Ixodes ricinus_Genome sequencing by RRL. Ixodes ricinus strain:natural population

Tick collections and DNA extraction The DNA libraries to be used to detect suitable SNPs were constructed by sampling two I. ricinus populations. Only adult females (the life cycle stage with the largest amount of DNA) were used. The T population corresponds to 10 partially engorged females that were collected on roe-deer in south-west France (‘Gardouch’; 43° 23' 27.88"N, 1° 41' 1.67"E) during the winter of 2010 and kept at -80°C until DNA extraction. The M population corresponds to 20 non-engorged females that were w collected in spring 2011 (and kept alive at 4°C in the lab until DNA extraction) as they were questing for a host on vegetation in north-west France (‘Malville’; 47° 21' 30.10" N, 1° 51' 41.59"W). Each individual was extracted and the DNA concentration measured separately. The ticks were frozen in liquid nitrogen and crushed with a pestle in individual tubes. DNA extraction was conducted according to manufacturer instructions using the Nucleospin tissue XS kit (Macherey-Nagel). Genome reduction and sequencing After testing several restriction enzymes, MseI (T ^ AAT; New England Biolabs) was selected because a high concentration of DNA fragments of the target size could be obtained (id est 500-600b, to maximize the efficiency of 454 pyrosequencing relative to reads length) and no discrete band (that would reveal the presence of repeat elements) was observed in the target range of DNA fragments selected. Each sample was digested for 8 hours (2.5U/µg of DNA) according to manufacturer’s instructions. The digested DNA was then separated on a 1% agarose gel (4h, 80v). A gel piece of each sample, containing DNA fragments from 500 to 600 bp (according to the 100 pb marker size ladder; Eurobio) was sheared under a UV lamp. The DNA was then extracted and cleaned with the gel clean-up kit (Macherey-Nagel), eluted in 40 µl of EB Buffer (3.33mM Tris, pH 8.5), then quantified using Qubit™ with the dsDNA HS Assay™ kit (Invitrogen). Each sample was then sent to the Biogenouest Genomic platform (Rennes, France) (where the 454 sequencing was conducted). The samples from each of the 2 populations (T and M) were pooled separately. Pools were prepared from an equimolar quantity of DNA from each of the 10 to 20 samples respectively, corresponding to 500ng of DNA in 10 μl. Each of the 2 pools was tagged with a unique barcode (multiplex identifier MID), and was then sequenced using the Roche 454 GS FLX and Titanium chemistry.