Anisakis pegreffii (Nematoda: Anisakidae) is the main cause of the emerging fish-borne zoonosis called anisakidosis. Humans are accidentally infected when they eat raw marine food contaminated by the infective third-stage larvae developing a gastrointestinal and allergic disease, and co-occurrence of larve with tumors. Despite its wide economic and medical relevance, most of the scientific efforts were focused so far on food safety, leaving subtle mechanisms of pathogenicity and tumorigenic potential as topics largely underinvestigated. In the last years the application of high-throughput sequencing methods are increasing and advances in computational biology and bioinformatics have greatly accelerated discovery within basic and biomedical research for some parasitic diseases. However, good quality genomes are missing for Anisakis pegreffii, thus impeding proper functional characterization of genes and other regulatory molecules as transcripts and miRNAs. For these reasons, we propose here to obtain the first highly accurate genome assembly of the zoonotic species A. pegreffii. To this aim, we will combine traditional Illumina sequencing coupled with a cutting-edge technology as the High-Fidelity long reads sequencing of PacBio. Furthermore, the highly accurate genome will assist the fine characterization and mapping of our already collected data on the transcriptomes and the miRNAs enriched in exosomal fraction of the extracellular vesicles, which will be experimentally validated by qPCR. Data will be publicly available and this will benefit the entire scientific community interested in food-borne zoonoses and in genomic and molecular aspects of pathogenicity.
More than one-third of humanity (two billion people) are infected with helminth parasites. These infections cause diseases with enormous morbidity and less mortality, physical impairment in children, loss of productivity and maintenance of poverty. The employment of high-throughput NGS methods is greatly recommended to describe the molecular profiles of studied organisms and discover their widest levels of diversity. NGS have been widely used in human medicine and other infectious diseases, and recently also parasitic helminths of medical, veterinary and economic concerns become subjects of intensive genome sequencing and annotation. These genome data will provide the basis for a comprehensive understanding of the molecular mechanisms involved in helminth metabolism, interactions with host, immune evasion, and more broadly they are likely also to predict new potential vaccine candidates and drug targets. Among helminths of interest, gastrointestinal nematodes are still under-investigated: only two attempts have been made to sequence the entire genomes in the Anisakid family and the first studies have encountered unexpected bioinformatic complexities, since parasitic helminths generally show much more complex genomes than their closely related models or free-living organisms. The two version of A. simplex available genomes are highly fragmented and only scaffolds are retrievable while annotation, well defined genes and chromosomes are completely missing. Moreover, species identities of parasites used to produce such data were not well characterized, as A. simplex is a complex of at least three cryptic species and one hybrid form.
Therefore, reference genomes are not suitable for curated gene prediction, as well as comparative and functional genomic analysis. An improved high-quality genome assembly of Anisakis pegreffii is urgently required.
SMRT sequencing technology from Pacific Biosciences (PacBio) can provide an opportunity to significantly improve genome assembly. In the present study, we will use PacBio and Illumina sequencing data from L3 worms to generate a highly accurate copy of A. pegreffii genome. Then, we will re-annotate the improved genome assembly by applying additional data on RNAseq from the same cryptic species (entire L3 transcriptomes and miRNAs). With this very highly accurate and reliable information resource, compiling data on sequenced genome and linking it to the wealth of associated functional data will assist a more precise characterization of genes and functions. Moreover, we will also be able to deepen previous observation on A. pegreffii miRNAs: once compared to other parasitic nematodes miRNAs, several showed complete homology with Ascaris miRNAs, known to interact with host gene targets related to immunity and inflammatory pathways (personal data ongoing project). Such evidences coupled with known data from other parasitic organisms will implement our ongoing and future research lines, open the intriguing and biologically fascinating hypothesis that manipulation of the vertebrate host may take place not only through the action of parasitic proteins but also through miRNA-mediated post-transcriptional regulation of host genes.