Ixodes ricinus genome shotgun sequencing
High molecular weight DNA was extracted according to a modified protocol from Hill and Gutierrez (Hill, C. A.; Gutierrez, J. A., A method for extraction and analysis of high quality genomic DNA from ixodid ticks. Medical and veterinary entomology 2003, 17 (2), 224-7). In brief, male ticks were frozen in liquid nitrogen and ground with a pestle. Powder was resuspended in 20 volumes of extraction buffer (10 mM Tris-Cl, pH 8.0, 100 mM EDTA and 1 % SDS) and incubated for 30 min at 37 °C under gentle mixing.  After centrifugation at 4,000 g for 10 min, proteinase K was added to the supernatant to a final concentration of 50 µg/ml and incubated at 37 °C overnight under gentle mixing. RNase cocktail (0.05 units RNase A and 2 units RNase T1 per mg tick tissue) was added and incubated at 50 °C for 3 hours. DNA was extracted using one volume 25:24:1 phenol:chloroform:isoamylalcohol mix and centrifuged for 5 min at 10,000 g and 4 °C. The aqueous phase was transferred to a fresh tube and the extraction repeated. A final extraction was done with 24:1 chloroform:isoamylalcohol. Extracted DNA was precipitated by adding 1/10 volume of 3 M sodium acetate (pH 5.5) and 2 volumes of 100 % ethanol and centrifugation for 10 min and 4 °C at 19,000 g. The pellet was washed with 70 % ice-cold ethanol, dried at room temperature and resuspended in 150 µl TE buffer (10 mM Tris, 1 mM EDTA). DNA was quantified with the Quant-iT Picogreen dsDNA Assay kit (Life technologies, Carlsbad, CA, USA) and integrity of high molecular weight DNA was confirmed by agarose gel electrophoresis.   A paired-end library was prepared according to the manufacturer’s paired-end sample preparation guide (Illumina, San Diego, CA, USA) and run on an Illumina HiSeqTM 2500 sequencer (Illumina, San Diego, CA, USA). Initial data processing and base calling of 100 base pair (bp) reads was done with RTA 1.17.20 (HiSeq Control Software 2.0.5, Illumina, San Diego, CA, USA) and sequence quality filtering script was executed in the Illumina CASAVA software (V1.8.2, Illumina, San Diego, CA, USA) by Ambry Genetics (Aliso Viejo, CA, USA).
Luxembourg Institute of Health
Genome_reads_Illumina