RFI_USGS_SOW_eDNA_Metabarcoding_Analysis_1.pdf

Statement of Work

The USGS Western Fisheries Research Center (WFRC) is conducting studies on salmon predators and non-native fish in the Yakima River in central Washington. As part of this research, WFRC staff have collected 80 environmental DNA (eDNA) samples using two sampling methods, pump sampling and passive filter sampling. This Statement of Work outlines the laboratory services that a contractor will provide for metabarcoding analysis of these eDNA samples, with the ultimate goal of comparing each of the two collection methods against results obtained with electrofishing of the same locations.

1. DNA Extraction The contractor will perform DNA extraction on 80 MCE filters provided by USGS WFRC. Filters will be labeled with sampling dates, method (pumped or passive), and study site location codes. Samples will be shipped overnight on blue ice during the work week to ensure staff availability for receipt.

2. PCR Amplification and Library Preparation Each extracted sample will undergo Polymerase Chain Reaction (PCR) using universal fish primers equivalent to what is described in Miya et al. (2015;

https://doi.org/10.1098/rsos.150088) multiplexed with a custom lamprey forward and reverse primer designed to enhance detection of Petromyzontidae. This step amplifies eDNA fragments from target taxa (i.e., teleost fishes and lamprey species).

A second PCR will be used to add sequencing adapters and index sequences to generate metabarcoding libraries.

3. Next-Generation Sequencing Prepared libraries will be sequenced using Next Generation Sequencing (NGS) platforms, as determined by the contractor. The assay will need to specify species that inhabit the Yakima River basin, and include species or subspecies identification of lamprey species of the general Lampetra and Entosphenus, as determined by the workflow of the contractor.

4. Bioinformatics Processing and Taxonomic Assignment Raw NGS data will be processed using bioinformatics pipelines developed by the contractor research laboratory. Sequences will be compared against taxonomic reference databases using standard methods. A sequence will be identified to species when ≥97% of its nucleotides match a reference sequence. Sequences with <97% similarity will be assigned to the genus or family level as appropriate.

5. Reporting The contractor will provide a written report(PDF form) summarizing methods, results, and primer references approximately 4–5 weeks after sample receipt. The data will be provided in a form suitable for data release by USGS upon publication of research results (excel spreadsheet).

https://doi.org/10.1098/rsos.150088