Preparation of 16S rDNA Amplicon Libraries and Sequencing

The V1–V2 region of the16 S rRNA gene was amplified using the forward primer mix 27f-YM+3, which consisted of four parts 27f-YM 5′-AGA GTT TGA TYM TGG CTC AG-3′ plus one part each of 27f-Bif 5′-AGG GTT CGA TTC TGG CTC AG-3′, 27f-Bor 5′-AGA GTT TGA TCC TGG CTT AG-3′, and 27f-Chl 5′-AGA ATT TGA TCT TGG TTC AG-3′ (21). V1V2rev 5′-CTG CTG CCT YCC GTA-3′ was used as reverse primer (22). All primers were tagged with unique nonameric (= conisting of nine subunits) barcodes to allow for multiplexing of samples (23). The predicted amplicon length was 380 base pairs (bp). The reaction was carried out in 50-μL reaction mixtures containing 5 μL of AmpliTaq Gold buffer 10x (Applied Biosystems, Branchburg, NJ, USA), 2 μL of mixed forward and reverse primer (20 μM), 1 μL of 10 mM deoxynucleoside triphosphates, 3 μL of 25 mM MgCl₂, 0.5 μL of AmpliTaq Gold polymerase (5 U/μL; Applied Biosystems), 37.5 μL of nuclease-free water, and 1 μL of DNA template. Cycling conditions were 94°C for 6 min; 35 cycles of 94°C for 45s, 57°C for 45 s, and 72°C for 90 s followed by a final elongation at 72°C for 10 min. The samples' respective extraction controls and one no-template control (NTC) were included in each PCR run. Purified DNA from C. burnetii, Chlamydia abortus, Chlamydia pecorum, Chlamydia psittaci, and Leptospira interrogans was amplified in the same way and was used as positive control: each species separately and additionally all five species together as one mixed positive control. DNA concentration and purity of the resulting PCR products were analyzed on an Agilent 2100 Bioanalyzer using the Agilent DNA 1000 kit (Agilent Technologies, Waldbronn, Germany). PCR products were pooled in equimolar ratios of 50 ng per sample. For the controls, all available PCR product up to 50 ng was used. Each pool consisted of a total of 49 samples and their respective controls. The pooled DNA was purified using the QiagenMinElute PCR purification kit (Qiagen) according to the manufacturer's instructions. The DNA pools were sequenced on a HiSeq 250PE platform (Illumina, San Diego, CA, USA) at the National High-Throughput DNA Sequencing Centre, University of Copenhagen, Denmark.