G2 lysis buffer

DNA extraction was performed daily using the EZ1 (Qiagen GmbH, Hilden, Germany) automated protocol with mechanical, chemical and enzymatic pre-treatment [15 (link)]. The extraction protocol was adapted for stool processing as follows: 200 mg of stool sample was added to 350 µl of G2 lysis buffer (Qiagen GmbH) in a tube containing glass powder (acid-washed glass beads 425–600 µm, Sigma-Aldrich, Saint Quentin Fallavier, France) and then disrupted in a FastPrep-24 grinder (MP Biomedicals, Illkirch-Graffenstaden, France) at maximum power for 40 s. After 10 min of incubation at 100 °C to allow complete lysis, tubes were centrifuged at 10 000 g for 1 min. A 200 µl of supernatant was then added to a tube containing 20 µl of Proteinase K, and incubated overnight at 56 °C. Finally, the automated protocol using the EZ1 Advanced XL extractor was performed as described by the manufacturer. Extracted DNA was eluted in 200 µl.
To control for both DNA extraction quality and the absence of PCR inhibitors, a eubacterial 16S rRNA qPCR was performed on each DNA, as previously described [16 (link)]. Extraction was repeated in the event of a negative result which indicates the presence of PCR inhibitors in the sample.

DNA extraction was performed using the EZ1^®DNA tissue kit (Qiagen, Hiden, Germany) on BIOROBOT EZ1 (Qiagen, Hiden, Germany), according to the manufacturer’s instructions. Initially, we mixed in tubes about 200 mg of stool with 360 µL of G2 lysis buffer (Qiagen, Hiden, Germany). This was mechanically lysed with tungsten beads (Qiagen, Hiden, Germany) using FastPrep-24TM 5G Grinder for 40 s. After 10 min of incubation at 100 °C to allow for complete lysis, tubes were centrifuged at 10,000× g for 1 min. Subsequently, 200 μL of supernatant was enzymatically digested using 20 μL of proteinase K (20 mg/mL, Qiagen) and incubated overnight at 56 °C. DNA was extracted from 200 µL of sample, eluted in 200 µL volume, then aliquoted in individual tubes of: pure extracted DNA, diluted to 1:10 and finally DNA diluted to 1:100.
To control the extraction quality and the absence of PCR inhibitors, universal eubacterial qPCR targeting the 16S rRNA bacterial genes, named “qPCR all bacteria” [70 (link)], was performed on pure DNA, dilutions to1:10 and to 1:100. By comparison of Ct values obtained for pure and diluted DNAs, the dilution to1:10 were chosen for parasite screening. DNA tubes were stored at −20 °C until use.

The half-ticks were incubated individually at 56 °C overnight in 180 μl of G2 lysis buffer (Qiagen, Hilden, Germany) and 20 μl of proteinase K (Qiagen), followed by extraction using the EZ1 DNA tissue kit (Qiagen) according to the manufacturer’s recommendations. The 15 tick specimens whose spectra were entered in our database as well as 43 ticks with LSVs > 1.8 and five specimens with LSVs < 1.8 randomly selected were subjected to standard PCR, followed by sequencing using the 16s, 12s rDNA genes and the mDNA Cox1 gene (Boyer et al. 2019 (link); Diarra et al. 2017 (link)). The forward and reverse reads of the targeted mtDNA sequences were assembled and analysed using ChromasPro v.1.34 software (Technelysium, Tewantin, Australia) and submitted for analysis to the NCBI BLAST website (http://blast.ncbi.nlm.nih.gov).
The different mtDNA gene sequences were aligned with BioEdit, and phylogenetic trees were inferred using TOPALi v.2.5 software (Biomathematics and Statistics Scotland, Edinburgh, UK) (Laroche et al. 2017b (link)). The model Maximum Likelihood (ML) phylogenetic tree proposed default in TOPALi v.2.5 software was used for the construction of phylogenetic trees. Node numbers are percentages of the bootstrap values obtained by repeating 100 iterations of the analysis to generate a majority consensus tree (only those with a value equal to or greater than 80 were retained).