Nitrate stable isotopes and major ions in snow and ice samples from four Svalbard sites

Increasing reactive nitrogen (N-r) deposition in the Arctic may adversely impact N-limited ecosystems. To investigate atmospheric transport of N-r to Svalbard, Norwegian Arctic, snow and firn samples were collected from glaciers and analysed to define spatial and temporal variations (1 10 years) in major ion concentrations and the stable isotope composition (delta N-15 and delta O-18) of nitrate (NO3-) across the archipelago. The delta N-15(NO3-) and delta O-18(NO3-) averaged -4 parts per thousand and 67 parts per thousand in seasonal snow (2010-11) and -9 parts per thousand and 74 parts per thousand in firn accumulated over the decade 2001-2011. East-west zonal gradients were observed across the archipelago for some major ions (non-sea salt sulphate and magnesium) and also for delta N-15(NO3-) and delta O-18(NO3-) in snow, which suggests a different origin for air masses arriving in different sectors of Svalbard. We propose that snowfall associated with long-distance air mass transport over the Arctic Ocean inherits relatively low delta N-15(NO3-) due to in-transport N isotope fractionation. In contrast, faster air mass transport from the north-west Atlantic or northern Europe results in snowfall with higher delta N-15(NO3-) because in-transport fractionation of N is then time-limited

Relative Abundance of Transcripts (RATs):Identifying differential isoform abundance from RNA-seq [version 1; referees: 1 approved, 2 approved with reservations]

The biological importance of changes in RNA expression is reflected by the wide variety of tools available to characterise these changes from RNA-seq data. Several tools exist for detecting differential transcript isoform usage (DTU) from aligned or assembled RNA-seq data, but few exist for DTU detection from alignment-free RNA-seq quantifications. We present the RATs, an R package that identifies DTU transcriptome-wide directly from transcript abundance estimates. RATs is unique in applying bootstrapping to estimate the reliability of detected DTU events and shows good performance at all replication levels (median false positive fraction < 0.05). We compare RATs to two existing DTU tools, DRIM-Seq & SUPPA2, using two publicly available simulated RNA-seq datasets and a published human RNA-seq dataset, in which 248 genes have been previously identified as displaying significant DTU. RATs with default threshold values on the simulated Human data has a sensitivity of 0.55, a Matthews correlation coefficient of 0.71 and a false discovery rate (FDR) of 0.04, outperforming both other tools. Applying the same thresholds for SUPPA2 results in a higher sensitivity (0.61) but poorer FDR performance (0.33). RATs and DRIM-seq use different methods for measuring DTU effect-sizes complicating the comparison of results between these tools, however, for a likelihood-ratio threshold of 30, DRIM-Seq has similar FDR performance to RATs (0.06), but worse sensitivity (0.47). These differences persist for the simulated drosophila dataset. On the published human RNA-seq dataset the greatest agreement between the tools tested is 53%, observed between RATs and SUPPA2. The bootstrapping quality filter in RATs is responsible for removing the majority of DTU events called by SUPPA2 that are not reported by RATs. All methods, including the previously published qRT-PCR of three of the 248 detected DTU events, were found to be sensitive to annotation differences between Ensembl v60 and v87

The Tetraodon nigroviridis reference transcriptome: Developmental transition, length retention and microsynteny of long non-coding RNAs in a compact vertebrate genome

Pufferfish such as fugu and tetraodon carry the smallest genomes among all vertebrates and are ideal for studying genome evolution. However, comparative genomics using these species is hindered by the poor annotation of their genomes. We performed RNA sequencing during key stages of maternal to zygotic transition of Tetraodon nigroviridis and report its first developmental transcriptome. We assembled 61,033 transcripts (23,837 loci) representing 80% of the annotated gene models and 3816 novel coding transcripts from 2667 loci. We demonstrate the similarities of gene expression profiles between pufferfish and zebrafish during maternal to zygotic transition and annotated 1120 long non-coding RNAs (lncRNAs) many of which differentially expressed during development. The promoters for 60% of the assembled transcripts result validated by CAGE-seq. Despite the extreme compaction of the tetraodon genome and the dramatic loss of transposons, the length of lncRNA exons remain comparable to that of other vertebrates and a small set of lncRNAs appears enriched for transposable elements suggesting a selective pressure acting on lncRNAs length and composition. Finally, a set of lncRNAs are microsyntenic between teleost and vertebrates, which indicates potential regulatory interactions between lncRNAs and their flanking coding genes. Our work provides a fundamental molecular resource for vertebrate comparative genomics and embryogenesis studies

Identification of Vascular and Hematopoietic Genes Downstream of etsrp by Deep Sequencing in Zebrafish

The transcription factor etsrp/Er71/Etv2 is a master control gene for vasculogenesis in all species studied to date. It is also required for hematopoiesis in zebrafish and mice. Several novel genes expressed in vasculature have been identified through transcriptional profiling of zebrafish embryos overexpressing etsrp by microarrays. Here we re-examined this transcriptional profile by Illumina RNA-sequencing technology, revealing a substantially increased number of candidate genes regulated by etsrp. Expression studies of 50 selected candidate genes from this dataset resulted in the identification of 39 new genes that are expressed in vascular cells. Regulation of these genes by etsrp was confirmed by their ectopic induction in etsrp overexpressing and decreased expression in etsrp deficient embryos. Our studies demonstrate the effectiveness of the RNA-sequencing technology to identify biologically relevant genes in zebrfish and produced a comprehensive profile of genes previously unexplored in vascular endothelial cell biology