Genome scaffolding and annotation for the pathogen vector Ixodes ricinus by ultra-long single molecule sequencing.

BACKGROUND: Global warming and other ecological changes have facilitated the expansion of Ixodes ricinus tick populations. Ixodes ricinus is the most important carrier of vector-borne pathogens in Europe, transmitting viruses, protozoa and bacteria, in particular Borrelia burgdorferi (sensu lato), the causative agent of Lyme borreliosis, the most prevalent vector-borne disease in humans in the Northern hemisphere. To faster control this disease vector, a better understanding of the I. ricinus tick is necessary. To facilitate such studies, we recently published the first reference genome of this highly prevalent pathogen vector. Here, we further extend these studies by scaffolding and annotating the first reference genome by using ultra-long sequencing reads from third generation single molecule sequencing. In addition, we present the first genome size estimation for I. ricinus ticks and the embryo-derived cell line IRE/CTVM19. RESULTS: 235,953 contigs were integrated into 204,904 scaffolds, extending the currently known genome lengths by more than 30% from 393 to 516 Mb and the N50 contig value by 87% from 1643 bp to a N50 scaffold value of 3067 bp. In addition, 25,263 sequences were annotated by comparison to the tick's North American relative Ixodes scapularis. After (conserved) hypothetical proteins, zinc finger proteins, secreted proteins and P450 coding proteins were the most prevalent protein categories annotated. Interestingly, more than 50% of the amino acid sequences matching the homology threshold had 95-100% identity to the corresponding I. scapularis gene models. The sequence information was complemented by the first genome size estimation for this species. Flow cytometry-based genome size analysis revealed a haploid genome size of 2.65Gb for I. ricinus ticks and 3.80 Gb for the cell line. CONCLUSIONS: We present a first draft sequence map of the I. ricinus genome based on a PacBio-Illumina assembly. The I. ricinus genome was shown to be 26% (500 Mb) larger than the genome of its American relative I. scapularis. Based on the genome size of 2.65 Gb we estimated that we covered about 67% of the non-repetitive sequences. Genome annotation will facilitate screening for specific molecular pathways in I. ricinus cells and provides an overview of characteristics and functions.

Where does transcription start? 5'-RACE adapted to next-generation sequencing.

The variability and complexity of the transcription initiation process was examined by adapting RNA ligase-mediated rapid amplification of 5' cDNA ends (5'-RACE) to Next-Generation Sequencing (NGS). We oligo-labelled 5'-m(7)G-capped mRNA from two genes, the simple mono-exonic Beta-2-Adrenoceptor (ADRB2R)and the complex multi-exonic Glucocorticoid Receptor (GR, NR3C1), and detected a variability in TSS location that has received little attention up to now. Transcription was not initiated at a fixed TSS, but from loci of 4 to 10 adjacent nucleotides. Individual TSSs had frequencies from <0.001% to 38.5% of the total gene-specific 5' m(7)G-capped transcripts. ADRB2R used a single locus consisting of 4 adjacent TSSs. Unstimulated, the GR used a total of 358 TSSs distributed throughout 38 loci, that were principally in the 5' UTRs and were spliced using established donor and acceptor sites. Complete demethylation of the epigenetically sensitive GR promoter with 5-azacytidine induced one new locus and 127 TSSs, 12 of which were unique. We induced GR transcription with dexamethasone and Interferon-γ, adding one new locus and 185 additional TSSs distributed throughout the promoter region. In-vitro the TSS microvariability regulated mRNA translation efficiency and the relative abundance of the different GRN-terminal protein isoform levels.

Integration of Ixodes ricinus genome sequencing with transcriptome and proteome annotation of the naïve midgut.

BACKGROUND: In Europe, Ixodes ricinus ticks are the most important vectors of diseases threatening humans, livestock, wildlife and companion animals. Nevertheless, genomic sequence information is missing and functional annotation of transcripts and proteins is limited. This lack of information is restricting studies of the vector and its interactions with pathogens and hosts. Here we present and integrate the first analysis of the I. ricinus genome with the transcriptome and proteome of the unfed I. ricinus midgut. METHODS: Whole genome sequencing was performed on I. ricinus ticks and the sequences were de novo assembled. In parallel, I. ricinus ticks were dissected and the midgut transcriptome sequenced. Both datasets were integrated by transcript discovery analysis to identify putative genes and genome contigs were screened for homology. An alignment-based and a motif-search-based approach were combined for the annotation of the midgut transcriptome. Additionally, midgut proteins were identified and annotated by mass spectrometry with public databases and the in-house built transcriptome database as references and results were cross-validated. RESULTS: The de novo assembly of 1 billion DNA sequences to a reference genome of 393 Mb length provides an unprecedented insight into the I. ricinus genome. A homology search revealed sequences in the assembled genome contigs homologous to 89% of the I. scapularis genome scaffolds indicating coverage of most genome regions. We identified moreover 6,415 putative genes. More than 10,000 transcripts from naïve midgut were annotated with respect of predicted function and/or cellular localization. By combining an alignment-based with a motif-search-based annotation approach, we doubled the number of annotations throughout all functional categories. In addition, 574 gel spots were significantly identified by mass spectrometry (p<0.05) and 285 distinct proteins expressed in the naïve midgut were annotated functionally and/or for cellular localization. Our systems approach reveals a midgut metabolism of the unfed tick that is prepared to sense and process an anticipated blood meal. CONCLUSIONS: This multiple-omics study vastly extends the publicly available DNA and RNA databases for I. ricinus, paving the way for further in-depth analysis of the most important European disease vector and its interactions with pathogens and hosts.

Bayesian inference of the evolution of HBV/E.

Despite its wide spread and high prevalence in sub-Saharan Africa, hepatitis B virus genotype E (HBV/E) has a surprisingly low genetic diversity, indicating an only recent emergence of this genotype in the general African population. Here, we performed extensive phylogeographic analyses, including Bayesian MCMC modeling. Our results indicate a mutation rate of 1.9 × 10(-4) substitutions per site and year (s/s/y) and confirm a recent emergence of HBV/E, most likely within the last 130 years, and only after the transatlantic slave-trade had come to an end. Our analyses suggest that HBV/E originated from the area of Nigeria, before rapidly spreading throughout sub-Saharan Africa. Interestingly, viral strains found in Haiti seem to be the result of multiple introductions only in the second half of the 20th century, corroborating an absence of a significant number of HBV/E strains in West Africa several centuries ago. Our results confirm that the hyperendemicity of HBV(E) in today's Africa is a recent phenomenon and likely the result of dramatic changes in the routes of viral transmission in a relatively recent past.