negative controls - PCR2_1
Genomic DNA extracted from ticks was amplified using a three-step PCR protocol. In PCR1, the full-length bacterial 16S rRNA gene was amplified using the 27F-YM and 1492R-HY primers in the presence of Rickettsia buchneri-specific blocking primers. PCR1 amplicons were then used as templates for PCR2 to amplify the V4 region using the 515F and 806R primers. Finally, PCR3 was performed to add Illumina sequencing adapters to the V4 amplicons prior to sequencing.