Bacterial endosymbionts of Dermacentor spp. ticks genomes and annotations

Here we provide the genome assemblies and genome annotations of rickettsial endosymbionts as well as CLE of Dermacentor spp. ticks. Hard ticks are ancient arachnid with unique physiology and evolution adaptability. Here we estimate the genetic diversity of bacterial endosymbionts of Dermacentor spp ticks. collected on large Eurasian territory (D. niveus, D. nuttalli, D. reticulatus, D. marginatus, D. silvarum). We have assembled and study rickettsial genomes from individual ticks. Additionally, we assembled D. reticulatus genome and developed genetic approach for determination of Dermacentor tick species. Adult ticks were subjected to DNA extraction. To remove soil bacteria and other microbial contaminants, each tick was thoroughly and individually washed in water solutions in a 2.0 ml plastic tube with a cap, first (i) in 0.25% Tween-20 and 1% sodium hypochlorite for five minutes, and then (ii) in mQ water three times for 1 min each with intensive pipetting. Each tick was immediately placed in 50 µl of DNA preservation solution. Fixing the tick with curved microsurgical tweezers, we made a transverse incision with microsurgical scissors and cut off part of the idiosome together with the gnathostome. Afterward, we isolated the tick organocomplex (soft tissues) using straight microsurgical tweezers and immediately placed the organocomplex with the solution in a tube containing an additional 50 µl of DNA preservation solution. Then, the organocomplexes were used for DNA extraction using a nucleic acid kit for metagenomic (microbiome) analysis via sequential steps: selective lysis of tick cells without damaging bacteria, treatment with nuclease to remove a large portion of eukaryotic nucleic acid, bacterial sedimentation, bacterial lysis and DNA isolation. Afterward, the DNA isolated from the ticks was subjected to ONT long-read sequencing as well as shotgun sequencing via the Illumina platform using 2300 paired-end reads, and the sequences were used to assemble genomes. To assemble rickettsial genomes, raw Illumina sequencing reads were processed for quality trimming (Phred score < 20) and adapter removal using Trim Galore (v0.6.10) available online at https://zenodo.org/records/7598955. Long reads obtained from ONT sequencing were adapter-trimmed using Porechop (v0.2.4) available online at https://github.com/rrwick/Porechop and quality-filtered with Filtlong (v0.2.1) available online at https://github.com/rrwick/Filtlong, applying a minimum read quality of 10 and a minimum read length of 100 bases. Taxonomic classification and reference genome selection were performed using Kraken2 (v2.1.3). Reads classified as belonging to the genera Rickettsia or CLE were extracted from metagenomic datasets using seqtk subseq (v1.4-r122) available online at https://github.com/lh3/seqtk. The reference genome for scaffolding was identified by NCBI BLAST. Hybrid reference-guided consensus assembly was then conducted using both Illumina and ONT sequencing data with SPAdes (v3.15.4) in hybrid mode (--nanopore). The resulting contigs were then scaffolded against a closely related reference genome using RagTag (v2.1.0). The largest scaffold was extracted and polished through multiple steps: Illumina reads were aligned to this scaffold using BWA-MEM (v0.7.18), and polishing was performed first with Pilon (v1.24), followed by Medaka (v1.2.3) available online at https://github.com/nanoporetech/medaka polishing using ONT reads. The final assembly was circularized using Simple-Circularise available online at https://github.com/ablab/simple-circularise, targeting sequences between 1 and 2.5 Mb in length. Genome completeness and assembly quality were assessed using BUSCO (v5.8.1) with the rickettsiales_odb10 lineage dataset at multiple steps, i.e., after RagTag scaffolding, Pilon polishing, and Medaka polishing. The assembly version with the highest BUSCO (5.8.1) completeness (more than 95%) and best overall quality metrics was selected as the final genome. For reference-guided consensus genome reconstruction of CLE genomes paired-end Illumina reads were aligned to the reference genome using minimap2 (v2.28; option -ax sr) , and ONT long reads were aligned with minimap2 (option -ax map-ont). The resulting BAM files were subsequently sorted and indexed with SAMtools (v1.21). Variant calling was performed using bcftools mpileup and bcftools call (v1.21) with --ploidy 1. Low-quality variants and low-coverage positions were masked, and a draft consensus genome was generated with bcftools consensus. The consensus was further polished in two rounds with racon (v1.5.0) using ONT reads, followed by a final polishing step with medaka_consensus (v1.2.3) available online at https://github.com/nanoporetech/medaka. The quality of the bacterial genomes was evaluated using QUAST v5.3.0. The completeness of the final assemblies was evaluated using BUSCO (v5.8.2, database “rickettsiaceae_odb12” for rickettsial genomes and the database “legionellales_odb12” CLE) based on the evolutionarily informed expectations of gene content from near-universal single-copy orthologs. All assembled bacterial genomes were annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP, version 2025-05-06.bulid 7983).