16s rRNA sequencing of Syngnathus_typhle : Male Frontgut
DNA from samples stored at -80C was extracted using the Qiagen DNeasy Blood & Tissue kit according to manufacture recommendations including a pre-treatment for Gram-positive bacteria (as in Tanger et al., 2024). Approximately the same amount of total DNA (expected to be primarily host DNA but also microbial DNA) per sample was then used for library preparation, which were then sequenced (V3-V4 region; forward primer 16s08fwd:5  3: AGAGTTTGATCMTGGCTCAG; reverse primer 16s1492rev: 5  3: ACCTTGTTACGACTT) on Illumina MiSeq sequencers by the Institute of Clinical molecular Biology (IKMB) at Kiel University, Germany. As the amount of input DNA was mostly comprised of host DNA and the ratio of microbial to host DNA in the input DNA likely varied among samples, microbial community results derived from these samples likely rather reflect the microbial community of a similarly sized piece of host tissue than a similar number of bacteria from a tissue. However, during library preparation sample may have been subjected to different PCR cycle numbers and thus absolute abundance values cannot necessarily be compared. Obtained read count files were demultiplexed and adapter sequences were trimmed in Qiime (v. 2020.8.0; Bolyen et al., 2019). Data were de-noised, merged, chimeric reads were removed and reads were trimmed using DADA2 in Qiime (similarly to Tanger et al., 2024). Taxonomy was assigned using the silva database (v.138SSUnr99-304.806; Pruesse et al., 2007), and mitochondria and chloroplast reads were removed. The dataset was than imported into R using the qiime2R package, where subsequent analyses were conducted (v.0.99.6; Bisanz, 2018; R Core Team, 2021).