DOI: 10.5061/dryad.83bk3jb1s
title: An environmental microbiome study of Loch Ryan: Taxonomic and abundance data

description: The microbiome plays a key role in animal health and is an important
 aspect of any natural or farmed ecosystem. Here we present the first
 environmental microbiome study of Ostrea edulis, as well as the first of a
 natural wild spawning event of any oyster species. Larval abundance was
 hypothesised to be correlated with specific microbial signatures. Water
 samples were collected throughout a natural spawning event of O. edulis at
 Loch Ryan, Scotland, UK. Samples were collected on 4 different dates from
 June to September 2019, across 8 different sampling sites on the Loch at
 mid, bottom, and surface levels within the water column to remove the
 effects of salinity and tidal fluctuations. Larval count data was obtained
 from these samples before full-length sequencing of the 16S rRNA gene
 using Oxford Nanopore Technologies. Significant microbial differences were
 only found between samples collected on different dates, and not at
 different sites or water column depths. Differences in the microbiome
 throughout the spawning season were driven by changes in the abundance of
 certain taxa, most notably those belonging to
 the Rhodobacteraceae family. Inverse abundance profiles of
 Rhodobacteraceae and Vibrio over time are also discussed. This study
 provides important microbial baseline data about the spawning environment
 of O. edulis.
description: <em>Sample collection: </em>
 Weekly water samples were collected from Loch Ryan between the
 7<sup>th</sup> June to 24<sup>th</sup> September
 2019 as described by Chapman et al, (2021). This sampling time frame was
 chosen to cover the native oyster spawning season as comprehensively as
 possible. Water samples were collected across 8 different sites in the
 Loch, covering 4 sites from known oyster fishing grounds off Lefnoll
 Point, and 4 sites from non-oyster bed habitats (see Figure 1a, Chapman et
 al, 2021). The location of all sample sites visited on Loch Ryan in
 relation to the oyster beds can be seen in Figure 1. Water was collected
 from the surface, mid and bottom points within the water column using the
 pump and trap method (see Fig.2. Chapman et al, 2021). Each sample was
 filtered through a 90µm mesh to capture plankton (but retain oyster
 larvae) within the water with 200 L of filtrate being measured from each
 sample. All samples were then used to conduct larval counts of both
 <em>O. edulis</em> and other bivalves such as cockles,
 mussels, and saddle oysters (Chapman et al, 2021). Water samples were then
 frozen in a -20°C freezer. To aid in the downstream
 analysis, count data of total bivalves and <em>O. edulis</em>
 larvae were used to assign each individual water sample a “low”, “medium”
 or “high” count category for these two factors. This was done by dividing
 the highest count number recorded for bivalves and <em>O.
 edulis</em> by 3 in order to set threshold values for each category.
 The majority of count data for both bivalves and <em>O.
 edulis</em> were categorised at low, with just 4 and 2 samples
 belonging in the high categories for bivalve and <em>O.
 edulis</em> counts respectively. A range of
 physicochemical parameters were recorded at the time of sample collection.
 Loggers measured water temperature, conductivity, and dissolved oxygen.
 The recording of daily sea surface temperature allowed for the calculation
 of ‘temperature sum’ as defined in Chapman et al, 2021. The tidal range
 and lunar cycle were also recorded for each of the sampling
 dates. <em>Molecular biology:
 </em> After the initial filtration and freezing,
 thawed water samples were thoroughly mixed by vortexing for 20 seconds. 15
 ml of each was added to a 50 ml falcon tube containing 33 ml of absolute
 ethanol and 1.5 ml of 3 M sodium acetate. After 1 hour incubation at
 -80°C, tubes were centrifuged at 4°C, 4000 rpm for 30 minutes to pellet
 any cellular material and free DNA. Supernatant was poured off and ethanol
 was allowed to evaporate for 15 mins before 2 ml Liftons buffer with 100
 µg/ml Proteinase K was added. This solution was lysed at 55°C for 1 hour,
 then spun down. 400 µl of supernatant was mixed with 800 µl of Qiagen AL
 buffer with ethanol, loaded onto a Qiagen spin column, and cleaned by
 following the remainder of the Qiagen blood and tissue kit tissue protocol
 (Qiagen, Hilden, Germany). In a 96-well plate setup, each DNA sample was
 then quantified using the QuantiFluor dsDNA system from Promega (Promega,
 Madison, US) before using as template for amplification of full-length 16S
 rRNA gene by PCR. This PCR was optimised by using universal 16S primers
 adapted from Callahan et al, (2019) to also detect bacteria of
 the<em> Vibrio</em> genus: F= AGRGTTYGATYMTGGCTCAG R=
 VGYTACCTTGTTACGACTT with Phusion High-Fidelity DNA polymerase from APExBIO
 (ApexBIO, Texas, US). Three positive controls were included by the use of
 <em>Streptococcus</em> bacterial DNA, alongside four negative
 controls of nuclease free water. The PCR reaction was run with an initial
 denaturation step at 98°C for 30 seconds, followed by 34 cycles of 98°C
 for 10 seconds, 65°C for 30 seconds, 72°C for 50 seconds in a PTC-225
 Peltier MJ Research Thermocycler (Marshall Scientific, Hampton, US). The
 subsequent PCR product was then visualised via gel electrophoresis to
 check for DNA fragments of 1466 base pairs before purifying with the High
 Prep PCR clean-up system (MagBio, Maryland, US). Each DNA sample was then
 re-quantified using Qubit dsDNA BR Assay kit (Invitrogen, Massachusetts,
 US) and standardised to ~200 fmol through dilution with nuclease-free
 water. The SQK-LSK110 sequencing ligation kit and EXP-NBD196 Native
 Barcode Expansion kit (Oxford Nanopore Technologies, Oxford, UK), were
 then used to perform library preparation with metabarcoding on each of the
 samples. The pooled library was then loaded across 4 separate ONT MinION
 R10 flow cells for 48 hours, obtaining a total of 98.681 GB of
 data. <em> </em>
 <em>Data handling and analysis: </em>
 High accuracy basecalling was performed using Guppy v.6.1.7 (ONT)
 and reads were quality controlled by inspecting the average quality and
 length with Nanoplot version 1.4.0 (De coster et al, 2018). Trimming of
 primers from reads was then performed using Cutadapt v.3.4 (Martin, 2011),
 with a maximum error rate of 0.15 and a minimum overlap of 20 base pairs
 (bp). Reads shorter than 1,300 bp and longer than 1,600 bp were also
 discarded. Porechop v0.2.4 (Wick et al, 2017) was then used to trim reads
 of chimeras and adapter sequences. Microbial taxonomy was then identified
 using Kraken2 v2.1.1 (Wood and Salzberg, 2014), where 2,112,739 reads were
 aligned against the SILVA138 database (Wood, Lu, and Langmead,
 2019). Downstream data analysis was carried out in R
 studio v4.2.0 (RStudio Team, 2022) using the Phyloseq (McMurdie and
 Holmes, 2013) and ggplot2 (Wickham, 2014) packages. This included the
 calculation of observed and Choa1 alpha diversity measures which were then
 tested for significant differences with a Kruskal-Wallis test. Control and
 outlying samples were identified from non-metric multidimensional scaling
 (NMDS) analysis and excluded, leaving 85 samples in the results presented.
 Use of the Vegan package (Oksanen, 2012) in R Studio also allowed for a
 PERMANOVA statistical test to be performed, and weighted phylogenetic
 tress (see Figures 3 and 6), were created using the Pavian web application
 tool (Breitwieser and Salzberg, 2020).
description: # An environmental microbiome study of Loch Ryan: Taxonomic and abundance
 data
 [https://doi.org/10.5061/dryad.83bk3jb1s](https://doi.org/10.5061/dryad.83bk3jb1s) ##### Description of the data and file structure The data linked here consists of one Excel sheet, containing three sheets: **Sheet 1**: An OTU (Organised Taxonomic Unit) table, listing the abundance numbers of each OTU within each sample. OTU's are listed in rows against individual samples in columns. The heading of each column contains the barcode number used during the metabarcoding of that sample. A total of 995 OTU's were identified.  **Sheet 2**: A list identifying each OTU at different taxonomic levels. Most OTU's were identified down to a genus level. Where a genus level of identification was not possible, 'Unknown Genus' is seen under the 'Genus' heading for that OTU. A total of 995 OTU's are listed (matching the abundance data shown on sheet 1).  **Sheet 3**: Metadata giving information on each water sample including the site location, depth, and date of their collections. Water samples were collected from 8 different locations around Loch Ryan (Scotland) during a natural spawning event of *O. edulis* (the European Flat oyster). Four of these sites were located on the oyster bed and 4 off the oyster bed. Samples were also collected from the bottom, mid, and surface points within the water column. Bottom samples were collected 6 cm off the seabed, surface samples at a depth of 30cm, and mid samples from half the water depth under the keel. A map of site locations from around Loch Ryan and additional information on the sampling process can be found in Chapman et al, 2021.  Prior to 16S sequencing, water samples were used to preform counts of *O. edulis* larvae per 200L of water.  Mean count values for each sample are shown under the heading 'Meanedulislarvae*'. *Each water sample was also used to conduct counts of general bivalve larvae which included *O. edulis* as well as cockles, mussels and saddle oysters. The mean count values for each sample are shown under the heading 'Meanbivalvelarvae'. To aid in the downstream analysis, count data of total bivalves and *O. edulis* larvae were used to assign each individual water sample a “low”, “medium” or “high” count category for these two factors. This was done by dividing the highest count number recorded for bivalves and *O. edulis* by 3 in order to set threshold values for each category. The majority of count data for both bivalves and *O. edulis* were categorised as 'low'. The larval count categories for each sample are shown under headings of 'Meanbivalvecat' and 'Meaneduliscat'. Sample collection and larval counts were performed during Chapman et al, 2021.  Erica C.N. Chapman, Ana Rodriguez-Perez, Tristan Hugh-Jones, Cass Bromley, Mark A. James, Karen Diele, William G. Sanderson. Optimising recruitment in habitat creation for the native European oyster (Ostrea edulis): Implications of temporal and spatial variability in larval abundance,Marine Pollution BulletinVolume 170,2021,112579,ISSN 0025-326X, [https://doi.org/10.1016/j.marpolbul.2021.112579](https://doi.org/10.1016/j.marpolbul.2021.112579).