Design, Validation and Annotation of Transcriptome-Wide Oligonucleotide Probes for the Oligochaete Annelid Eisenia fetida

High density oligonucleotide probe arrays have increasingly become an important tool in genomics studies. In organisms with incomplete genome sequence, one strategy for oligo probe design is to reduce the number of unique probes that target every non-redundant transcript through bioinformatic analysis and experimental testing. Here we adopted this strategy in making oligo probes for the earthworm Eisenia fetida, a species for which we have sequenced transcriptome-scale expressed sequence tags (ESTs). Our objectives were to identify unique transcripts as targets, to select an optimal and non-redundant oligo probe for each of these target ESTs, and to annotate the selected target sequences. We developed a streamlined and easy-to-follow approach to the design, validation and annotation of species-specific array probes. Four 244K-formatted oligo arrays were designed using eArray and were hybridized to a pooled E. fetida cRNA sample. We identified 63,541 probes with unsaturated signal intensities consistently above the background level. Target transcripts of these probes were annotated using several sequence alignment algorithms. Significant hits were obtained for 37,439 (59%) probed targets. We validated and made publicly available 63.5K oligo probes so the earthworm research community can use them to pursue ecological, toxicological, and other functional genomics questions. Our approach is efficient, cost-effective and robust because it (1) does not require a major genomics core facility; (2) allows new probes to be easily added and old probes modified or eliminated when new sequence information becomes available, (3) is not bioinformatics-intensive upfront but does provide opportunities for more in-depth annotation of biological functions for target genes; and (4) if desired, EST orthologs to the UniGene clusters of a reference genome can be identified and selected in order to improve the target gene specificity of designed probes. This approach is particularly applicable to organisms with a wealth of EST sequences but unfinished genome

Novel Bioinformatic Approaches for Analyzing Next-Generation Sequencing Data

In general, DNA reconstruction is deemed as the key of molecular biology since it makes people realize how genotype affects phenotypes. The DNA sequencing technology emerged exactly towards this and has greatly promoted molecular biology’s development. The traditional method, Sanger, is effective but extremely expensive on a cost-per-base basis. This shortcoming of Sanger method leads to the rapid development of next-generation sequencing technologies. The NGS technologies are widely used by virtue of their low-cost, high-throughput, and fast nature. However, they still face major drawbacks such as huge amounts of data as well as relatively short read length compared with traditional methods. The scope of the research mainly focuses upon a quick preliminary analysis of NGS data, identification of genome-wide structural variations (SVs), and microRNA prediction. In terms of preliminary NGS data analysis, the author developed a toolkit named SeqAssist to evaluate genomic library coverage and estimate the redundancy between different sequencing runs. Regarding the genome-wide SV detection, a one-stop pipeline was proposed to identify SVs, which integrates the components of preprocessing, alignment, SV detection, breakpoints revision, and annotation. This pipeline not only detects SVs at the individual sample level, but also identifies consensus SVs at the population and cross-population levels. At last, miRDisc, a pipeline for microRNA discovery, was developed for the identification of three categories of miRNAs, i.e., known, conserved, and novel microRNAs

Genetic, epigenetic and microbiome characterisation of an earthworm species (Octolasion lacteum) along a radiation exposure gradient at Chernobyl

The effects of exposure to different levels of ionising radiation were assessed on the genetic, epigenetic and microbiome characteristics of the “hologenome” of earthworms collected at sites within the Chernobyl exclusion zone (CEZ). The earthworms Aporrectodea caliginosa (Savigny, 1826) and Octolasion lacteum (Örley, 1881) were the two species that were most frequently found at visited sites, however, only O. lacteum was present at sufficient number across different exposure levels to enable comparative hologenome analysis. The identification of morphotype O. lacteum as a probable single clade was established using a combination of mitochondrial (cytochrome oxidase I) and nuclear genome (Amplified Fragment Length Polymorphism (AFLP) using MspI loci). No clear site associated differences in population genetic structure was found between populations using the AFLP marker loci. Further, no relationship between ionising radiation exposure levels and the percentage of methylated loci or pattern of distribution of DNA methylation marks was found. Microbiome structure was clearly site dependent, with gut microbiome community structure and diversity being systematically associated with calculated site-specific earthworm dose rates. There was, however, also co-correlation between earthworm dose rates and other soil properties, notably soil pH; a property known to affect soil bacterial community structure. Such co-correlation means that it is not possible to attribute microbiome changes unequivocally to radionuclide exposure. A better understanding of the relationship between radionuclide exposure soil properties and their interactions on bacterial microbiome community response is, therefore, needed to establish whether these the observed microbiome changes are attributed directly to radiation exposure, other soil properties or to an interaction between multiple variables at sites within the CEZ

Vector-pathogen interactomics: connecting the dots

As carraças e doenças associadas a carraças têm um impacto negativo considerável na saúde humana e animal. Rhipicephalus bursa é uma carraça multihospedeiro hematófaga e é o principal vetor de Babesia ovis, um hemoparasita altamente patogénico em pequenos ruminantes, que pode levar a uma taxa de mortalidade de 30- 50% em animais suscetíveis e, indiretamente contribuir para um impacto socioeconómico negativo na sociedade humana. O controlo de carraças e doenças associadas depende principalmente do uso de fármacos, que apresentam grandes desvantagens, como a contaminação de alimentos e ambiente e o aumento da resistência, reforçando assim a necessidade de medidas alternativas, como a vacinação. Com base na premissa de que as glândulas salivares da carraça têm um papel crucial no comportamento hematófago e na transmissão de agentes patogénicos, o objetivo principal deste trabalho é aumentar o conhecimento sobre a interação R. bursa-B. ovis neste tecido, de forma a identificar novos candidatos a antigénios protetores para o desenvolvimento de vacinas. Assim sendo, os sialotranscritos e as sialoproteínas de R. bursa foram analisados em diferentes condições, para compreender melhor os processos de alimentação e infeção e contribuir para o desenvolvimento de novas vacinas anti-carraça e doenças associadas. A análise comparativa dos transcriptomas e proteomas revelou que a alimentação por sangue induz a produção de moléculas por parte da carraça, o que se traduziu no aumento da expressão genética e da síntese proteica. Além disso, os dados mostram que a combinação de estímulos (alimentação e infeção) influenciou positivamente a expressão genética, mas negativamente a tradução, podendo sugerir a manipulação de B. ovis no sialoma de R. bursa. Estes resultados aliados a diferentes metodologias como RNA de interferência (in vitro e in vivo) e vacinologia reversa, permitem explorar a maquinaria celular da carraça e identificar vários alvos como potenciais antigénios para vacinas. Os ensaios de silenciamento revelaram o impacto direto de algumas moléculas na sobrevivência da carraça e a sua fixação ao hospedeiro (como a putativa “Vitelogenin-3” e uma proteína do “cement”), enquanto que outros demonstraram um efeito duplo divergente na sobrevivência do vetor e do agente patogénico (como a “lachesin” e a “UB2N”). A análise imunoinformática dos dados anteriores de sequenciação permitiu a identificação de proteínas/peptídeos capazes de induzir, no hospedeiro vertebrado, uma resposta imunológica forte e robusta contra o vetor e o agente patogénico. Nesta análise, uma proteína membranar (proteína contendo domínios “Marvel”) e duas secretórias (uma “Evasin” e uma proteína contendo domínios de “Ricin”) foram selecionadas e promissores "immunological kernels" foram encontrados, contendo características ideais de uma vacina baseada em peptídeos, sem causar alergia e toxicidade. Além disso, a integração de diferentes análises ómicas de diferentes espécies de carraças foi usada como uma estratégia para pesquisar e caracterizar vias biológicas conservadas, a fim de selecionar novos alvos capazes de impactar uma ampla gama de espécies de carraças e bloquear a transmissão de vários agentes patogénicos transmitidos por estas. Deste estudo, destacou-se a via de biossíntese de folato, ao observar que durante a infeção da carraça, quer por bactéria quer por protozoário, a expressão de genes relacionados com esta via era aumentada. No entanto, ensaios de silenciamento numa linha celular de carraça mostraram que, a curto prazo, a redução da expressão de um gene relacionado ao folato (gch-I), não exorta alterações significativas nas células de carraça ou no comportamento do agente patogénico em termos de invasão ou multiplicação. Estudos aplicados e ensaios de vacinação precisam ser conduzidos para validar o potencial desses alvos promissores para o desenvolvimento de abordagens anti-carraça e de bloqueio de transmissão de doenças.health. Rhipicephalus bursa is a hematophagous multi-host tick and the main vector of Babesia ovis, a highly pathogenic hemoparasite in small ruminants, which leads to a 30-50 % of mortality rate in susceptible animals and, indirectly, to a negative socioeconomic impact in human society. Tick and disease control rely mainly in the use of chemotherapy and acaracides, which has major drawbacks including food and environment contamination and the increase of resistance, reinforcing the need for alternatives measures, such as vaccination. Based on the premise that tick salivary glands have a crucial role on hematophagous behaviour and on pathogen transmission, the main objective of this research was to increase the understanding on the Rhipicephalus bursa- Babesia ovis interaction in this organ, in order to find new protective antigen candidates for vaccine design. Thus, the R. bursa sialotranscripts and sialoproteins were screened under different conditions, to better understand the feeding and infection processes and contribute for the development of new anti-tick and tick-borne diseases. The comparative analyses of the transcriptomes and proteomes revealed that blood feeding induces the production of tick molecules, which was translated by the increased gene expression and protein synthesis. Moreover, the data unveiled that the combination of stimuli (feeding and infection) influenced positively gene expression but negatively translation, suggesting that B. ovis might manipulate R. bursa sialome. These results allied to interference RNA (in vitro and in vivo) and reverse vaccinology, allowed to explore the tick cellular machinery and pinpointed several targets as potential vaccine antigens. The silencing assays revealed the direct impact of some molecules in tick survival and attachment to the host (such as putative Vitellogenin-3 and a Cement protein), while others demonstrated a divergent dual-effect on both vector and parasite survival (such as Lachesin and UB2N). Immunoinformatic analysis of the previous sequencing data allowed the identification of proteins/peptides capable of elicit, in the vertebrate host, a strong and robust immune response against both vector and pathogen. In this experiment, one membrane-related (Marvel-containing protein) and two secreted (a Evasin and a ricin-containing protein) proteins were selected and promising “immunological kernels” were found to have ideal characteristics for an anti-tick peptide-based vaccine, without causing allergy and toxicity. Furthermore, the integration of different omics analyses from different tick species was used as a strategy to search and characterize conserved biological pathways in order to select new targets able to impact a wide range of tick vectors and block the transmission of several transmitted pathogens. From this study the folate biosynthesis pathway stood out by observing that during tick infection, by either bacteria or protozoan, the expression of genes related to this pathway were increased. However, silencing assays in a tick cell line demonstrate that, in a short term, the reduction of expression of a folate-related gene (gch-I), did not lead to significant changes in tick cells or pathogen behaviour of invasion or multiplication. Applied studies and vaccination trials need to be conducted to validate the potencial of these promising targets for the development of anti-tick and transmission blocking approaches

Unearthing the genome of the earthworm Lumbricus rubellus

The earthworm has long been of interest to biologists, most notably Charles Darwin, who was the first to reveal their true role as eco-engineers of the soil. However, to fully understand an animal one needs to combine observational data with the fundamental building blocks of life, DNA. For many years, sequencing a genome was an incredibly costly and time-consuming process. Recent advances in sequencing technology have led to high quality, high throughput data being available at low cost. Although this provides large amounts of sequence data, the bioinformatics knowledge required to assemble and annotate these new data are still in their infancy. This bottleneck is slowly opening up, and with it come the first glimpses into the new and exciting biology of many new species. This thesis provides the first high quality draft genome assembly and annotation of an earthworm, Lumbricus rubellus. The assembly process and resulting data highlight the complexity of assembling a eukaryotic genome using short read data. To improve assembly, a novel approach was created utilising transcripts to scaffold the genome (https://github.com/elswob/SCUBAT). The annotation of the assembly provides the draft of the complete proteome, which is also supported by the first RNA-Seq generated transcriptome. These annotations have enabled detailed analysis of the protein coding genes including comparative analysis with two other annelids (a leech and a polychaete worm) and a symbiont (Verminephrobacter). This analysis identified four key areas which appear to be either highly enhanced or unique to L. rubellus. Three of these may be related to the unique environment from which the sequenced worms originated and add to the mounting evidence for the use of earthworms as bioindicators of soil quality. All data is stored in relational databases and available to search and browse via a website at www.earthworms.org. It is hoped that this genome will provide a springboard for many future investigations into the earthworm and continue research into this wonderful animal

Introduction to RNAi and miRNA pathways

Dráhy malých RNA jsou skupinou drah využívajících malé RNA k sekvenčně specifické represi. Tento soubor článků o drahách malých RNA má původ ve zprávě vypracované pro European Food and Safety Authority (EFSA) v letech 2016 a 2017. Text byl nově rozčleněn do dvanácti kapitol a doplněn úvody; vypuštěn byl naopak materiál podléhající autorskému právu třetích stran. Devět kapitol je věnováno drahám malých RNA ve zvířatech a rostlinách, zbývající tři připadají na obecný úvod a problematiku extracelulární RNA.Small RNA pathways or RNA silencing is a group of pathways, which utilize small RNAs as guides for sequence-specific repression. This collection of texts has an origin in a report prepared for the EFSA in 2016 and 2017. The original text was reorganized into twelve chapters, which were reformatted and revised in order to remove copyrighted material from third parties and provide a introductory parts for stand alone chapters. Nine of the chapters focus on small RNA pathways in animals and plants. The remaining three chapters include a general introduction and reviews of important phenomena – off-targeting and extracellular small RNAs

Relación entre el silenciamiento de RNA y la patogénesis inducida por un viroide con replicación nuclear

Interés del estudio: Los viroides son patógenos exclusivos de plantas que infectan un gran número de especies de interés agronómico. Sin capacidad descrita para codificar proteínas, todas las fases de su ciclo vital son estrictamente dependientes de su interacción con factores del huésped. Históricamente se ha asumido que la patogénesis es consecuencia de la competencia huésped-patógeno por factores celulares implicados en el ciclo vital del viroide. En los últimos años se ha propuesto que la respuesta de silenciamiento de RNA (RNAi) del huésped frente a estos patógenos es la responsable de la respuesta patogénica de los viroides. En el presente trabajo se ha tratado de estudiar en profundidad la relación entre el mecanismo de silenciamiento de RNA y la patogénesis viroidal (en concreto del viroide del enanismo del lúpulo, HSVd). Objetivos: 1- Estudiar el proceso de patogénesis inducido por HSVd y la maquinaria de RNAi 2- Caracterizar mediante secuenciación masiva los sRNAs derivados de HSVd (vd-sRNAs). 4- Analizar la distribución diferencial de vd-sRNAs en distintos tejidos (hoja y floema). 5- Determinar las alteraciones inducidas por la infección en las vías endógenas de RNAi. Elementos de la metodología a destacar: - Uso de un sistema transgénico para estudiar el fenómeno de la patogénesis. - Uso de técnicas de secuenciación masiva para la caracterización de sRNAs. - Análisis bioinformático de los datos generados por la secuenciación masiva. Resultados logrados: - Primera publicación en la literatura de la relación de un componente de las rutas de RNAi en la patogénesis viroidal ocasionada por HSVd. - Publicación de una de las primeras secuenciaciones masivas de sRNAs derivados del HSVd. - Primera caracterización de las poblaciones de sRNAs endógenos de C.sativus, incluyendo la primera caracterización de microRNAs en esta especie. - Identificación y detección de nuevos microRNAs en C.sativus.Martínez Arias, GE. (2011). Relación entre el silenciamiento de RNA y la patogénesis inducida por un viroide con replicación nuclear [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/11302Palanci

Comparative genomics and computational biology in the basal metazoan Hydra

Cnidaria stellen als basale Metazoen klassische Modellsysteme in der Entwicklungs- und Evolutionsbiologie dar. Im Zeitalter von Genom- und Transkriptomanalyse sind auch für einzelne Vertreter der Cnidaria, wie z. B. der Anemone Nematostella vectensis oder dem Süßwasserpolypen Hydra magnipapillata, neue molekulare Werkzeuge und Datenressourcen entwickelt worden, die neue Perspektiven für wissenschaftliche Fragestellungen ergeben. Mit dem Ziel, die bereits vorhandenen Ressourcen für Hydra zu erweitern, wurde eine bioinformatische Analyse-Plattform für vergleichende Genomanalyse in basalen Metazoen entwickelt und in verschiedenen Fallstudien eingesetzt, die sich mit der Evolution von basalen Immunsystemen, der Genomevolution in Hydra, sowie mit der Hochdurchsatz-Transkriptionsanalyse von Hydra ESTs beschäftigten