DNA extraction, amplicon, and metagenomic sequencing

DNA extraction was performed using the DNeasy PowerSoil® Kit (Qiagen, USA) following the manufacturer’s instructions, using 0.25–0.35 g of sinter or sediment per sample (with a no-sample negative control included). DNA quality and quantity were checked via gel electrophoresis, a NanoPhotometer (Implen, Germany), and Qubit 3.0 fluorometric quantitation (Invitrogen, USA). 16S rRNA gene amplification was performed in triplicate using 515′F/926′R primers, and 2 × 250 bp paired-end Illumina MiSeq sequencing was performed with the MiSeq Reagent Kit V2, as previously described [7], at Auckland Genomics (University of Auckland). To explore the genomic features of the TVZ Acidithiobacillus, 4 representative sinter and 18 representative sediment samples were selected for whole genome sequencing (WGS). Five to seven g of DNA was extracted per sample using the DNeasy PowerMax Soil Kit® (Qiagen, USA). Due to low DNA concentrations, multiple extractions were performed for each sample, and DNA was concentrated and cleaned by ethanol precipitation using 2× absolute ethanol, 3 M sodium acetate, and 20 μl/μg glycogen. Samples were then incubated at − 80 °C overnight before DNA pellets were washed with 70% ethanol and resuspended in 25 μl of nuclease-free water. Final DNA quality and quantity were checked using an Implen NanoPhotometer, Qubit 3.0 fluorometric quantitation with the Qubit™ dsDNA HS Assay Kit, and gel electrophoresis. WGS sequencing was performed at the Otago Genomics Facility (University of Otago, Dunedin, New Zealand). Thruplex DNA-Seq libraries (Takara Bio USA, Inc, USA) were prepared with 500–600 bp insert sizes and sequenced using the Illumina HiSeq 2500 platform with the HiSeq Rapid SBS Kit V2, yielding 2 × 250 bp reads, for the 4 sinter samples, and the HiSeq 2500 SBS Kit V4, yielding 2 × 125 bp reads, for the 18 sediment samples.