Next Generation Sequencing and Tick Systematics

Ticks (Arachnida: Ixodida) are important vectors of a variety of pathogens. There are ~900 species that can be placed in three main families, hard ticks (Ixodidae), soft ticks (Argasidae) and the monotypic Nuttalliellidae. Current systematics uses mainly the nuclear 18S and 28S rRNA genes and mitochondrial genes. Next generation sequencing (Illumina) strategies were investigated to determine its usefulness for the assembly of these main markers and the discovery of new markers from fresh as well as museum specimens using genomic DNA sequencing. For this study, 25 different specimens (21 fresh, 4 museum) were investigated. Total genomic DNA yields varied from 2-450ng/ul. Samples were prepared using Nextera kits and sequenced using an Illumina HiScan. Data generated ranged from 0.5-4Gbp of ~100bp single read and paired end reads. Data were de novo assembled using CLC Genomics Workbench. The majority of assemblies yielded full-length 18S rRNA, 28S rRNA and mitochondrial genomes encoded in one contig. These generally corresponded with contigs that showed the highest coverage in the assemblies (37-2200 fold, depending on the sample). Assembly of fragmented de novo assembled contigs could be attained by mapping approaches. Coverage and assembly length for museum samples were generally better for mitochondrial genomes, while nuclear genes were more fragmented. Other highly abundant nuclear markers that could be identified in all samples included the core histone cassette (H4-H3-H2A-H2B). A next generation sequencing approach to identify markers for systematic analysis of single tick specimens is therefore a feasible alternative to labour some cloning and conventional sequencing approaches.