Gadoid eggs from haddock spawning ground survey 2022 identified to species by DNA analysis

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).本数据集的鱼卵样本采集自黑线鳕（haddock）产卵场调查工作，具体细节参见Johannesen等人（2022）的研究。样本在调查船上通过实体显微镜（stereomicroscope）进行分拣，将直径介于1.2 mm至1.6 mm之间的鱼卵判定为鳕形目鱼卵（gadoid eggs）。依据Thompson与Riley（1981）的分类标准，将鱼卵划分为不同发育阶段。样本在船上完成标准化处理后，使用98%酒精进行固定保存。所有被归类为鳕形目鱼卵的样本，均被送至挪威海洋研究所弗勒迪维根（Flødevigen）站实验室开展遗传学分析。采用汉密尔顿Star机器人（Hamilton Star robot），以96孔板形式完成DNA提取前的样本制备。每块96孔板设置2个阴性对照，以随机化方式排布，用于后续分析的板标识与方向确认。本次实验所用的化学试剂与提取方法由Omega Biotek提供，基于Mag-Bind® Blood and Tissue DNA HDQ磁珠技术：通过将微小磁珠与溶液中经酶解处理的细胞DNA结合，实现DNA分离，后续经进一步纯化后与磁珠分离，最终得到DNA提取物。采用荧光定量法对DNA提取物的浓度进行测定：使用ThermoFisher Fluoroskan微孔板荧光光度计，搭配Quan IT dsDNA高灵敏度试剂盒（检测范围0.2-50 ng）。将样本浓度归一化至≤5 ng/µl，用于后续DNA文库制备以开展测序。目标序列为线粒体COI（细胞色素c氧化酶亚基1）的295 bp通用迷你条形码序列，由Sultana等人（2018）开发，用于鱼类物种鉴定。文库制备采用两轮PCR的两步法流程：第一轮PCR（PCR1）扩增COI基因片段；第二轮PCR（PCR2）为每个样本添加特异性条形码（i5），同时加入代表板孔信息的索引编码（i7），以此实现测序后对单一样本的识别。本次文库制备使用定制条形码与索引编码，并非源自商业试剂盒，而是基于Campbell等人（2015）发表的实验方案开发。文库质检通过Agilent Bioanalyzer完成：通过凝胶电泳获取文库片段分布图谱，并使用Qubit荧光光度计测定文库浓度。测序工作在Illumina MiSeq平台上开展，采用MiSeq Reagent Kit v3（600循环）的双端测序（paired end sequencing）模式，同时添加5%的PhiX作为测序对照。原始测序数据以fastq格式存储，通过R软件（R Core Team 2023）搭配DADA2模块（Callahan等人2016）进行处理。将经过质量过滤的序列与鳕形目鱼类COI序列数据库进行比对，比对工具为R模块rBLAST（Hahsler 2019）。物种鉴定成功的判定标准为：与数据库中单一物种实现100%序列匹配。关于2022年鳕鱼产卵场调查采样鱼卵所采用的相同实验方法的详细信息，参见Fuglebakk等人（待刊）的研究。