A shared microbiome between wild and captive fathead minnows.

GeoNetwork record:www.gwfnet.net/geonetwork/srv/eng/catalog.search#/metadata/caa25857-949b-43d5-8465-268302e609e9
Tracking ID under eDNA project: UofS-eDNA-dataset-metadata-2

Intestinal samples were collected from fathead minnows. Fish collected from the field were collected in minnow traps set overnight. All samples were collected with sterile dissection tools, and samples were immediately placed on ice prior to being transferred to a -80C freezer for long-term storage.

16s amplicon sequencing
Total genomic DNA was extracted from guts using the DNeasy PowerSoil Kit (Qiagen Inc., Mississauga, ON). Concentrations were measured using a Qubit 4 Fluorometer and dsDNA HS assay kit (ThermoFisher Scientific, Waltham, MA). The V3-V4 variable region of the 16S rRNA gene was amplified using the Bact-0341 forward primer (CCTACGGGNGGCWGCAG) (Klindworth et al., 2013) and the Bact-806 reverse primer (GGACTACNVGGGTWTCTAAT) (Apprill et al., 2015). Samples were dual indexed to increase throughput of sequencing (Fadrosh et al., 2014). Samples were amplified with a 50 ?L PCR reaction including Phusion green polymerase (ThermoFisher Scientific) using a SimpliAmp thermal cycler (ThermoFisher Scientific) under the following conditions: initial denaturation at 98?C for 30s, followed by 25 cycles of 98?C for 30s, 58?C for 30s, and 72?C for 30s, with a final extension at 72?C for 10 min. PCR products were assessed for size and specificity using electrophoresis on a 1.2% w/v agarose gel and purified using the Qiagen QIAquick PCR Purification Kit (Qiagen Inc.). All purified products were quantified with the Qubit dsDNA HS assay kit and concentrations were adjusted to 1 ng/ ?L with molecular-grade water. Purified products were pooled, and libraries were constructed using the NEBNext? DNA Library Prep Master Mix Set for Illumina? (New England BioLabs Inc., Whitby, ON). Libraries were quantified prior to sequencing using the NEBNext? Library Quant Kit for Illumina?. Sequencing was performed on an Illumina? MiSeq instrument (Illumina, San Diego, CA) using a 2x300 base pair kit.

The gut microbiota of animals has been described as an additional host ?organ' with beneficial roles. However, little is known about the potential for a core microbiome, particularly in fish. Obtaining a baseline for a core microbiome allows us to determine how the microbiome shifts following exposure to a contaminant. This study seeks to determine whether or not a core microbiome exists within fathead minnows (Pimephales promelas) from both field- and lab-raised fish.

This study sought to determine if fathead minnows have a core community of gut bacteria. In order to fully understand how a chemical alters an ecological community, it is helpful to first understand the baseline. The gut microbiome is a relatively new area of research in toxicology. It is believed that several toxicant can impair the gut bacteria and result in decreased health of an organism, therefore gathering baseline data is crucial. To answer some of this question, we collected fish from our aquatics facility and from a nearby lake and examined the community structure. This study will tell us if a shared community exists or not.

University of Saskatchewan Aquatic Toxicology Research Facility (ATRF)
https://toxicology.usask.ca/atrf/facilities-and-equipment.php

Duck Lake, Saskatchewan

Tracking ID under eDNA project: UofS-eDNA-dataset-metadata-2

Sample Collection
Intestinal samples were collected from fathead minnows. Fish collected from the field were collected in minnow traps set overnight. All samples were collected with sterile dissection tools, and samples were immediately placed on ice prior to being transferred to a -80C freezer for long-term storage.
16s amplicon sequencing
Total genomic DNA was extracted from guts using the DNeasy PowerSoil Kit (Qiagen Inc., Mississauga, ON). Concentrations were measured using a Qubit 4 Fluorometer and dsDNA HS assay kit (ThermoFisher Scientific, Waltham, MA). The V3-V4 variable region of the 16S rRNA gene was amplified using the Bact-0341 forward primer (CCTACGGGNGGCWGCAG) (Klindworth et al., 2013) and the Bact-806 reverse primer (GGACTACNVGGGTWTCTAAT) (Apprill et al., 2015). Samples were dual indexed to increase throughput of sequencing (Fadrosh et al., 2014). Samples were amplified with a 50 ?L PCR reaction including Phusion green polymerase (ThermoFisher Scientific) using a SimpliAmp thermal cycler (ThermoFisher Scientific) under the following conditions: initial denaturation at 98?C for 30s, followed by 25 cycles of 98?C for 30s, 58?C for 30s, and 72?C for 30s, with a final extension at 72?C for 10 min. PCR products were assessed for size and specificity using electrophoresis on a 1.2% w/v agarose gel and purified using the Qiagen QIAquick PCR Purification Kit (Qiagen Inc.). All purified products were quantified with the Qubit dsDNA HS assay kit and concentrations were adjusted to 1 ng/ ?L with molecular-grade water. Purified products were pooled, and libraries were constructed using the NEBNext? DNA Library Prep Master Mix Set for Illumina? (New England BioLabs Inc., Whitby, ON). Libraries were quantified prior to sequencing using the NEBNext? Library Quant Kit for Illumina?. Sequencing was performed on an Illumina? MiSeq instrument (Illumina, San Diego, CA) using a 2x300 base pair kit.

Data processing
Sequences were trimmed, cleaned, and demultiplexed using a combination of Trimmomatic (Bolger et al., 2014), USEARCH v11 (Edgar 2010), and QIIME1 (Caporaso et al., 2010). Paired-end sequences were merged with DADA2 (Callahan et al., 2016) in QIIME2 (Bolyen et al., 2019) after truncating the forward read to 280 nucleotides and the reverse read to 230 nucleotides in order to ensure maximum quality and percentage of reads retained. The DADA2 package generates sequence variants (SVs) that are used to infer different bacterial species. Chimeric sequences were subsequently removed, and SVs were compared to the Silva rRNA database release 132 for taxonomic identification in QIIME2. Samples were rarefied to a sequencing depth of 10,533 reads prior to downstream analyses. Statistical analyses were performed in PRIMER-e v7 (Auckland, NZ) and R (R Core Team, 2013).

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