title: Hot spring water
design: V3 regions of all bacterial 16S rRNA genes present in an environmental DNA preparation were PCR amplified using domain-specific universal oligonucleotides, following the fusion primer protocol described previously (Ghosh et al., 2015; Roy et al., 2016; Fernandes et al., 2018). Amplification was carried out using a 16S forward primer prefixed with an Ion Torrent adapter and a unique sample-specific barcode or multiplex identifier in the following 5 to 3 organization: (a) a 26-mer A-linker followed by a 4-mer A-linker key common for all sample-specific primers (see bases in bold fonts in the sequences given below), (b) a 10-mer barcode unique to each sample-specific primer, which is followed by a common 3-mer barcode adaptor (all denoted as stars in the sequences below), and then (c) the relevant domain-specific universal forward primer in its 5 to 3 direction (see underlined bases in the sequences given below). A reverse primer, in contrast, had (a) a common trP1 adapter (see bases represented in italics), followed by (ii) the relevant domain-specific universal reverse primer in its 5 to 3 direction (see underlined bases in the sequences given below). In this way, V3 portions of all bacterial 16S rRNA genes present in a sample DNA were amplified using the forward primer 5 - CCA TCT CAT CCC TGC GTG TCT CCG ACT CAG *** *** *** *** *CC TAC GGG AGG CAG CAG - 3 and the reverse primer 5' - CCT CTC TAT GGG CAG TCG GTG ATA TTA CCG CGG CTG CTG G - 3 (where the underlined segments represent the universal primers 341f and 515r respectively).PCR products from all the samples were pooled up at equal concentrations for sequencing on Ion PGM (Thermo Fisher Scientific). Prior to sequencing, size distribution and DNA concentration within the amplicon pool was checked using a Bioanalyzer 2100 (Agilent Technologies) and adjusted to 26 pM. Amplicons were then attached to the surface of Ion Sphere Particles (ISPs) using an Ion Onetouch 200 Template kit (Thermo Fisher Scientific). Manually enriched, templated-ISPs were then sequenced by PGM on an Ion 316 Chip for 500 flows. Before retrieval from the sequencing machine, all reads were filtered by the inbuilt Torrent Suite 5.8.0 software to remove low-quality and polyclonal-sequences; sequences matching the PGM 3 adaptor were also trimmed.