Integrated de novo transcriptome of Culex pipiens mosquito larvae as a resource for genetic control strategies.

We present a de novo transcriptome of the mosquito vector Culex pipiens, assembled by sequences of susceptible and insecticide resistant larvae. The high quality of the assembly was confirmed by TransRate and BUSCO. A mapping percentage until 94.8% was obtained by aligning contigs to Nr, SwissProt, and TrEMBL, with 27,281 sequences that simultaneously mapped on the three databases. A total of 14,966 ORFs were also functionally annotated by using the eggNOG database. Among them, we identified ORF sequences of the main gene families involved in insecticide resistance. Therefore, this resource stands as a valuable reference for further studies of differential gene expression as well as to identify genes of interest for genetic-based control tools.

De novo transcriptome assembly of an Antarctic nematode for the study of thermal adaptation in marine parasites.

Understanding the genomic underpinnings of thermal adaptation is a hot topic in eco-evolutionary studies of parasites. Marine heteroxenous parasites have complex life cycles encompassing a free-living larval stage, an ectothermic intermediate host and a homeothermic definitive host, thus representing compelling systems for the study of thermal adaptation. The Antarctic anisakid Contracaecum osculatum sp. D is a marine parasite able to survive and thrive both at very cold and warm temperatures within the environment and its hosts. Here, a de novo transcriptome of C. osculatum sp. D was generated for the first time, by performing RNA-Seq experiments on a set of individuals exposed to temperatures experienced by the nematode during its life cycle. The analysis generated 425,954,724 reads, which were assembled and then annotated. The high-quality assembly was validated, achieving over 88% mapping against the transcriptome. The transcriptome of this parasite will represent a valuable genomic resource for future studies aimed at disentangling the genomic architecture of thermal tolerance and metabolic pathways related to temperature stress.

De novo transcriptome assembly and annotation for gene discovery in Salamandra salamandra at the larval stage.

Dispersal is a key process in ecology and evolutionary biology, as it shapes biodiversity patterns over space and time. Attitude to disperse is unevenly distributed among individuals within populations, and that individual personality can have pivotal roles in the shaping of this attitude. Here, we assembled and annotated the first de novo transcriptome of the head tissues of Salamandra salamandra from individuals, representative of distinct behavioral profiles. We obtained 1,153,432,918 reads, which were successfully assembled and annotated. The high-quality of the assembly was confirmed by three assembly validators. The alignment of contigs against the de novo transcriptome led to a mapping percentage higher than 94%. The homology annotation with DIAMOND led to 153,048 (blastx) and 95,942 (blastp) shared contigs, annotated on NR, Swiss-Prot and TrEMBL. The domain and site protein prediction led to 9850 GO-annotated contigs. This de novo transcriptome represents reliable reference for comparative gene expression studies between alternative behavioral types, for comparative gene expression studies within Salamandra, and for whole transcriptome and proteome studies in amphibians.

Brain de novo transcriptome assembly of a toad species showing polymorphic anti-predatory behavior.

Understanding the genomic underpinnings of antipredatory behaviors is a hot topic in eco-evolutionary research. Yellow-bellied toad of the genus Bombina are textbook examples of the deimatic display, a time-structured behavior aimed at startling predators. Here, we generated the first de novo brain transcriptome of the Apennine yellow-bellied toad Bombina pachypus, a species showing inter-individual variation in the deimatic display. Through Rna-Seq experiments on a set of individuals showing distinct behavioral phenotypes, we generated 316,329,573 reads, which were assembled and annotated. The high-quality assembly was confirmed by assembly validators and by aligning the contigs against the de novo transcriptome with a mapping percentage higher than 91.0%. The homology annotation with DIAMOND (blastx) led to 77,391 contigs annotated on Nr, Swiss Prot and TrEMBL, whereas the domain and site protein prediction made with InterProScan led to 4747 GO-annotated and 1025 KEGG-annotated contigs. The B. pachypus transcriptome described here will be a valuable resource for further studies on the genomic underpinnings of behavioral variation in amphibians.

De novo transcriptome assembly and annotation of the third stage larvae of the zoonotic parasite Anisakis pegreffii.

OBJECTIVES: Anisakis pegreffii is a zoonotic parasite requiring marine organisms to complete its life-history. Human infection (anisakiasis) occurs when the third stage larvae (L3) are accidentally ingested with raw or undercooked infected fish or squids. A new de novo transcriptome of A. pegreffii was here generated aiming to provide a robust bulk of data to be used for a comprehensive "ready-to-use" resource for detecting functional studies on genes and gene products of A. pegreffii involved in the molecular mechanisms of parasite-host interaction. DATA DESCRIPTION: A RNA-seq library of A. pegreffii L3 was here newly generated by using Illumina TruSeq platform. It was combined with other five RNA-seq datasets previously gathered from L3 of the same species stored in SRA of NCBI. The final dataset was analyzed by launching three assembler programs and two validation tools. The use of a robust pipeline produced a high-confidence protein-coding transcriptome of A. pegreffii. These data represent a more robust and complete transcriptome of this species with respect to the actually existing resources. This is of importance for understanding the involved adaptive and immunomodulatory genes implicated in the "cross talk" between the parasite and its hosts, including the accidental one (humans).

RNA Editing Detection in HPC Infrastructures.

RNA editing by A-to-I deamination is a relevant co/posttranscriptional modification carried out by ADAR enzymes. In humans, it has pivotal cellular effects and its deregulation has been linked to a variety of human disorders including neurological and neurodegenerative diseases and cancer. Despite its biological relevance, the detection of RNA editing variants in large transcriptome sequencing experiments (RNAseq) is yet a challenging computational task. To drastically reduce computing times we have developed a novel REDItools version able to identify A-to-I events in huge amount of RNAseq data employing High Performance Computing (HPC) infrastructures.Here we show how to use REDItools v2 in HPC systems.

RAP: A Web Tool for RNA-Seq Data Analysis.

Since 1950 main studies of RNA regarded its role in the protein synthesis. Later insights showed that only a small portion of RNA codes for proteins where the rest could have different functional roles. With the advent of Next Generation Sequencing (NGS) and in particular with RNA-seq technology the cost of sequencing production dropped down. Among the NGS application areas, the transcriptome analysis, that is, the analysis of transcripts in a cell, their quantification for a specific developmental stage or treatment condition, became more and more adopted in the laboratories. As a consequence in the last decade new insights were gained in the understanding of both transcriptome complexity and involvement of RNA molecules in cellular processes. For what concerns computational advances, bioinformatics research developed new methods for analyzing RNA-seq data. The comparison among transcriptome profiles from several samples is often a difficult task for nonexpert programmers. Here, in this chapter, we introduce RAP (RNA-Seq Analysis Pipeline), a completely automated web tool for transcriptome analysis. It is a user-friendly web tool implementing a detailed transcriptome workflow to detect differential expressed genes and transcript, identify spliced junctions and constitutive or alternative polyadenylation sites and predict gene fusion events. Through the web interface the researchers can get all this information without any knowledge of the underlying High Performance Computing infrastructure.