Trans-splicing of leader sequences onto the 5′ends of mRNAs is a widespread phenomenon in protozoa, nematodes and some chordates. Using parallel sequencing we have developed a method to simultaneously map 5′splice sites and analyze the corresponding gene expression profile, that we term spliced leader trapping (SLT). The method can be applied to any organism with a sequenced genome and trans-splicing of a conserved leader sequence. We analyzed the expression profiles and splicing patterns of bloodstream and insect forms of the parasite Trypanosoma brucei. We detected the 5′ splice sites of 85% of the annotated protein-coding genes and, contrary to previous reports, found up to 40% of transcripts to be differentially expressed. Furthermore, we discovered more than 2500 alternative splicing events, many of which appear to be stage-regulated. Based on our findings we hypothesize that alternatively spliced transcripts present a new means of regulating gene expression and could potentially contribute to protein diversity in the parasite. The entire dataset can be accessed online at TriTrypDB or through: http://splicer.unibe.ch/.

In recent years, unprecedented DNA sequencing capacity provided by next generation sequencing (NGS) has revolutionized genomic research. Combining the Illumina sequencing platform and a scFv library designed to confine diversity to both CDR3, >1.9 × 107 sequences have been generated. This approach allowed for in depth analysis of the library’s diversity, provided sequence information on virtually all scFv during selection for binding to two targets and a global view of these enrichment processes. Using the most frequent heavy chain CDR3 sequences, primers were designed to rescue scFv from the third selection round. Identification, based on sequence frequency, retrieved the most potent scFv and valuable candidates that were missed using classical in vitro screening. Thus, by combining NGS with display technologies, laborious and time consuming upfront screening can be by-passed or complemented and valuable insights into the selection process can be obtained to improve library design and understanding of antibody repertoires.

Deep-sea floors represent one of the largest and most complex ecosystems on Earth but remain essentially unexplored. The vastness and remoteness of this ecosystem make deep-sea sampling difficult, hampering traditional taxonomic observations and diversity assessment. This problem is particularly true in the case of the deep-sea meiofauna, which largely comprises small-sized, fragile, and difficult-to-identify metazoans and protists. Here, we introduce an ultra-deep sequencing-based metagenetic approach to examine the richness of benthic foraminifera, a principal component of deep-sea meiofauna. We used Illumina sequencing technology to assess foraminiferal richness in 31 unsieved deep-sea sediment samples from five distinct oceanic regions. We sequenced an extremely short fragment (36 bases) of the small subunit ribosomal DNA hypervariable region 37f, which has been shown to accurately distinguish foraminiferal species. In total, we obtained 495,978 unique sequences that were grouped into 1,643 operational taxonomic units, of which about half (841) could be reliably assigned to foraminifera. The vast majority of the operational taxonomic units (nearly 90%) were either assigned to early (ancient) lineages of soft-walled, single-chambered (monothalamous) foraminifera or remained undetermined and yet possibly belong to unknown early lineages. Contrasting with the classical view of multichambered taxa dominating foraminiferal assemblages, our work reflects an unexpected diversity of monothalamous lineages that are as yet unknown using conventional micropaleontological observations. Although we can only speculate about their morphology, the immense richness of deep-sea phylotypes revealed by this study suggests that ultra-deep sequencing can improve understanding of deep-sea benthic diversity considered until now as unknowable based on a traditional taxonomic approach.DNA barcoding | next-generation sequencing | small subunit ribosomal RNA | microbial eukaryote | cosmopolitanism D eep-sea sediments are home for a wide range of small-sized metazoan and protistan taxa. The diversity of this meiofaunal community is difficult to estimate because its study suffers from undersampling, difficult access, and the problems involved in culturing deep-sea organisms. Additionally, most of the deep-sea species are tiny, fragile, and difficult to identify. Benthic foraminifera form one of the most abundant and diverse groups of deepsea meiofauna, found even in the deepest ocean trenches (1). Particularly in abyssal areas, a large proportion of deep-sea foraminifera belongs to early lineages characterized by simple, singlechambered (monothalamous), organic-walled or agglutinated tests, which are poorly preserved in the fossil record (2). These early monothalamous lineages traditionally classified in orders Allogromiida and Astrorhiza have been proposed to form a large radiation in the Neoproterozoic, well before the first multichambered foraminifera appeared (3). However, the assessment of their diversity is hampered by the fragment...

Toxic metals polluting aquatic ecosystems are taken up by inhabitants and accumulate in the food web, affecting species at all trophic levels. It is therefore important to have good tools to assess the level of risk represented by toxic metals in the environment. Macrophytes are potential organisms for the identification of metal-responsive biomarkers but are still underrepresented in ecotoxicology. In the present study, we used next-generation sequencing to investigate the transcriptomic response of Elodea nuttallii exposed to enhanced concentrations of Hg and Cd. We de novo assembled more than 60 000 contigs, of which we found 170 to be regulated dose-dependently by Hg and 212 by Cd. Functional analysis showed that these genes were notably related to energy and metal homeostasis. Expression analysis using nCounter of a subset of genes showed that the gene expression pattern was able to assess toxic metal exposure in complex environmental samples and was more sensitive than other end points (e.g., bioaccumulation, photosynthesis, etc.). In conclusion, we demonstrate the feasibility of using gene expression signatures for the assessment of environmental contamination, using an organism without previous genetic information. This is of interest to ecotoxicology in a wider sense given the possibility to develop specific and sensitive bioassays.

In most eukaryotes, small RNA (sRNA) molecules such as miRNAs, siRNAs and piRNAs regulate gene expression and repress transposons and viruses. AGO/PIWI family proteins sort functional sRNAs based on size, 5'-nucleotide and other sequence features. In plants and some animals, viral sRNAs are extremely diverse and cover the entire viral genome sequences, which allows for de novo reconstruction of a complete viral genome by deep sequencing and bioinformatics analysis of viral sRNAs. Previously, we have developed a tool MISIS to view and analyze sRNA maps of viruses and cellular genome regions which spawn multiple sRNAs. Here we describe a new release of MISIS, MISIS-2, which enables to determine and visualize a consensus sequence and count sRNAs of any chosen sizes and 5'-terminal nucleotide identities. Furthermore we demonstrate the utility of MISIS-2 for identification of single nucleotide polymorphisms (SNPs) at each position of a reference sequence and reconstruction of a consensus master genome in evolving viral quasispecies. MISIS-2 is a Java standalone program. It is freely available along with the source code at the website http://www.fasteris.com/apps.