DOI: 10.5061/dryad.xd2547dh6
title: Data from: Metabarcoding under Brine: Microbial ecology of five hyper-saline lakes at Rottnest Island (WA, Australia)

description: Hypersaline ecosystems - aquatic environments where concentration of salt
 exceeds 35 g/L - host microbial communities which are highly specialized
 to cope with these extreme conditions. However, our knowledge on the
 taxonomic diversity and functional metabolisms characterising microbial
 communities in the water columns of hypersaline ecosystems is still
 limited, and this lack of knowledge may compromise the future preservation
 of these unique environments. DNA metabarcoding provides a reliable and
 affordable tool to investigate environmental dynamics of aquatic
 ecosystems, and its use in brine can be highly informative. Here, we make
 use of bacterial 16S metabarcoding techniques combined with hydrochemical
 analyses to investigate the microbial patterns (diversity and functions)
 from five hypersaline lakes located at Rottnest Island (WA). Our results
 indicate lake-driven microbial aquatic assemblages characterised by
 taxonomically and functionally moderately to extremely halophilic groups,
 with TDS (Total Dissolved Solids) and alkalinity amongst the most
 influential parameters driving the community assemblages. Overall, our
 findings suggest that DNA metabarcoding allows rapid but reliable
 ecological assessment of the hypersaline aquatic microbial communities at
 Rottnest Island. Further studies involving different hypersaline lakes
 across multiple seasons will help elucidate the full extent of the
 potential of this tool in brine.
description: Information extracted from the section '2.2.2. Genetic
 Investigations' from the source manuscript titled 'Metabarcoding
 under Brine: Microbial Ecology of Five Hyper-saline Lakes at Rottnest
 Island (WA, Australia)': Water samples were used
 for bacterial 16S metabarcoding and microbial functional analysis. Five 1
 litre water sample replicates (<i>n</i>=50) from the five
 lakes (Garden, Vincent, Serpentine, Herschel and Baghdad; Figure 1) were
 investigated. Water samples were filtered using two Sentino peristaltic
 microbiology pumps (Pall Life 126 Sciences, New York, USA), through 0.45
 μm sterile membrane filters (Pall Life Sciences, New York, USA). All water
 filtering equipment was soaked for a minimum of 10 minutes in 10% sodium
 hypochlorite solution and treated with UV light prior to use and between
 sample replicates (<i>n</i>=5) for each SP. Immediately
 post-filtering, half of the filter membrane was used for DNA extraction,
 while the remaining half was frozen at -20°C.Water membranes, inclusive of
 laboratory controls, were extracted using DNeasy Blood and Tissue Kit
 (Qiagen; Venlo, Netherlands), with the following modifications to the
 manufacturer’s protocol. For the DNA digest from water
 samples, both the ATL buffer (360 μL) and Proteinase K (40 μL) solutions
 were doubled to ensure that the samples were adequately exposed to the
 lysis solution to optimise DNA yield. The DNA digests were incubated
 (56°C) overnight in a rotating hybridisation oven. The digest was
 transferred into a clean tube and loaded into a QIAcube (Qiagen; Venlo,
 Netherlands) automated DNA extraction system for the remainder of the
 extraction process. The DNA was eluted off the silica column in 100 μL AE
 buffer. The quality and quantity of DNA extracted from
 each sample was measured using quantitative PCR (qPCR), targeting the
 bacterial 16S gene. The PCR master mixes used to assess the quality and
 quantity of the DNA target of interest via qPCR (Applied Biosystems [ABI],
 USA) were carried out in 25 μL reaction volumes consisting of 2 mM
 MgCl<sub>2</sub> (Fisher Biotec, Australia), 1 x PCR Gold
 Buffer (Fisher Biotec, Australia), 0.4 μM dNTPs (Astral Scientific,
 Australia), 0.1 mg bovine serum albumin (Fisher Biotec, Australia), 0.4 μM
 of each primer (Bact16S_515F and Bact16S_806R; [36,37]), 0.2 μL of
 AmpliTaq Gold (AmpliTaq Gold, ABI, USA) and 2 μL of template DNA (Neat,
 1/10, 1/100 dilutions). The cycling conditions were: initial denaturation
 at 95°C for 5 minutes, followed by 40 cycles of 95°C for 30 seconds, 52°C
 for 30 seconds, 72°C for 30 seconds, and a final extension at 72°C for 10
 minutes. Extracts that successfully yielded DNA of
 sufficient quality, free of inhibition, as determined by the initial qPCR
 screen (detailed above), were assigned a unique 6-8bp multiplex identifier
 tag (MID-tag) with the bacterial 16S primer set. Independent MID-tag qPCRs
 for each sample were carried out in 25μL reactions containing 1 x PCR Gold
 Buffer, 2.5 mM MgCl<sub>2</sub>, 0.4 mg/mL BSA, 0.25 mM of
 each dNTP, 0.4 μM of each primer, 0.2μL AmpliTaq Gold and 2–4 μL of DNA as
 determined by the initial qPCR screen. The cycling conditions for qPCR
 using the MID-tag primer sets were as described above. MID-tag PCR
 amplicons were generated in duplicate for each sample and the bacterial
 16S library was pooled in equimolar ratio post-PCR for DNA sequencing. The
 final library was size selected (160-600bp) using Pippin Prep (Sage
 Sciences, USA) to remove any MID-tag primer-dimer products that may have
 formed during amplification. The final library concentration was
 determined using a QuBitTM 4 Fluorometer (Thermofischer, Australia) and
 sequenced using a 500 cycle V2 kit on an Illumina MiSeq platform
 (Illumina, USA). MID-tag bacterial 16S sequence reads
 obtained from the MiSeq were sorted (filtered) back to the water sample
 based on the MID-tags assigned to each DNA extract using Geneious v10.2.5
 [38]. MID-tag and primer sequences were trimmed from the sequence reads
 allowing for no mismatch in length or base composition.