Alberto Rastrojo

The transcriptome of Leishmania major in the axenic promastigote stage: transcript annotation and relative expression levels by RNA-seq

BackgroundAlthough the genome sequence of the protozoan parasite Leishmania major was determined several years ago, the knowledge of its transcriptome was incomplete, both regarding the real number of genes and their primary structure.ResultsHere, we describe the first comprehensive transcriptome analysis of a parasite from the genus Leishmania. Using high-throughput RNA sequencing (RNA-seq), a total of 10285 transcripts were identified, of which 1884 were considered novel, as they did not match previously annotated genes. In addition, our data indicate that current annotations should be modified for many of the genes. The detailed analysis of the transcript processing sites revealed extensive heterogeneity in the spliced leader (SL) and polyadenylation addition sites. As a result, around 50% of the genes presented multiple transcripts differing in the length of the UTRs, sometimes in the order of hundreds of nucleotides. This transcript heterogeneity could provide an additional source for regulation as the different sizes of UTRs could modify RNA stability and/or influence the efficiency of RNA translation. In addition, for the first time for the Leishmania major promastigote stage, we are providing relative expression transcript levels.ConclusionsThis study provides a concise view of the global transcriptome of the L. major promastigote stage, providing the basis for future comparative analysis with other development stages or other Leishmania species.

Global variability in gene expression and alternative splicing is modulated by mitochondrial content

Noise in gene expression is a main determinant of phenotypic variability. Increasing experimental evidence suggests that genome-wide cellular constraints largely contribute to the heterogeneity observed in gene products. It is still unclear, however, which global factors affect gene expression noise and to what extent. Since eukaryotic gene expression is an energy demanding process, differences in the energy budget of each cell could determine gene expression differences. Here, we quantify the contribution of mitochondrial variability (a natural source of ATP variation) to global variability in gene expression. We find that changes in mitochondrial content can account for ∼50% of the variability observed in protein levels. This is the combined result of the effect of mitochondria dosage on transcription and translation apparatus content and activities. Moreover, we find that mitochondrial levels have a large impact on alternative splicing, thus modulating both the abundance and type of mRNAs. A simple mathematical model in which mitochondrial content simultaneously affects transcription rate and splicing site choice can explain the alternative splicing data. The results of this study show that mitochondrial content (and/or probably function) influences mRNA abundance, translation, and alternative splicing, which ultimately affects cellular phenotype.

Ecological connectivity shapes quasispecies structure of RNA viruses in an Antarctic lake

RNA viruses exist as complex mixtures of genotypes, known as quasispecies, where the evolution potential resides in the whole community of related genotypes. Quasispecies structure and dynamics have been studied in detail for virus infecting animals and plants but remain unexplored for those infecting micro-organisms in environmental samples. We report the first metagenomic study of RNA viruses in an Antarctic lake (Lake Limnopolar, Livingston Island). Similar to low-latitude aquatic environments, this lake harbours an RNA virome dominated by positive single-strand RNA viruses from the order Picornavirales probably infecting micro-organisms. Antarctic picorna-like virus 1 (APLV1), one of the most abundant viruses in the lake, does not incorporate any mutation in the consensus sequence from 2006 to 2010 and shows stable quasispecies with low-complexity indexes. By contrast, APLV2-APLV3 are detected in the lake water exclusively in summer samples and are major constituents of surrounding cyanobacterial mats. Their quasispecies exhibit low complexity in cyanobacterial mat, but their run-off-mediated transfer to the lake results in a remarkable increase of complexity that may reflect the convergence of different viral quasispecies from the catchment area or replication in a more diverse host community. This is the first example of viral quasispecies from natural aquatic ecosystems and points to ecological connectivity as a modulating factor of quasispecies complexity.

Complex tissue-specific patterns and distribution of multiple RAGE splice variants in different mammals

The receptor for advanced glycosylation end products (RAGE) is a multiligand receptor involved in diverse cell signaling pathways. Previous studies show that this gene expresses several splice variants in human, mouse, and dog. Alternative splicing (AS) plays an important role in expanding transcriptomic and proteomic diversity, and it has been related to disease. AS is also one of the main evolutionary mechanisms in mammalian genomes. However, limited information is available regarding the AS of RAGE in a wide context of mammalian tissues. In this study, we examined in detail the different RAGE mRNAs generated by AS from six mammals, including two primates (human and monkey), two artiodactyla (cow and pig), and two rodentia (mouse and rat) in 6–18 different tissues including fetal, adult, and tumor. By nested reverse transcription-polymerase chain reaction (RT-PCR) we identified a high number of splice variants including noncoding transcripts and predicted coding ones with different potential protein modifications affecting mainly the transmembrane and ligand-binding domains that could influence their biological function. However, analysis of RNA-seq data enabled detecting only the most abundant splice variants. More than 80% of the detected RT-PCR variants (87 of 101 transcripts) are novel (different exon/intron structure to the previously described ones), and interestingly, 20–60% of the total transcripts (depending on the species) are noncoding ones that present tissue specificity. Our results suggest that RAGE undergoes extensive AS in mammals, with different expression patterns among adult, fetal, and tumor tissues. Moreover, most splice variants seem to be species specific, especially the noncoding variants, with only two (canonical human Tv1-RAGE, and human N-truncated or Tv10-RAGE) conserved among the six different species. This could indicate a special evolution pattern of this gene at mRNA level.

Aquatic viral metagenomics: Lights and shadows

Viruses are the most abundant biological entities on Earth, exceeding bacteria in most of the ecosystems. Specially in oceans, viruses are thought to be the major planktonic predators shaping microorganism communities and controlling ocean biological capacity. Plankton lysis by viruses plays an important role in ocean nutrient and energy cycles. Viral metagenomics has emerged as a powerful tool to uncover viral diversity in aquatic ecosystems through the use of Next Generation Sequencing. However, many of the commonly used viral sample preparation steps have several important biases that must be considered to avoid a misinterpretation of the results. In addition to biases caused by the purification of virus particles, viral DNA/RNA amplification and the preparation of genomic libraries could also introduce biases, and a detailed knowledge about such protocols is required. In this review, the main steps in the viral metagenomic workflow are described paying special attention to the potential biases introduced by each one.

ChimPipe: accurate detection of fusion genes and transcription-induced chimeras from RNA-seq data

BackgroundChimeric transcripts are commonly defined as transcripts linking two or more different genes in the genome, and can be explained by various biological mechanisms such as genomic rearrangement, read-through or trans-splicing, but also by technical or biological artefacts. Several studies have shown their importance in cancer, cell pluripotency and motility. Many programs have recently been developed to identify chimeras from Illumina RNA-seq data (mostly fusion genes in cancer). However outputs of different programs on the same dataset can be widely inconsistent, and tend to include many false positives. Other issues relate to simulated datasets restricted to fusion genes, real datasets with limited numbers of validated cases, result inconsistencies between simulated and real datasets, and gene rather than junction level assessment.ResultsHere we present ChimPipe, a modular and easy-to-use method to reliably identify fusion genes and transcription-induced chimeras from paired-end Illumina RNA-seq data. We have also produced realistic simulated datasets for three different read lengths, and enhanced two gold-standard cancer datasets by associating exact junction points to validated gene fusions. Benchmarking ChimPipe together with four other state-of-the-art tools on this data showed ChimPipe to be the top program at identifying exact junction coordinates for both kinds of datasets, and the one showing the best trade-off between sensitivity and precision. Applied to 106 ENCODE human RNA-seq datasets, ChimPipe identified 137 high confidence chimeras connecting the protein coding sequence of their parent genes. In subsequent experiments, three out of four predicted chimeras, two of which recurrently expressed in a large majority of the samples, could be validated. Cloning and sequencing of the three cases revealed several new chimeric transcript structures, 3 of which with the potential to encode a chimeric protein for which we hypothesized a new role. Applying ChimPipe to human and mouse ENCODE RNA-seq data led to the identification of 131 recurrent chimeras common to both species, and therefore potentially conserved.ConclusionsChimPipe combines discordant paired-end reads and split-reads to detect any kind of chimeras, including those originating from polymerase read-through, and shows an excellent trade-off between sensitivity and precision. The chimeras found by ChimPipe can be validated in-vitro with high accuracy.

Resequencing and assembly of seven complex loci to improve the Leishmania major (Friedlin strain) reference genome

BackgroundLeishmania parasites cause severe human diseases known as leishmaniasis. These eukaryotic microorganisms possess an atypical chromosomal architecture and the regulation of gene expression occurs almost exclusively at post-transcriptional levels. Accordingly, sequencing of the genome of Leishmania major, and subsequently the genome of other related species, was paramount for highlighting these peculiar molecular aspects. Recently, we carried out an analysis of gene expression by massive sequencing of RNA in the L. major promastigote, and data derived from that analysis were suggestive of possible errors in the current genome assembly for this Leishmania species.ResultsDuring the analysis by RNA-Seq of the transcriptome for L. major Friedlin strain, 163,714 reads could not be aligned with the reference genome. Thus, de novo assembly with these reads was carried out and the resulting contigs were further analyzed. After detailed homology searches using available databases, it was postulated that 15 contigs might correspond to genomic sequences lost during the initial genome assembly of the L. major Friedlin strain. This was experimentally confirmed by PCR amplification, cloning and sequencing of the new genomic regions. As a result, we have identified seven regions of the L. major (Friedlin) genome that were lost during the sequence assembly. This led to the uncovering of six new genes (LmjF.15.1475, LmjF.15.0285, LmjF.24.0765, LmjF.14.0860, LmjF.19.0305, and LmjF.27.2035), and correction of the annotation for two others (LmjF.15.1480 and LmjF.27.2030). Our data suggest that these genomic regions probably collapsed during the genome assembly due to the existence of gene duplications and/or repeated regions surrounding the missed genes.ConclusionRNA-seq data helped to reconstruct some genomic regions misassembled during the L. major Friedlin genome assembly, which is otherwise quite robust. On the other hand, this study shows that data derived from massive sequencing approaches, including RNA-Seq, should be carefully inspected to improve current genome definition and gene annotations.

Genomic cartography and proposal of nomenclature for the repeated, interspersed elements of the Leishmania major SIDER2 family and identification of SIDER2-containing transcripts.

The genomes of most eukaryotic organisms contain a large number of transposable elements that are able to move from one genomic site to another either by transferring of DNA mobile elements (transposons) or transpose via reverse transcription of an RNA intermediate (retroposons). An exception to this rule is found in protists of the subgenus Leishmania, in which active retroposons degenerated after a flourishing era, leaving only retroposon remains; these have been classified into two families: SIDER1 and SIDER2. In this work, we have re-examined the elements belonging to the family SIDER2 present in the genome of Leishmania major with the aim of providing a nomenclature that will facilitate a future reference to particular elements. According to sequence conservation, the 1100 SIDER2 elements have been grouped into subfamilies, and the inferred taxonomic relationships have also been incorporated into the nomenclature. Additionally, we are providing detailed data regarding the genomic distribution of these elements and their association with specific transcripts, based on the recently established transcriptome for L. major. Thus, the presented data can help to study and better understand the roles played by these degenerated retroposons in both regulation of gene expression and genome plasticity.

Genome Sequence of Herpes Simplex Virus 1 Strain SC16

ABSTRACT Herpes simplex virus 1 (HSV-1), also known as Human herpesvirus 1, is a highly prevalent human neurotropic pathogen that causes a variety of diseases, including lethal encephalitis. Here, we report the genome sequence of the HSV-1 strain SC16.

Resequencing of the Leishmania infantum (strain JPCM5) genome and de novo assembly into 36 contigs

Leishmania parasites are the causative of leishmaniasis, a group of potentially fatal human diseases. Control strategies for leishmaniasis can be enhanced by genome based investigations. The publication in 2005 of the Leishmania major genome sequence, and two years later the genomes for the species Leishmania braziliensis and Leishmania infantum were major milestones. Since then, the L. infantum genome, although highly fragmented and incomplete, has been used widely as the reference genome to address whole transcriptomics and proteomics studies. Here, we report the sequencing of the L. infantum genome by two NGS methodologies and, as a result, the complete genome assembly on 36 contigs (chromosomes). Regarding the present L. infantum genome-draft, 495 new genes have been annotated, a hundred have been corrected and 75 previous annotated genes have been discontinued. These changes are not only the result of an increase in the genome size, but a significant contribution derives from the existence of a large number of incorrectly assembled regions in current chromosomal scaffolds. Furthermore, an improved assembly of tandemly repeated genes has been obtained. All these analyses support that the de novo assembled L. infantum genome represents a robust assembly and should replace the currently available in the databases.