DOI: 10.5061/dryad.pg4f4qrs4
title: Environmental nucleic acids: a field-based comparison for monitoring freshwater habitats using eDNA and eRNA

description: Nucleic acids released by organisms and isolated from environmental
 substrates are increasingly being used for molecular biomonitoring. While
 environmental DNA (eDNA) has received attention recently, the potential of
 environmental RNA as a biomonitoring tool remains less explored. Several
 recent studies using paired DNA and RNA metabarcoding of bulk samples
 suggest that RNA might better reflect “metabolically active” parts of the
 community. However, such studies mainly capture organismal eDNA and eRNA.
 For larger eukaryotes, isolation of extra-organismal RNA will be
 important, but viability needs to be examined in a field-based setting. In
 this study we evaluate (a) whether extra-organismal eRNA release from
 macroeukaryotes can be detected given its supposedly rapid degradation,
 and (b) if the same field collection methods for eDNA can be applied to
 eRNA. We collected eDNA and eRNA from water in lakes where fish community
 composition is well documented, enabling a comparison between the two
 nucleic acids in two different seasons with monitoring using conventional
 methods. We found that eRNA is released from macroeukaryotes and can be
 filtered from water and metabarcoded in a similar manner as eDNA to
 reliably provide species composition information. eRNA had a small but
 significantly greater true positive rate than eDNA, indicating that it
 correctly detects more species known to exist in the lakes. Given
 relatively small differences between the two molecules in describing fish
 community composition, we conclude that if eRNA provides significant
 advantages in terms of lability, it is a strong candidate to add to the
 suite of molecular monitoring tools.
description: Sampling was conducted at the IISD
 Experimental Lakes Area (IISD-ELA), a remote research and monitoring
 facility in north-western Ontario, Canada. We sampled two lakes in summer
 and autumn of 2017 and repeated the summer and autumn sampling in five
 lakes in 2018. There are 14 species of fish across all the study lakes
 (mean 8, range 6-10 species per lake). All lakes have overlapping
 community compositions, including Salvelinus namaycush, a cold-water top
 predator, in every lake. Water samples were taken at six depths, dispersed
 vertically throughout the water column at the deepest centre point of each
 lake. The sampling points were distributed at six evenly spaced intervals,
 but because the lakes were different depths, absolute measurements differ
 between the lakes. One 500 ml water sample was taken per depth (for a
 total of 6 samples per lake per season) using an electrical pump and
 Jayflex PVC tubing (Winnipeg Johnston Plastics, MB, Canada) secured to a
 weight. In total, 84 water samples were taken throughout the entire study
 (6 samples x 2 lake states x 7 lake replicates). Water was filtered onto
 47 mm 0.7 μm pore GF/F filters using an electric vacuum pump and filtering
 manifold (Pall Corporation, ON, Canada). The filters were divided into two
 with scissors which had been bleached, rinsed and autoclaved. Half of each
 filter was immediately stored in screw-cap tubes at -20 ⁰C. The other half
 was preserved in 370μl RLT buffer (Qiagen) with 1% β-mercaptoethanol and
 then frozen at -20°C for RNA analysis. One negative control of 500 ml
 distilled water was stored in the cooler and filtered in the same way as
 the field samples for each lake. In total, 84 eDNA and 84 eRNA samples
 were taken across the entire study. Filters were shipped on dry ice to
 McGill University, Montréal for molecular analysis. DNA was extracted from filters using the
 Qiagen Blood and Tissue kit following the manufacturer’s instructions
 except that 370μl of ATL buffer was added to the filter used in an initial
 overnight incubation step. After incubation, 325μl of fluid was
 transferred into an autoclaved microtube for the downstream extraction
 protocol. RNA was extracted from the filter halves preserved in RLT and
 β-mercaptoethanol. Extractions of eRNA were performed from the first half
 of the filter using the Qiagen RNEasy Mini kit with some modifications to
 accommodate the filter. Filters were vortexed for 20 seconds and
 centrifuged in the RLT/ β-mercaptoethanol buffer for 3 minutes at
 14,000rpm. A total of 325μl of this buffer was mixed with 325μl ethanol
 and the rest of the procedure followed the kit protocol intended for
 extracting total RNA from animal cells. The eRNA was resuspended in two
 elutions of 30μl RNAse free water to give a final volume of
 60μl. To avoid DNA
 contamination in the eRNA samples, DNA was digested from 20µl eRNA
 extracts with the DNA-free™ DNA Removal Kit (ThermoFisher Scientific)
 following the manufacturer’s instructions and using 2µl DNase I Buffer,
 1µl rDNase I and 2µl DNase Inactivation reagent. RNA samples were checked
 for residual contaminating DNA using PCR amplification using the MiFish-U
 primers tagged with Illumina sequencing adapters (Miya et al., 2015).
 These primers target a hypervariable region of the 12S rRNA locus
 (163-185bp in length). We used the following PCR chemistry: 7.4µl nuclease
 free water (Qiagen), 1.25µl 10X buffer (Genscript), 1 mM MgCl2
 (ThermoFisher Scientific), 0.2mM GeneDirex dNTPs, 0.05mg bovine serum
 albumen (ThermoFisher Scientific), 0.25mM each primer, 1U taq (Genscript)
 and 2µl DNA in a final volume of 12.5µl. We followed a touchdown
 thermocycling protocol which we have found reduces the amount of
 non-specific amplification (bacterial taxa) at this locus: 95°C for 3
 minutes, 12 cycles of touchdown PCR (98°C for 20 seconds, 66°C for 15
 seconds decreasing by 0.2°C each time, 72°C for 15 seconds) followed by 28
 cycles with an annealing temperature of 64°C, 72°C extension for 5
 minutes. We then visualised the resulting PCR products on 1% agarose gels
 stained with SYBR Safe. No residual contaminating DNA was found in eRNA
 samples. A total of 10μl of sample was therefore reverse transcribed into
 cDNA using the High-Capacity cDNA Reverse Transcription kit (ThermoFisher
 Scientific) in 20µl reactions following the kit instructions.
 We then amplified the cDNA and eDNA
 in triplicate 12.5µl reactions following the MiFish-U PCR protocol laid
 out above, and checked amplification using 1% agarose gels with SYBR Safe.
 We then combined the triplicate reactions into one sample and performed a
 cleanup with AMPure beads using a ratio of 1 part PCR product to 1.2 parts
 beads. Cleaned amplicons were then dual-indexed using the Nextera v2 DNA
 indexes, and cleaned again with a ratio of 1 part PCR product to 0.8 parts
 beads. The second clean-up was performed using a Biomek FX Laboratory
 liquid handling Automation Workstation (Beckman Coulter, CA). Samples were
 equimolarised to 3ng/μl and sent for sequencing at Génome Québec,
 Montréal. Samples were sequenced using 2 x 250bp paired end sequencing
 with an Illumina MiSeq. We
 used custom scripts to remove adapters, merge paired sequences, check
 quality and generate amplicon sequencing variants (ASVs). Samples were
 received as demultiplexed fastq files from Génome Québec. Non-biological
 nucleotides were removed (primers, indices and adapters) using cutadapt.
 Paired reads were merged using PEAR. Quality scores for sequences were
 analysed with FASTQC. Amplicon sequencing variants (ASVs) were generated
 using the UNOISE3 package, which uses a denoising pipeline to remove
 sequencing error and to cluster sequences into single variants (100%
 similarity). The full pipeline is available from
 https://github.com/CristescuLab/YAAP. After ASVs were generated, we
 assigned taxonomy using BLAST+ and BASTA, a last common ancestor
 algorithm. We used a custom reference database which contained only fish
 known to exist in the Lake of the Woods region (Ontario, CA), downloaded
 from the NCBI database on 12 August 2018.
description: See ReadMe file.