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Fish mock community with 41 species from 13 orders

Citation

Hleap, Jose Sergio et al. (2020), Fish mock community with 41 species from 13 orders , Dryad, Dataset, https://doi.org/10.5061/dryad.05qfttf0n

Abstract

Tissue extracts of 41 North American fish species were obtained from the Ministère des Forêts, de la Faune et des Parcs (Québec). The selected species were chosen to represent a diversity of families within the Actinopterygii, and to include some species known to be present at the Experimental Lakes Area. Muscle or fin tissue was extracted using Qiagen Blood and Tissue kits and equimolarised to 15ng/µl. Library preparation and next-generation sequencing (NGS) was performed by equimolarising the DNA and combining two replicate mock community libraries which were then PCR amplified and sequenced. Sequencing was conducted using 2x300bp Illumina MiSeq at the McGill University and Génome Québec Innovation Centre, Montréal.
 

Methods

Tissue extracts of 41 North American fish species were obtained from the Ministère des Forêts, de la Faune et des Parcs (Québec). The selected species were chosen to represent a diversity of families within the Actinopterygii. Tissues were stored in ethanol at -20C before extraction. Muscle or fin tissue was extracted using Qiagen Blood and Tissue kits according to the manufacturer’s instructions and equimolarised to 15ng/µl using the Quant-iT Picogreen dsDNA assay kit.

Library preparation and next-generation sequencing (NGS) 
Equimolar amounts of DNA were combined to make two replicate mock communities. The mixture of mock community DNA was amplified in triplicate with the Leray primer with the following PCR chemistry: 7.875µl nuclease free water, 1.25µl 10X buffer (Genscript), 1mM MgCl2 (ThermoFisher Scientific), 0.1mM GeneDirex dNTPs, 0.0125mg bovine serum albumen (ThermoFisher Scientific), 0.2mM each 10mM primer, 1.25U taq (GenScript) and 2µl DNA in a final volume of 12.5µl. The thermocycling regime followed that of the original paper. Amplicons were run on 1% agarose gel stained with SYBR™ Safe DNA Gel Stain (Thermo-Fisher Scientific) and visualized with UV light. Triplicate PCR amplicons for each sample were combined, cleaned with AMPure beads in a 0.75 ratio and indexed with the Nextera DNA indexing kit for 96 samples (Illumina). A second clean-up with AMPure beads was performed, and libraries were quantified and normalised to 5ng/ul. Sequencing was conducted using 2x300bp Illumina MiSeq at the McGill University and Génome Québec Innovation Centre, Montréal.

Leray, M., Yang, J.Y., Meyer, C.P. et al. A new versatile primer set targeting a short fragment of the mitochondrial COI region for metabarcoding metazoan diversity: application for characterizing coral reef fish gut contents. Front Zool 10, 34 (2013). https://doi.org/10.1186/1742-9994-10-34

Funding

Food from Thought: Agricultural Systems for a Healthy Planet Initiative

Canada First Research Excellence Fund

Food from Thought: Agricultural Systems for a Healthy Planet Initiative