Enumeration Potential of Environmental DNA for Pacific Salmon Stock Assessments using qPCR based methodologies converting Cq into copy number using linear regression and the eLowQuant method

The field of environmental DNA has advanced over the past decade, with multiple approaches available for a variety of sampling media and species. Here, we quantify salmon environmental DNA upstream of a fish counting fence along with river velocity, and together, use these values to determine the correlation between the number of salmon passing by the fish fence daily with daily eDNA rates in water before, during and after the salmon spawning season for 4 Pacific salmonids (Oncorhynchus gorbuscha, O. kisutch, O. tshawytscha, O. nerka; pink, coho, Chinook, sockeye respectively).
Hydrogeological data of streamflow and water temperature were measured in 10 minutes intervals by automatic barologgers and levelloggers and averaged over a 24-hour period between 9pm the previous day and 9pm to get daily temperature and streamflow rate.
Triplicate daily water samples were 50 m upstream of the fish counting fence in bleach sterilised glass bottles and filtered on 0.45 micron cellulose filters before storing in envelopes placed in desiccant. DNA is then extracted from these filters with stringent contamination protocols using DNeasy Blood & Tissue kits. qPCR is performed using primers and probes for each of the 4 salmonid species and the Cq values generated are converted to copies/L (S ̂, sHat) using either linear regression using parameters derived from n=24 replicate tests or by publicly available eLowQuant method (https://github.com/mlespera/eLowQuant) depending on the number of non-zero reads out of each 8 technical replicate test. No amplification producing no Cq value is an absence of target DNA.
The copies/L readings for each daily triplicate water sample are then averaged and multiplied by the daily stream flow rate to get flow adjusted eDNA rate. This can then by log transformed by doing log_10⁡〖(S ̂ 〗+1) as to not lose readings of 0 copies/L.

环境DNA领域在过去十年中取得了显著进展，为各种采样介质和物种提供了多种方法。本研究中，我们量化了鱼计数栅栏上游的鲑鱼环境DNA，并测量了河流流速，将这两个值结合使用，以确定每日通过鱼栅栏的鲑鱼数量与鲑鱼产卵季节前后每日水中的eDNA频率之间的相关性，研究对象包括四种太平洋鲑鱼（大马哈鱼Oncorhynchus gorbuscha、红大马哈鱼O. kisutch、大鳞鲑O. tshawytscha、红点鲑O. nerka，分别称为粉红鲑、狗鱼、王鲑、红鲑）。通过自动气压计和水位计以10分钟为间隔测量水文地质数据，并在前一天晚上9点至当天晚上9点之间对24小时的平均值进行计算，以获取每日温度和流量速率。在鱼计数栅栏上游50米处，使用漂白消毒的玻璃瓶采集三份每日水样，并在0.45微米的纤维素滤膜上进行过滤，之后存放在装有干燥剂的信封中。然后，使用DNeasy Blood & Tissue试剂盒，按照严格的污染控制程序从这些滤膜中提取DNA。使用针对每种鲑鱼物种的引物和探针进行qPCR，并将生成的Cq值转换为每升的拷贝数（Ŝ，sHat），具体方法为根据n=24的重复测试参数进行的线性回归，或者根据每个8个技术重复测试中非零读数的数量使用公开可用的eLowQuant方法（https://github.com/mlespera/eLowQuant）。没有扩增且没有Cq值的读数表示目标DNA不存在。然后，将每个每日三份水样的拷贝/L读数平均，并乘以每日流量速率，以获得流量调整后的eDNA频率。为了防止丢失0拷贝/L的读数，可以通过log_10(Ŝ+1)进行对数转换。