In the few years since its initial application, massively parallel cDNA sequencing, or RNA-seq, has allowed many advances in the characterization and quantification of transcriptomes. Recently, several developments in RNA-seq methods have provided an even more complete characterization of RNA transcripts. These developments include improvements in transcription start site mapping, strand-specific measurements, gene fusion detection, small RNA characterization and detection of alternative splicing events. Ongoing developments promise further advances in the application of RNA-seq, particularly direct RNA sequencing and approaches that allow RNA quantification from very small amounts of cellular materials.Over the past 10 years we have come to appreciate the dynamic state of genomes, including both DNA modifications and RNA quantitative and qualitative changes, which have been characterized in species ranging from simple model organisms to humans. This advance has occurred through the use of various genomic measurements, including comprehensive © 2010 Macmillan Publishers Limited. All rights reserved fozsolak@helicosbio.com; pmilos@helicosbio.com. Competing interests statementThe authors declare competing financial interests; see Web version for details. Next generation DNA sequencing (Often abbreviated to NGS.) Non-Sanger-based high-throughput DNA sequencing technologies. Compared to Sanger sequencing, NGS platforms sequence as many as billions of DNA strands in parallel, yielding substantially more throughput and minimizing the need for the fragment-cloning methods that are often used in Sanger sequencing of genomes. FURTHER INFORMATION Semisuppressive PCRA PCR strategy that aims to reduce primer dimer accumulation by preferentially amplifying longer DNA fragments. Spike poolInternal controls added to RNA samples, consisting of RNA elements of known sequence and composition. Paired-end readsA strategy involving sequencing of two different regions that are located apart from each other on the same DNA fragment. This strategy provides elevated physical coverage and alleviates several limitations of NGS platforms that arise because of their relatively short read length.Laser capture microdissection (Often abbreviated to LCM.) A method allowing cells of interest that are chosen by the operator using a microscope to be specifically captured from heterogeneous tissue samples. The isolated cells can be used for various analyses including of protein and nucleic acid.Quantitative real-time polymerase chain reaction A PCR application that enables the measurement of nucleic acid quantities in samples. Nucleic acid of interest is amplified with PCR. The level of the amplified product accumulation during PCR cycles are measured in real time. This data is used to infer starting nucleic acid quantities. Initial transcriptomics studies largely relied on hybridization-based microarray technologies and offered a limited ability to fully catalogue and quantify the diverse RNA molecules that are expressed from genomes over ...

5-hydroxymethylcytosine (5hmC) is a modified base present at low levels in diverse cell types in mammals1–5. 5hmC is generated by the TET family of Fe(II) and 2-oxoglutarate-dependent enzymes through oxidation of 5-methylcytosine (5mC)1,2,4–7. 5hmC and TET proteins have been implicated in stem cell biology and cancer1,4,5,8,9, but information on the genome-wide distribution of 5hmC is limited. Here we describe two novel and specific approaches to profile the genomic localization of 5hmC. The first approach, termed GLIB (glucosylation, periodate oxidation, biotinylation) uses a combination of enzymatic and chemical steps to isolate DNA fragments containing as few as a single 5hmC. The second approach involves conversion of 5hmC to cytosine 5-methylenesulphonate (CMS) by treatment of genomic DNA with sodium bisulphite, followed by immunoprecipitation of CMS-containing DNA with a specific antiserum to CMS5. High-throughput sequencing of 5hmC-containing DNA from mouse embryonic stem (ES) cells showed strong enrichment within exons and near transcriptional start sites. 5hmC was especially enriched at the start sites of genes whose promoters bear dual histone 3 lysine 27 trimethylation (H3K27me3) and histone 3 lysine 4 trimethylation (H3K4me3) marks. Our results indicate that 5hmC has a probable role in transcriptional regulation, and suggest a model in which 5hmC contributes to the ‘poised’ chromatin signature found at developmentally-regulated genes in ES cells.

Circulating tumour cells (CTCs) shed into blood from primary cancers include putative precursors that initiate distal metastases1. While these cells are extraordinarily rare, they may identify cellular pathways contributing to the blood-borne dissemination of cancer. Here, we adapted a microfluidic device2 for efficient capture of CTCs from an endogenous mouse pancreatic cancer model3 and subjected CTCs to single molecule RNA sequencing4, identifying Wnt2 as a candidate gene enriched in CTCs. Expression of Wnt2 in pancreatic cancer cells suppresses anoikis, enhances anchorage-independent sphere formation, and increases metastatic propensity in vivo. This effect is correlated with fibronectin upregulation and suppressed by inhibition of Map3k7 (Tak1) kinase. In humans, formation of non-adherent tumour spheres by pancreatic cancer cells is associated with upregulation of multiple Wnt genes, and pancreatic CTCs revealed enrichment for Wnt signaling in 5 of 11 cases. Thus, molecular analysis of CTCs may identify candidate therapeutic targets to prevent the distal spread of cancer.

Summary The emerging discoveries on the link between polyadenylation and disease states underline the need to fully characterize genome-wide polyadenylation states. Here, we report comprehensive maps of global polyadenylation events in human and yeast generated using refinements to the Direct RNA Sequencing technology. This direct approach provides a quantitative view of genome-wide polyadenylation states in a strand-specific manner and requires only attomole RNA quantities. The polyadenylation profiles revealed an abundance of unannotated polyadenylation sites, alternative polyadenylation patterns, and regulatory element-associated polyA+ RNAs. We observed differences in sequence composition surrounding canonical and non-canonical human polyadenylation sites, suggesting novel non-coding RNA-specific polyadenylation mechanisms in humans. Furthermore, we observed the correlation level between sense and antisense transcripts to depend on gene expression levels, supporting the view that overlapping transcription from opposite strands may play a regulatory role. Our data provide a comprehensive view of the polyadenylation state and overlapping transcription.

Haematopoietic stem cells (HSCs) can convert between growth states that have marked differences in bioenergetic needs. Although often quiescent in adults, these cells become proliferative upon physiological demand. Balancing HSC energetics in response to nutrient availability and growth state is poorly understood, yet essential for the dynamism of the haematopoietic system. Here we show that the Lkb1 tumour suppressor is critical for the maintenance of energy homeostasis in haematopoietic cells. Lkb1 inactivation in adult mice causes loss of HSC quiescence followed by rapid depletion of all haematopoietic subpopulations. Lkb1-deficient bone marrow cells exhibit mitochondrial defects, alterations in lipid and nucleotide metabolism, and depletion of cellular ATP. The haematopoietic effects are largely independent of Lkb1 regulation of AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) signalling. Instead, these data define a central role for Lkb1 in restricting HSC entry into cell cycle and in broadly maintaining energy homeostasis in haematopoietic cells through a novel metabolic checkpoint.The metabolic control systems in HSCs are poorly understood although these cells have been shown to be highly sensitive to energetic and oxidative stress and must be able to shift Author Information Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Readers are welcome to comment on the online version of this article at www.nature.com/nature. NIH Public Access Author ManuscriptNature. Author manuscript; available in PMC 2011 March 1. Lkb1 mutant animals displayed progressive pancytopenia as well as rapid loss of bone marrow myeloid, B lymphoid and erythroid cells (Fig. 1c, d and Supplementary Fig. 2a), and markedly decreased cellularity of the thymus and spleen ( Supplementary Fig. 2c, d and data not shown). Notably, in the bone marrow and thymus, immature lymphoid cells declined at a faster rate than the more differentiated cells ( Supplementary Fig. 2b, c). The Lkb1 mutants also exhibited a pronounced loss of HSC and multipotent progenitor populations at day 5 after pIpC treatment (Fig. 1e, f and Supplementary Fig. 2f). Furthermore, Lkb1 mutant bone marrow cells formed fewer and smaller colonies in in vitro colony forming assays ( Fig. 1g and Supplementary Fig. 2e). Comparable in vivo and in vitro phenotypes were seen using a second model system in which Lkb1 was deleted using the tamoxifen-inducible Rosa26-creERt2 strain ( Supplementary Fig. 2g, h, and not shown). These results show that Lkb1 is critically required for haematopoiesis and for the maintenance of HSCs and progenitor cells. Lkb1 function in bone marrow is cell intrinsicBoth theMx1-cre and Rosa26-creERt2 systems induce Cre recombinase activity in many cell types; thus bone marrow transplants were used to determine whether Lkb1 has a cellautonomous role in haematopoiesis. We performed non-competitive transplants of CD45.1 whole bone marrow from contr...