Bacteria 16S rRNA amplicon sequencing of A. variegatum: Adult female SO1
Total nucleic acid was isolated from the whole tick with the Quick-DNA/RNA Miniprep Kit (Zymo Research, Irvine, CA, US) following the manufacturers protocol. RNase treatment was performed for each sample to obtain pure genomic DNA (gDNA) using NucleoSpin Clean-up XS kit (Takara Bio. Inc, Shiga, Japan) according to the manufacturer's protocol. The concentration and purity of the gDNA was measured using the Nanodrop 2000 (Thermo Scientific, Delaware, ME, USA). The V3-V4 variable region of the bacteria 16SrRNA were paired-end sequenced on the Illumina platform (250 bp paired-end) using the primers 341F (5'-CCT AYG GGR BGC ASC AG-3') and 806R (5'-GGA CTA CNN GGG TAT CTA AT-3') at the Novogene Bioinformatics Technology Co., Ltd (Japan K.K., Tokyo, Japan). A total of 1,754,133 demultiplexed paired-end reads were obtained following sequencing and initial quality trimming. Data viewing and further sequence processing were performed with QIIME 2023.7. A table of amplicon sequence variants (ASVs) and representative sequences were obtained following sequence dereplication, joining of paired reads and chimera filtering with dada2 plugin. Taxonomic classification was performed using a custom nave bayes classifier that was extracted from SILVA 138.1 SSU reference based on the primers used. The resulting taxonomic table and representative sequences were filtered to exclude mitochondria, chloroplasts, Archaea and Eukaryota.