Individual haplotyping of whale sharks from seawater environmental DNA

Water samples were collected over two weeks directly behind individual whale sharks at Ningaloo Reef, Australia in May, 2019. A total of 56 seawater samples were collected, filtered immediately through sterile 0.22 μm filters, and stored at -80°C. Tissue biopsy samples were then taken from the same animal using a hand spear with a dart head, totalling 77 individual samples.

Individual whale sharks were identified by their unique spot and stipe patters through photographs analysed by I³S Classic Software. Re-sightings of the same individual were removed from the dataset. Following photo-identification, a total of 48 unique individuals were confirmed, of which 42 consisted of both tissue and seawater samples. Additional samples were later discarded from the study, resulting in libraries for 28 seawater samples, and whale shark d-loop haplotypes.

DNA extractions of the tissue samples were carried out and PCR amplification of the mitochondrial control region and reaction protocol verified. Seawater samples were processed and environmental DNA was extracted. Statistical analysis was performed with R Studio.

2019年5月，研究团队于澳大利亚宁格鲁礁（Ningaloo Reef），在两周内直接采集各头鲸鲨正后方的海水样本。本次实验共收集56份海水样本，经无菌0.22 μm滤膜即刻过滤后，于-80℃条件下低温保存。随后使用带有镖头的手持鱼叉，从对应鲸鲨个体身上采集组织活检样本，总计77份独立样本。

通过I³S Classic Software分析拍摄照片，依据鲸鲨独特的斑点与条纹图案对个体鲸鲨进行识别，并将同一个体的重识别数据从数据集中剔除。经照片识别流程后，共确认48头独特鲸鲨个体，其中42头同时具备组织样本与海水样本。后续研究中又舍弃了部分额外样本，最终得到28份海水样本的测序文库，以及鲸鲨d-loop单倍型数据。

对组织样本完成DNA提取，对线粒体控制区进行PCR扩增并验证反应流程。对海水样本进行处理并提取环境DNA。所有统计分析工作均依托R Studio开展。