RNA editing by members of the ADAR (adenosine deaminases acting on RNA) family leads to site-specific conversion of adenosine to inosine (A-to-I) in precursor messenger RNAs. Editing by ADARs is believed to occur in all metazoa, and is essential for mammalian development. Currently, only a limited number of human ADAR substrates are known, whereas indirect evidence suggests a substantial fraction of all pre-mRNAs being affected. Here we describe a computational search for ADAR editing sites in the human transcriptome, using millions of available expressed sequences. We mapped 12,723 A-to-I editing sites in 1,637 different genes, with an estimated accuracy of 95%, raising the number of known editing sites by two orders of magnitude. We experimentally validated our method by verifying the occurrence of editing in 26 novel substrates. A-to-I editing in humans primarily occurs in noncoding regions of the RNA, typically in Alu repeats. Analysis of the large set of editing sites indicates the role of editing in controlling dsRNA stability.

The sweet melon fruit is characterized by a metabolic transition during its development that leads to extensive accumulation of the disaccharide sucrose in the mature fruit. While the biochemistry of the sugar metabolism pathway of the cucurbits has been well studied, a comprehensive analysis of the pathway at the transcriptional level allows for a global genomic view of sugar metabolism during fruit sink development. We identified 42 genes encoding the enzymatic reactions of the sugar metabolism pathway in melon. The expression pattern of the 42 genes during fruit development of the sweet melon cv Dulce was determined from a deep sequencing analysis performed by 454 pyrosequencing technology, comprising over 350,000 transcripts from four stages of developing melon fruit flesh, allowing for digital expression of the complete metabolic pathway. The results shed light on the transcriptional control of sugar metabolism in the developing sweet melon fruit, particularly the metabolic transition to sucrose accumulation, and point to a concerted metabolic transition that occurs during fruit development.

SUMMARY Mitochondria undergo fission and fusion to maintain homeostasis, and tumors exhibit the dysregulation of mitochondrial dynamics. We recently demonstrated that ectopic HRas G12V promotes mitochondrial fragmentation and tumor growth through Erk phosphorylation of the mitochondrial fission GTPase Dynamin-related protein 1 (Drp1). However, the role of Drp1 in the setting of endogenous oncogenic KRas remains unknown. Here, we show that Drp1 is required for KRas-driven anchorage-independent growth in fibroblasts and patient-derived pancreatic cancer cell lines, and it promotes glycolytic flux, in part through the regulation of hexokinase 2 (HK2). Furthermore, Drp1 deletion imparts a significant survival advantage in a model of KRas-driven pancreatic cancer, and tumors exhibit a strong selective pressure against complete Drp1 deletion. Rare tumors that arise in the absence of Drp1 have restored glycolysis but exhibit defective mitochondrial metabolism. This work demonstrates that Drp1 plays dual roles in KRas-driven tumor growth: supporting both glycolysis and mitochondrial function through independent mechanisms.

We introduce a massively parallel novel sequencing platform that combines an open flow cell design on a circular wafer with a large surface area and mostly natural nucleotides that allow optical end-point detection without reversible terminators. This platform enables sequencing billions of reads with longer read length (~300bp) and fast runs times (<20hrs) with high base accuracy (Q30 > 85%), at a low cost of $1/Gb. We establish system performance by whole-genome sequencing of the Genome-In-A-Bottle reference samples HG001-7, demonstrating high accuracy for SNPs (99.6%) and Indels in homopolymers up to length 10 (96.4%) across the vast majority (>98%) of the defined high-confidence regions of these samples. We demonstrate scalability of the whole-genome sequencing workflow by sequencing an additional 224 selected samples from the 1000 Genomes project achieving high concordance with reference data.

The melon (Cucumis melo L.) fruit is an important crop and model system for the genomic study of both fl eshy fruit development and the Cucurbitaceae family. To obtain an accurate representation of the melon fruit transcriptome based on expressed sequence tag (EST) abundance in 454-pyrosequencing data, we prepared double-stranded complementary DNA (cDNA) of melon without the usual amplifi cation and normalization steps. A purifi cation step was also included to eliminate small fragments. Complementary DNAs were obtained from 14 individual fruit libraries derived from two genotypes, separated into fl esh and peel tissues, and sampled throughout fruit development. Pyrosequencing was performed using Genome Sequencer FLX (GS FLX) technology, resulting in 1,215,359 reads, with mean length of >200 nucleotides. The global digital expression data was validated by comparative reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) of 40 selected genes and expression patterns were similar for the two methods. The results indicate that high-quality, nonbiased cDNA for nextgeneration sequencing can be prepared from mature, fl eshy fruit, which are notorious for diffi culties in ribonucleic acid (RNA) preparation.