title: tick metagenome
design: To identify the bacterial diversity in the tick, we used a community profiling based amplification MiSeq Illumina sequencing approach of amplicon of bacterial 16S rDNA. Ticks were surface sterilised in 3% Hydrogen Peroxide, 70% (w/v) ethanol and 2 min in Phosphate Buffered Saline (PBS, pH = 7.4). Each tick was longitudinally bisected using a sterile disposable scalpel blade and each half was subjected to a different DNA extraction method. Method 1 exploited a mechanical maceration protocol using a high-speed benchtop homogeniser FastPrep-24 (MP Biomedicals, Australia) followed by extraction using the ISOLATE Fecal DNA Kit (Bioline, Australia). Method 2 consisted of enzymatic cell lysis followed by the ISOLATE II Genomic DNA Kit (Bioline, Australia).  Tick DNA samples were subjected to 16S rRNA gene diversity profiling assays at the Australian Genome Research Facility (Brisbane, Australia). Sequencing of the V1-V3 and V3-V4 16S rRNA hypervariable regions was performed on the Ilumina MiSeq (300-nt pair-end) using the following assay; 16S (V1-V3): 27F (AGA GTT TGA TCM TGG CTC AG) with 519R (GWA TTA CCG CGG CKG CTG) and 16S (V3-V4) 341F (CCT AYG GGR BGC ASC AG) with 806R (GGA CTA CNN GGG TAT CTA AT). The barcoded PCR amplicon was pooled and sequenced on the Ilumina Miseq.
identifiers.namespace: SUB3200459
identifiers.id: JS2950-16S-V3-V4