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Abstract:
Eggs were sampled at the haddock spawning ground survey for details se Johannesen et al (2022). The eggs were sorted on board using a stereomicroscope, and eggs with diameter of 1.2-1.6 mm were assumed to be gadoid eggs. The eggs were classified into egg development stages based on Thompson and Riley (1981). The samples were fixed in 98% alcohol after standard processing on board. All collected eggs classified as gadoid eggs, were subjected to genetic analysis at the laboratory at the Institute of Marine Research's station in Flødevigen. Eggs were set up for DNA extraction in a 96-well plate format using the Hamilton Star robot. Each plate had two negative controls randomized to ensure plate identity and orientation for further analyses. The chemistry and extraction method were provided by Omega Biotek and based on Mag-Bind® Blood and Tissue DNA HDQ Mag-Bind technology isolates DNA from enzymatically degraded cells in solution by binding small magnetic beads to DNA. This is further purified and eventually separated from the beads, resulting in a DNA extract. The concentration of DNA extracts was then measured using a fluorometric method on a ThermoFisher Fluoroskan Microplate Fluorometer with Quan IT kit dsDNA High Sensitivity (0.2-50ng). The samples were normalized to a concentration of <= 5 ng/µl for further preparation of DNA libraries for sequencing. The target sequence is a universal mini barcode 295 bp sequence of the mitochondrial COI (cytochrome c oxidase subunit 1) developed for species identification of fish by Sultana et al (2018). Library preparation involves a two-step process with two rounds of PCR. In PCR1, the COI fragment is amplified, and in PCR2, each individual sample is tagged with a barcode (i5) while also receiving an index code (i7) representing the plate. This allows the identification of each sample after sequencing. Library preparation uses custom barcodes and indexes, not part of a commercial kit but developed based on a protocol described in Campbell et al (2015). The library underwent quality control by running it on an Agilent Bioanalyzer, providing a profile of the library through gel electrophoresis, and measuring concentrations on a Qubit fluorometer. Sequencing of the library was performed on an Illumina MiSeq using "paired end sequencing" with a MiSeq Reagent Kit v3 (600-cycle), including 5% PhiX as a control. Raw sequencing data in fastq format were processed in the R software (R Core Team 2023) and using the DADA2 module (Callahan et al. 2016). Quality-filtered sequences were then compared with a database consisting of COI sequences for gadoid fish using the R module rBLAST (Hahsler 2019). Successful species identification required a 100% match in the database for a single species.
For further details on the same methods applied to eggs sampled at the cod spawning survey in 2022, see Fuglebakk et al (in prep).