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. 2017 Feb 8;10(1):71.
doi: 10.1186/s13071-017-2008-9.

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

Affiliations

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

Wibke J Cramaro et al. Parasit Vectors. .

Abstract

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.

Keywords: Annotation; Flow cytometry; Genome; Haploid genome size estimation; Ixodes ricinus; Single molecule real time sequencing; Tick; Tick cell line.

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Figures

Fig. 1
Fig. 1
Genome size estimation for I. ricinus tick cells by flow cytometry. Cellular DNA was quantitatively stained with propidium iodide (PI). Chicken red blood cells (CRBCs) were stained with CFSE and 1301 tetraploid T cells (1301) with BV421 coupled CD45-antibody. Fluorescence was analyzed for Ixodes ricinus nuclei (blue; a) and IRE/CTVM19 cells (blue; b) as well as CRBCs (green; a, b) and 1301 cells (red; a, b). Abbreviation: AFU, artificial fluorescence units. Graphs are representative of 3 independent biological replicates per sample
Fig. 2
Fig. 2
Genome size estimates for Ixodes ricinus tick cells from different origins by flow cytometry. The genome sizes of laboratory colony ticks from Insect Services (Germany) and Charles River (Ireland), field-collected ticks from Luxembourg as well as the I. ricinus embryo-derived cell line IRE/CTVM19 were analyzed by propidium iodide staining and flow cytometry. Error bars correspond to the standard deviation from 3 independent biological replicates
Fig. 3
Fig. 3
Most prevalent protein categories annotated in the Ixodes ricinus genome. Annotations were based on homology to annotated I. scapularis proteins. Annotative categories with at least 50 assigned sequences are shown. The number of assigned sequences is given in brackets. The most abundant hits were hypothetical protein (21% of total) and conserved hypothetical protein (13% of total), which were not included into the graph as they do not contain annotative information. A table detailing all annotations and their frequency is provided as Additional file 3

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