac is formed within each ovule. The mature embryo sac contains three distinct cell types: the synergids and the two female gametes: the egg and the central cell <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029685#pone.0029685-Schneitz1" target="_blank">[13]</a>. The mature embryo sac of <i>Arabidopsis thaliana</i>, accession Landsberg <i>erecta</i>, is around long and wide <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029685#pone.0029685-Christensen1" target="_blank">[44]</a>. The nuclei of the cells of the embryo sac are drawn as black circles, the vacuoles as white regions.</p