DOI: 10.5061/dryad.2rbnzs7tb
title: Drop it all: Extraction-free detection of non-indigenous marine species through optimized direct-droplet digital PCR

description: Molecular biosecurity surveillance programs increasingly use environmental
 DNA (eDNA) for detecting marine non-indigenous species (NIS). However, the
 current molecular detection workflow is cumbersome, prone to errors and
 delays, and thereby can hinder management efforts and restrict the
 “opportunity window” for a rapid response to new marine NIS incursions.
 Here, we describe the first method to use direct digital droplet PCR
 (direct-ddPCR) to detect species-specific free-floating extra-cellular
 eDNA (free-eDNA) signals, i.e. detection of species-specific eDNA without
 the need for filtration or DNA extraction, with seawater sample. This
 first proof-of-concept aquarium study was conducted with three distinct
 marine NIS: the Mediterranean fanworm Sabella spallanzanii, the
 ascidian clubbed tunicate Styela clava, and the brown bryozoan Bugula
 neritina to evaluate the detectability of free-eDNA in seawater. The
 detectability of targeted free-eDNA was assessed by directly analysing
 aquarium marine water samples using an optimized species-specific ddPCR
 assay. The results demonstrated the consistent detection
 of Sabella spallanzanii and Bugula neritina free-eDNA when these
 organisms were present in high abundance. Once organisms were removed, the
 free-eDNA signal exponentially declined, noting that free-eDNA persisted
 between 24-72 hours. Results indicate that organism biomass, specimen
 characteristics (e.g., stress and viability), and species-specific
 biological differences may influence free-eDNA detectability. This study
 represents the first step in assessing the feasibility of direct-ddPCR
 technology for the detection of marine species. Our results provide
 information that could aid in the development of new technology, such as a
 field development of ddPCR systems which could allow for automated
 continuous monitoring for marine biosurveillance, enabling point-of-need
 detection and rapid management response to biosecurity threats.
description: An aquarium experiment was conducted to assess the feasibility of
 digital polymerase chain reaction (ddPCR) for detecting free-floating
 extra-cellular environmental DNA (free-eDNA) from three marine invasive
 species: <em>Sabella spallanzanii</em> (Mediterranean
 fanworm), <em>Styela clava</em> (Ascidian clubbed tunicate),
 and <em>Bugula neritina</em> (brown bryozoan). The experiment
 aimed to achieve the following objectives:
 i. Investigate the immediate detection of marine invasive species
 using ddPCR technology and free-eDNA in seawater samples.
 ii. Determine the influence of species characteristics and
 biomass on the detection of free-eDNA. iii. Assess the
 longevity of free-eDNA signals in the system after the removal of
 organisms. In brief, the organisms were placed in three
 tanks with varying biomass, determined by the total weight and number of
 organisms. Water samples (1 mL) were collected using sterile dual filter
 tips from each tank, 5-7 cm below the surface. Sampling occurred
 immediately after adding the organisms and at various time points (4, 8,
 24, 48, 72, 96, and 192 hours). Six replicates were randomly taken from
 different locations within each tank. The samples were collected in
 microcentrifuge tubes, kept on ice, and processed using optimized
 species-specific ddPCR reactions. After removing the organisms, additional
 samples were collected at time points 0,4,8,24,48 and 72, as described
 above. The removed organisms were weighed and photographed. Each ddPCR
 plate run included at least one negative control (RNA/DNA-free water) and
 one positive control (targeted species genomic DNA). Note Based on our
 experience and observation of ddPCR noise (e.g., proportions of
 fluorescing droplets in water blanks), the detection for all assays was
 set above the maximum value of the negative controls in the experiment,
 i.e., 0.08 copies/µL for the <em>B. neritina</em> and
 <em>S. clava</em> ddPCR assays and 0.130 copies/µL for the
 <em>S. spallanzanii</em> ddPCR assay.
 Furthermore, the ddPCR reactions were assessed by determining the
 limit of detection (LOD) and quantification (LOQ) for the ddPCR assay.
 Serial dilutions of the positive control (genomic DNA extracted from
 either <em>S. clava</em> and <em>S.
 spallanzanii</em> or <em>B. neritina</em>) were
 performed and analyzed. The two assays' ten-fold series of the 2x
 dilution (e.g., 1/100 to 1/102400) began with the genomic DNA diluted to
 200 pg (1/100 dilution) and ended with a final concentration of 0.195 pg.
 Six replicates of each dilution and negative control were included in both
 series, and all dilutions were performed with fresh seawater from the
 aquarium experiment.
description: <strong>Data overview:</strong> The two spreadsheets
 contain raw data resulting from the aquarium experiment and the ddPCR
 reaction assessment. To open files it requires the use of software that
 opens xlsx such as microsoft office excel.
 <em>aquarium_ddPCR raw_data:</em>
 "Sample_ID" – Unique identifier for each
 sample. "Tank" – The tank from which the
 sample was collected. "Hours" – The
 time-point at which the sample was collected.
 "Organism_Present" – Indicates whether the marine NIS
 was present ("Yes") or absent ("No") in the tank at
 the time of sample collection. "Biomass" –
 Categorical biomass category of the targeted species. Categories include
 high, medium, and low, based on the relative weight within each
 species. "Weight" – Combined weight, in
 grams, of the target species in the tank, measured after the removal of
 organisms. "Target_Species" – The specific
 marine NIS organism targeted during the species-specific ddPCR
 assays. "Concentration" – Concentration of
 the Cytochrome c oxidase subunit 1 (COI) gene, reported by ddPCR, in
 copies per microliter (copies/µL). 
 <em>ddPCR_assessment raw_data:</em>
 "Dilution" – Dilution factor used to dilute
 the standard curve, record as 1/X, where x is the dilution
 "Loaded_DNA_pg" – Amount of DNA loaded as a standard,
 recorded in picograms (pg). "Target" – The
 specific marine NIS organism targeted during the species-specific ddPCR
 assays. "Reported_Conc" – Concentration of
 the Cytochrome c oxidase subunit 1 (COI) gene, reported by ddPCR, in
 copies per microliter (copies/µL).  Note that the
 "Blank" variable under dilutions represents the negative control
 for the standard curve, where no standard (genomic DNA) was
 loaded.
description: # Title of Dataset: Raw data from manuscript Drop it all: extraction-free
 detection of non-indigenous marine species through optimized direct
 droplet digital. See details below for information on experiment and the
 data included in the raw data files ### Manuscript authors \*Scriver,
 Michelle \*von Ammon, Ulla \*Youngbull, Cody \*Pochon, Xavier \*Stanton,
 Jo-Ann L. \*Gemmell, Neil \*Zaiko, Anastasija ## Description of the Data
 and experiment ### Abstract: 1. Molecular biosecurity surveillance
 programs increasingly use environmental DNA (eDNA) for detecting marine
 non-indigenous species (NIS). However, the current molecular detection
 workflow is cumbersome, prone to errors and delays, and thereby can hinder
 management efforts and restrict the “opportunity window” for a rapid
 response to new marine NIS incursions. 2. Here, we describe the first
 method to use direct digital droplet PCR (direct-ddPCR) to detect
 species-specific free-floating extra-cellular eDNA (free-eDNA) signals,
 i.e. detection of species-specific eDNA without the need for filtration or
 DNA extraction, with seawater sample. This first proof-of-concept aquarium
 study was conducted with three distinct marine NIS: the Mediterranean
 fanworm *Sabella spallanzanii,* the ascidian clubbed tunicate *Styela
 clava,* and the brown bryozoan *Bugula neritina* to evaluate the
 detectability of free-eDNA in seawater. The detectability of targeted
 free-eDNA was assessed by directly analysing aquarium marine water samples
 using an optimized species-specific ddPCR assay. 3. The results
 demonstrated the consistent detection of *Sabella spallanzanii* and
 *Bugula neritina* free-eDNA when these organisms were present in high
 abundance. Once organisms were removed, the free-eDNA signal exponentially
 declined, noting that free-eDNA persisted between 24-72 hours. Results
 indicate that organism biomass, specimen characteristics (e.g., stress and
 viability), and species-specific biological differences may influence
 free-eDNA detectability. 4. This study represents the first step in
 assessing the feasibility of direct-ddPCR technology for the detection of
 marine species. Our results provide information that could aid in the
 development of new technology, such as a field development of ddPCR
 systems which could allow for automated continuous monitoring for marine
 biosurveillance, enabling point-of-need detection and rapid management
 response to biosecurity threats. ### Methods An aquarium experiment was
 conducted to assess the feasibility of digital polymerase chain reaction
 (ddPCR) for detecting free-floating extra-cellular environmental DNA
 (free-eDNA) from three marine invasive species: Sabella spallanzanii
 (Mediterranean fanworm), Styela clava (Ascidian clubbed tunicate), and
 Bugula neritina (brown bryozoan). The experiment aimed to achieve the
 following objectives: i. Investigate the immediate detection of marine
 invasive species using ddPCR technology and free-eDNA in seawater samples.
 ii. Determine the influence of species characteristics and biomass on the
 detection of free-eDNA. iii. Assess the longevity of free-eDNA signals in
 the system after the removal of organisms. In brief, the organisms were
 placed in three tanks with varying biomass, determined by the total weight
 and number of organisms. Water samples (1 mL) were collected using sterile
 dual filter tips from each tank, 5-7 cm below the surface. Sampling
 occurred immediately after adding the organisms and at various time points
 (4, 8, 24, 48, 72, 96, and 192 hours). Six replicates were randomly taken
 from different locations within each tank. The samples were collected in
 microcentrifuge tubes, kept on ice, and processed using optimized
 species-specific ddPCR reactions. After removing the organisms, additional
 samples were collected at time points 0,4,8,24,48 and 72, as described
 above. The removed organisms were weighed and photographed. Each ddPCR
 plate run included at least one negative control (RNA/DNA-free water) and
 one positive control (targeted species genomic DNA). Note Based on our
 experience and observation of ddPCR noise (e.g., proportions of
 fluorescing droplets in water blanks), the detection for all assays was
 set above the maximum value of the negative controls in the experiment,
 i.e., 0.08 copies/L for the B. neritina and S. clava ddPCR assays and
 0.130 copies/L for the S. spallanzanii ddPCR assay. Furthermore, the ddPCR
 reactions were assessed by determining the limit of detection (LOD) and
 quantification (LOQ) for the ddPCR assay. Serial dilutions of the positive
 control (genomic DNA extracted from either S. clava and S. spallanzanii or
 B. neritina) were performed and analyzed. The two assays' ten-fold
 series of the 2x dilution (e.g., 1/100 to 1/102400) began with the genomic
 DNA diluted to 200 pg (1/100 dilution) and ended with a final
 concentration of 0.195 pg. Six replicates of each dilution and negative
 control were included in both series, and all dilutions were performed
 with fresh seawater from the aquarium experiment. ## Description of the
 Data and file structure The two spreadsheets contain raw data resulting
 from the aquarium experiment and the ddPCR reaction assessment. ### Data
 overview: The two spreadsheets contain raw data resulting from the
 aquarium experiment and the ddPCR reaction assessment. To open files it
 requires the use of software that opens xlsx such as microsoft office
 excel. ##### aquarium\_ddPCR raw\_data: * "Sample\_ID" Unique
 identifier for each sample. * "Tank" The tank from which the
 sample was collected. * "Hours" The time-point at which the
 sample was collected. * "Organism\_Present" Indicates whether
 the marine NIS was present ("Yes") or absent ("No") in
 the tank at the time of sample collection. * "Biomass"
 Categorical biomass category of the targeted species. Categories include
 high, medium, and low, based on the relative weight within each species. *
 "Weight" Combined weight, in grams, of the target species in the
 tank, measured after the removal of organisms. *
 "Target\_Species" The specific marine NIS organism targeted
 during the species-specific ddPCR assays. * "Concentration"
 Concentration of the Cytochrome c oxidase subunit 1 (COI) gene, reported
 by ddPCR, in copies per microliter (copies/L). ##### ddPCR\_assessment
 raw\_data: * "Dilution" Dilution factor used to dilute the
 standard curve, record as 1/X, where x is the dilution *
 "Loaded\_DNA\_pg" Amount of DNA loaded as a standard, recorded
 in picograms (pg). * "Target" The specific marine NIS organism
 targeted during the species-specific ddPCR assays. *
 "Reported\_Conc" Concentration of the Cytochrome c oxidase
 subunit 1 (COI) gene, reported by ddPCR, in copies per microliter
 (copies/L). * Note that the "Blank" variable under dilutions
 represents the negative control for the standard curve, where no standard
 (genomic DNA) was loaded. ## Funding This study was supported by New
 Zealand Ministry of Business, Innovation and Employment funding (CAWX1904
 A toolbox to underpin and enable tomorrows marine biosecurity system). ##
 Sharing/access Information All raw data asscoiated with this experiment
 and analysis is contained within the excel files attached. No custom
 computer code or algorithm was used to generate results. The software
 environment R was used for all data exploration, statistical analyses and
 model development described in this manuscript using freely available R
 packages.