Table 1.
Samples analyzed in this study summarized by sample purpose (type of sample), station location (latitude, longitude), and number of samples by depth.
Fig 1.
Final workflow proposed after methods evaluation.
Water samples are collected at different depths using Niskin bottles on a CTD rosette and filtered immediately after. The filters are preserved in tubes with Longmire’s buffer and the DNA is extracted using a phase-lock/phenol-chloroform extraction. Target DNA is analyzed in a multiplex qPCR assay (details in S3 File). Illustration by Su Kim, NWFSC/NOAA Fisheries.
Table 2.
List of species-specific primers, hydrolysis probes and targeted fragment (gBlocks) of the multiplex qPCR assay.
Table 3.
List of species used for the specificity and reproducibility tests and number of positive results in the multiplex assay.
Fig 2.
Comparison of total and target DNA quantification for five different DNA extraction methods (n = 20) and for the phenol-chloroform method with and without the phase lock with a different set of samples (n = 6, three filters split in halves).
[Total DNA] was measured on the Qubit; [Merluccius productus] was measured by qPCR.
Fig 3.
Initial tests show the results of the qPCR with contaminated samples and final specificity is ascertained after sequencing the resulting amplicons. Color of the tiles shows the rate of the true (orange) or false positive or negative observation (light yellow). Numbers in the labels in the tiles are the number of samples accounted for in the observation at > 1 copy μl-1.
Fig 4.
Comparison of the performance of the standards of the triple assay in a multiplex with 1 and 3 gBlocks per species.
The performance for Merluccius productus in a singleplex is also represented.
Fig 5.
Amplification plot in logarithmic view.
A. Comparison of the amplification of the Pacific hake gBlocks standards in a multiplex with the other two species combined and individually. B. Comparison of the amplification of the IPC for the non-template controls and the gBlocks standards combined at different concentrations (in copies μl-1) and some eDNA samples.