14Environmental DNA (eDNA) analysis has gained traction as a precise and cost effective method 15 for species and waterways management. To date, publications on eDNA protocol optimization have 16 focused primarily on DNA yield. Therefore, it has not been possible to evaluate the cost and speed of 17 specific components of the eDNA protocol, such as water filtration and DNA extraction method when 18 designing or choosing an eDNA pipeline. At the same time, these two parameters are essential for the 19 experimental design of a project. Here we evaluate and rank different eDNA protocols in the context of 20 Chinook salmon (Oncorhynchus tshawytscha) eDNA detection in an aquatic environment, the San 21 Francisco Estuary. We present a comprehensive evaluation of multiple eDNA protocol parameters, 22 balancing time, cost and DNA yield. For estuarine waters, which are challenging for eDNA studies due to 2 23 high turbidity, variable salinity, and the presence of PCR inhibitors, we find that a protocol combining glass 24 filters and magnetic beads, along with an extra step for PCR inhibitor removal, is the method that best 25 balances time, cost, and yield. In addition, we provide a generalized decision tree for determining the 26 optimal eDNA protocol for other studies on aquatic systems. Our findings should be applicable to most 27 aquatic environments and provide a clear guide for determining which eDNA pipeline should be used for 28 a given environmental condition.29 30 Author Summary
31The use of environmental DNA (eDNA) analysis for monitoring wildlife has steadily grown in recent 32 years. Though, due to differences in the ecology of the environment studied and the novelty of the 33 technique, eDNA currently shows a lack of standards compared to other fields. Here we take a deep look 34 into each step of an eDNA assay, looking at common protocols and comparing their efficiencies in terms 35 of time to process the samples, cost and how much DNA is recovered. We then analyze the data to provide 36 a concise interpretation of best practices given different project constraints. For the conditions of the San 37 Francisco Estuary we suggest the use of glass fiber filtration, the use of paramagnetic beads for DNA 38 extraction and the use of a secondary inhibitor removal. We expect our findings to provide better support 39 for managers to decide their standards ahead of project submission not only for estuarine conditions but 40 for other waterine conditions alike. 41 42 Introduction 43 Environmental management policies rely heavily on measurements of the spatial distribution of 44 habitat occupancy of species. In the past decade, environmental DNA (eDNA) has gained traction as one 45 of the most sensitive and cost effective monitoring methods [1], allowing researchers to better estimate 46 species occupancy rates in a given habitat. Due to high variability in the studied environments, currently, 3 47 there are no clear guidelines to assist investigators in choosing an optimal protocol for their particular 48 eDNA monitor...