title: Sequence analysis of bacterial tick-borne pathogens in cattle from two provinces of South Africa using 16S rRNA PacBio approach
design: Genomic DNA was extracted from 200 l of blood using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) following the manufacturers instructions. The presence of bacterial communities in the blood samples was analyzed using single molecule real-time PacBio sequencing technology (Pacific Biosciences, Menlo Park, CA, USA). The full-length 16S rRNA gene was amplified from genomic DNA using bacterial-specific primer 27F 5'-AGAGTT TGA TCM TGG CTC AG-3' and 1492R 5-TAC GGY TAC CTT GTT ACG ACT T-3. For multiplexing of amplicons, the 5`-ends of the 16S rRNA forward and reverse primers were tagged with the universal M13F (TGTAAAACGACGGCCAGT) and M13R (GGAAACAGCTATGACCATG) sequences respectively. Two amplification reactions contained water as a template were used as negative controls. SMRTbell libraries were created using SMRTbell Template Prep Kit 1.0 (Pacific Biosciences, CA, USA) following instructions in the protocol Procedure & Checklist  Amplicon Template Preparation and Sequencing (part number 100-801-600-04). Sequencing was done using the Sequel Sequencing Kit 2.1 (Pacific Biosciences, CA, USA) with on-plate loading concentration of 4 pM.