We have mapped and quantified mouse transcriptomes by deeply sequencing them and recording how frequently each gene is represented in the sequence sample (RNA-Seq). This provides a digital measure of the presence and prevalence of transcripts from known and previously unknown genes. We report reference measurements composed of 41-52 million mapped 25-base-pair reads for poly(A)-selected RNA from adult mouse brain, liver and skeletal muscle tissues. We used RNA standards to quantify transcript prevalence and to test the linear range of transcript detection, which spanned five orders of magnitude. Although >90% of uniquely mapped reads fell within known exons, the remaining data suggest new and revised gene models, including changed or additional promoters, exons and 3' untranscribed regions, as well as new candidate microRNA precursors. RNA splice events, which are not readily measured by standard gene expression microarray or serial analysis of gene expression methods, were detected directly by mapping splice-crossing sequence reads. We observed 1.45 x 10(5) distinct splices, and alternative splices were prominent, with 3,500 different genes expressing one or more alternate internal splices.

Abstract. We explore connections between metagenomic read assignment and the quantification of transcripts from RNA-Seq data. In particular, we show that the recent idea of pseudoalignment introduced in the RNA-Seq context is suitable in the metagenomics setting. When coupled with the Expectation-Maximization (EM) algorithm, reads can be assigned far more accurately and quickly than is currently possible with state of the art software.

Efficient sequencing of animal and plant genomes by next-generation technology should allow many neglected organisms of biological and medical importance to be better understood. As a test case, we have assembled a draft genome of Caenorhabditis sp. 3 PS1010 through a combination of direct sequencing and scaffolding with RNA-seq. We first sequenced genomic DNA and mixed-stage cDNA using paired 75-nt reads from an Illumina GAII. A set of 230 million genomic reads yielded an 80-Mb assembly, with a supercontig N50 of 5.0 kb, covering 90% of 429 kb from previously published genomic contigs. Mixed-stage poly(A) + cDNA gave 47.3 million mappable 75-mers (including 5.1 million spliced reads), which separately assembled into 17.8 Mb of cDNA, with an N50 of 1.06 kb. By further scaffolding our genomic supercontigs with cDNA, we increased their N50 to 9.4 kb, nearly double the average gene size in C. elegans. We predicted 22,851 protein-coding genes, and detected expression in 78% of them. Multigenome alignment and data filtering identified 2672 DNA elements conserved between PS1010 and C. elegans that are likely to encode regulatory sequences or previously unknown ncRNAs. Genomic and cDNA sequencing followed by joint assembly is a rapid and useful strategy for biological analysis.

One proposed strategy for controlling the transmission of insect-borne pathogens uses a drive mechanism to ensure the rapid spread of transgenes conferring disease refractoriness throughout wild populations. Here, we report the creation of maternal-effect selfish genetic elements in Drosophila that drive population replacement and are resistant to recombination-mediated dissociation of drive and disease refractoriness functions. These selfish elements use microRNA-mediated silencing of a maternally expressed gene essential for embryogenesis, which is coupled with early zygotic expression of a rescuing transgene.

SummaryGenetically engineered human pluripotent stem cells (hPSCs) have been proposed as a source for transplantation therapies and are rapidly becoming valuable tools for human disease modeling. However, many applications are limited due to the lack of robust differentiation paradigms that allow for the isolation of defined functional tissues. Here, using an endogenous LGR5-GFP reporter, we derived adult stem cells from hPSCs that gave rise to functional human intestinal tissue comprising all major cell types of the intestine. Histological and functional analyses revealed that such human organoid cultures could be derived with high purity and with a composition and morphology similar to those of cultures obtained from human biopsies. Importantly, hPSC-derived organoids responded to the canonical signaling pathways that control self-renewal and differentiation in the adult human intestinal stem cell compartment. This adult stem cell system provides a platform for studying human intestinal disease in vitro using genetically engineered hPSCs.

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Supplementary data are available at Bioinformatics online.

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