Whole genome sequencing

To investigate the clinical features of C. burnetii, we sequenced the genomes of human isolates causing various clinical symptoms, in addition to five strains isolated from ticks and six from animals. Among 67 strains sequenced in our laboratory, 15 had been sequenced using SOLiD 4_Life technology while the remaining 52 were sequenced using the Illumina Miseq technology. For the SOLiD 4_Life sequencing, the paired-end library was constructed from 1 μg of purified genomic DNA. The DNA was fragmented on the Covaris device. Libraries were pooled in equimolar ratios and size selected on the E-Gel iBase system at 240–270 bp. Sequencing was carried out to 50 × 35 bp using SOLiD™ V4 chemistry on one full slide of 96 barcoded projects on an Applied Biosystems SOLiD 4 sequencer. The output read length was as expected 85 bp.

The paired-end strategy was used for the 52 strains sequenced using MiSeq Technology (Illumina Inc., San Diego, CA, USA). The genomic DNA was quantified by a Qubit assay with the high sensitivity kit (Life Technologies, Carlsbad, CA, USA) and was barcoded in order to be mixed with 14 other projects with the Nextera XT DNA sample prep kit (Illumina Inc., San Diego, CA, USA).

Purified, normalized and barcoded DNA were pooled for sequencing on the MiSeq sequencer. The pooled single strand library was loaded onto the reagent cartridge and then onto the instrument along with the flow cell. Automated cluster generation and paired-end sequencing with dual index reads were performed in a single 39-h run in 2 × 250-bp.