DOI: 10.5061/dryad.vhhmgqnw8
title: Assessing the performance and efficiency of environmental DNA/RNA capture methodologies under controlled experimental conditions

description: Growing interest and affordability of environmental DNA and RNA (eDNA and
 eRNA) approaches for biodiversity assessments and monitoring of complex
 ecosystems have led to the emergence of manifold protocols for nucleic
 acids (NAs) isolation and processing. Although there is no consensus on a
 standardized workflow, the common practice for water samples is to
 concentrate NAs via filtration using varying pore size membranes. Using
 the smallest pore is assumed to be most efficient for NAs capture from a
 wide range of material (including sub-cellular particles), however a
 trade-off must occur between detection of a meaningful molecular signal
 and cost/time effort when processing samples using fine pore membranes.
 Comparative studies involving formal efficiency assessments are lacking,
 which restricts informed decision-making around an optimized sampling
 approach for applications such as biosurveillance (i.e., detection and
 monitoring of target taxa - nuisance organisms, endangered and indicator
 taxa or other species of economic or cultural importance). Here, we
 present an experimental study using an easily cultured microalgal species
 (Alexandrium pacificum) to test different filter membranes for capturing
 NAs in the context of cost/time effort and cell fractions encountered in
 nature (whole cells, partially lysed, and naked NAs). The results showed
 no statistically significant difference between membrane types for
 capturing target eDNA signal from intact and partially lysed cell
 treatments. In terms of time effort and volume processed, higher
 efficiency ratings were obtained with the larger pore size (5 µm)
 cellulose membranes. Positively charged nylon demonstrated enhanced
 capture of naked NAs, and especially eRNA signal, across treatments. Our
 findings support using coarse pore size filters for adequate capture of
 target NA signal (from both eDNA and eRNA) with less processing time. The
 framework presented here can provide a quick and robust feasibility check
 and comparative assessment of new and existing NA processing technologies,
 and allows sufficient control over multiple parameters, including
 physical-chemical water properties, temporal scales, and concentration and
 type of input material.
description: A two-tier experimental set-up was applied in this study. In the
 first experimental round, the performance of different filter membranes
 for capturing various fractions of eDNA/eRNA was assessed. We selected
 cellulose acetate (CA) membranes (repeatedly reported performing well in
 eDNA studies) of three pore sizes (5 μm, 1.2 μm, 0.45 μm) and a positively
 charged nylon (PCN) 1.2 μm pore size membrane (as a potential alternative
 to commonly used filter types). A crossed experimental design was applied
 with three experimental factors manipulated: 
 eDNA/eRNA fractions, four levels: 1) intact (whole) cells of
 microalgal culture, 2) broken (sonicated) cells, 3) naked DNA, and 4)
 naked RNA. Seawater, two levels: 1) pre-filtered
 (0.35 μm) and 2) non-filtered <em>A. pacificum</em> free
 seawater (determined via light microscopy and ddPCR assay).
 Filter type, four levels: 1) 5 μm cellulose acetate (CA)
 membrane, 2) 1.2 μm CA, 3) 0.45 μm CA, 4) 1.2 μm positively charged nylon
 (PCN) membrane. The membranes (three
 replicates for each filter type) with the captured material were
 immediately transferred to sterile centrifuge tubes and kept frozen at -80
 °C until processed for dual DNA/RNA extraction. The
 second experiment was focused on establishing input and output parameters
 for an efficiency model and was run with two concentrations of sonicated
 <em>A. pacificum</em> cells spiked into target-free
 (determined via light microscopy) seawater. The spiked water was filtered
 through the same four types of membrane filters as outlined above (six
 replicates each), while recording a) the time required to filter 250 mL of
 water sample and b) the volume filtered in 20 min (through continued
 filtering past the 250 mL mark in [a]). The filters from step (b) were
 immediately frozen at -80 °C until simultaneous DNA/RNA
 extraction. RNA and DNA were extracted simultaneously
 from each filter using the Zymo Quick-DNA/RNA™ Miniprep Kit (Zymo
 Research, California USA). The filters were placed into individual ZR
 BashingBead Lysis Tubes and 0.8 mL of lysis buffer from
 the kit was added to each tube which was adequate volume to cover the
 filter. These were then homogenized by bead beating (1,500 RPM, 2 min;
 1600 MiniG Spex SamplePrep NJ, USA) such that material on all parts of the
 filter was exposed to the buffer and beads. After centrifugation (3,000 ×
 g, 5 min, 20 °C). DNA and RNA were co-extracted following the
 manufacturer’s protocol. Trace DNA in isolated RNA
 samples was eliminated by two sequential DNase (TURBO
 DNA-free<sup>TM</sup> Kit, Thermo Fisher Scientific, MA,
 United States) treatments following Langlet et al. (2013). Treated RNA was
 diluted to 10 ng/µL equimolar concentrations and reverse transcribed into
 cDNA using the SuperScript<sup>TM</sup> III reverse
 transcriptase (Thermo Fisher Scientific, MA, United States). All extracted
 products were stored frozen (−20 °C) for DNA and cDNA, and at −80 °C for
 RNA until analysis. <em>Alexandrium
 pacificum</em>-specific copy numbers were quantified using ddPCR for
 all samples, including all negative extraction controls (DNA and RNA) on a
 QX200 Droplet Digital PCR SystemTM (Bio-Rad, CA, United States). For the
 ddPCR assays, <em>A. pacificum</em> specific primers
 (ACTA-416-F and ACTA-605-R); targeting 204bp from the large ribosomal
 subunit) and Taqman probe (ACTA-456-P) were used as described in Ruvindy
 et al. (2018). Each ddPCR reaction included 0.5, 1.0, and 0.05 µM of the
 forward primer, reverse primer and probe respectively, 1 × BioRad ddPCR
 Supermix for probes, 1 µL pre-diluted DNA (1:25 for the first experiment
 and 1:50 for the second experiment) or 1 µL undiluted cDNA, and sterile
 water in a total reaction volume of 21 µL. The BioRad QX200 droplet
 generator partitioned each reaction mixture into approximately 20,000
 nanodroplets by combining 20 µL of the reaction mixture with 70 µL of
 BioRad droplet generation oil. After processing, this resulted in a total
 droplet volume of 40 µl, which was transferred to a PCR plate for
 amplification using the following cycling protocol: hold at 95 °C for 10
 min, 40 cycles of 94 °C for 30 sec and 54 °C for 1 min, and a final enzyme
 deactivation step at 98 °C for 10 min. Each well of the plate was then
 individually analyzed on the QX200 instrument to establish the threshold
 value separating negative and positive droplets and perform absolute
 quantification of target DNA or cDNA. A positive control of extracted DNA
 from the <em>A. pacificum</em> CAWD234 culture and negative
 (sterile water) control was included on each plate. For the first
 experiment, the results were converted to copies per sample using the
 formula: number of copies per µL × 22 [the initial volume of the PCR
 reaction (µL)] × 50 [DNA elution volume] or 25 [RNA elution volume] (µL).
 Since various sample volumes were processed through filter membranes as
 part of the second experiment, the ddPCR results were standardized to
 number of copies per “cell” of input material (<em>A.
 pacificum</em>), applying the following formula. Total volume (mL)
 of material that passed through the filter in 20 min multiplied by the
 “cell” concentration (equivalent to 9.8 cells/ml or 91 cells/ml, see above
 for details) to calculate total number of “cells” on the filter. This
 number was divided by 50 µL (DNA elution volume) or 25 µL (RNA elution
 volume) to calculate number of “cells” per µL. Copies per µL from ddPCR
 results were divided by “cells” per µL to obtain copies per
 “cell”.