Bacterial microbiota of salivary gland from engorged female ticks after 2 days of water treatment
The V3-V4 region of the bacterial 16S ribosomal RNA (rRNA) gene was amplified by PCR with barcode-indexed primers (338F: 5'- ACT CCT ACG GGA GGC AGC AG-3' and 806R: 5'- GGA CTA CHV GGG TWT CTA AT-3'), using TransStart Fastpfu DNA Polymerase (TransGen, CityBeijing, China). PCRs were performed on GeneAmp 9700 PCR instrument (Applied Biosystems, Foster City, CACity, State abbreviation, USA). This primer set, resulteding in a 420- to 460 -bp PCR products. Amplicons were then purified by gel extraction (AxyPrep DNA GelExtraction Kit;, Axygen Biosciences, Union City, CA, City, State abbreviation, USA) and were quantified using QuantiFluor-ST (Promega, Madison, WI,City, State abbreviation, USA). The purified amplicons were pooled in equimolar concentrations, and paired-end sequencing was performed on an Illumina MiSeq PE344 platform (Shanghai Majorbio Bio-pharm Technology Co., Ltd, City,Shanghai, China) using standard protocols.