4.6. Whole Genome Sequencing (WGS)

To further characterise the strain involved in the abortion cases, C. pecorum from a previous abortion case that had been cultured in McCoy cells in [12] was subjected to whole genome sequencing using an Ion Torrent S5. DNA was extracted from tissue culture supernatant using the KingFisher Flex purification system (ThermoFisher, Waltham, MA, USA) and eluted in molecular grade water prior to quantification on a Qubit Fluorometer (Thermofisher). A library was prepared from 100 ng of genomic DNA by fragmentation of the sample using the Ion Xpress^TM Plus Fragment Library Kit (Thermofisher catalog no. 4471269) and incubating for 7 min at 37 °C before magnetic bead purification using ethanol washes. DNA was eluted in low TE buffer. DNA was ligated and barcodes added at 25 °C for 15 min followed by 72 °C for 5 min. The library was purified again using the magnetic bead method and products of approximately 450 bp in length were recovered using the E-gel precast system. Amplification of the library was achieved using the following conditions: 1 × cycle of 95 °C for 5 min followed by 9 × cycles of 95 °C for 15 s; 58 °C for 15 s and 70 °C for 60 s. The amplified library was purified and diluted 1:100 before real-time PCR quantification on a ViiA 7 against Escherichia coli standards using the Ion Universal Library Quantitation kit (ThermoFisher catalog no. A26217). The cycling conditions for the PCR were as follows: 50 °C for 2 min; 95 °C for 20 s followed by 40 × cycles of 95 °C for 1 s and 60 °C for 20 s. The quantified library was diluted to a concentration of 100 pM and loaded onto an Ion 520 chip using the Ion Chef system. The prepared chip was then run on the Ion Torrent S5 next generation sequencing system. To ensure adequate coverage of the C. pecorum assembly two libraries were generated from one sample of purified DNA using the above method.