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.