In-field genetic stock identification of overwintering coho salmon in the Gulf of Alaska: Evaluation of Nanopore sequencing for remote real-time deployment

Genetic stock identification (GSI) by single nucleotide polymorphism (SNP) sequencing has become the gold standard for stock identification in Pacific salmon, which are found in mixed-stocks during the oceanic phase of their lifecycle. Sequencing platforms currently applied require large batch sizes and multi-day processing in specialized facilities to perform genotyping by the thousands. However, recent advances in third-generation single-molecule sequencing platforms, like the Oxford Nanopore minION, provide base calling on portable, pocket-sized sequencers and hold promise for the application of real-time, in-field stock identification on variable batch sizes. Here we report and evaluate utility and comparability of at-sea stock identification of coho salmon Oncorhynchus kisutch based on targeted SNP amplicon sequencing on the minION platform during the International Year of the Salmon Signature Expedition to the Gulf of Alaska in the winter of 2019. Long read sequencers are not optimized for short amplicons, therefore we concatenate amplicons to increase coverage and throughput. Nanopore sequencing at-sea yielded stock assignment for 50 of the 80 assessed individuals. Nanopore-based SNP calls agreed with Ion Torrent based genotypes in 83.25%, but assignment of individuals to stock of origin only agreed in 61.5% of individuals highlighting inherent challenges of Nanopore sequencing, such as resolution of homopolymer tracts and indels. However, poor representation of assayed coho salmon in the queried baseline dataset contributed to poor assignment confidence on both platforms. Future improvements will focus on lowering turnaround time, accuracy, throughput, and cost, as well as augmentation of the existing baselines, specifically in stocks from coastal northern BC and Alaska. If successfully implemented, Nanopore sequencing will provide an alternative method to the large-scale laboratory approach. Genotyping by amplicon sequencing in the hands of diverse stakeholders could inform management decisions over a broad expanse of the coast by allowing the analysis of small batches in remote areas in near real-time.

Large-scale parentage-based tagging and genetic stock identification applied in assessing mixed-stock fisheries and hatchery brood stocks for coho salmon in British Columbia, Canada

Direct DNA sequencing is powering a revolution in the application of genetics to resource management, with parentage-based tagging (PBT) increasingly applied to salmon fisheries and hatchery brood stock management and assessment. Genetic stock identification (GSI) and PBT were applied to assessment of 2018 coho salmon (Oncorhynchus kisutch) ocean fisheries and hatchery brood stocks in British Columbia (BC), Canada, with 6391 individuals successfully genotyped in fishery samples and 7805 individuals genotyped in 40 hatchery brood stocks. Population-specific contributions to mixed-stock fisheries and exploitation rates were estimated with coded-wire tags (CWTs) and GSI–PBT technologies for six populations. PBT assignments, verified by CWTs, were 100% accurate for 308 individuals with respect to population of origin and age. There was generally reasonably close agreement of estimated population-specific exploitation rates between CWT and genetic methods. We conclude that a genetic approach can improve upon the results available from the current CWT program for assessment and management of coho salmon fisheries and hatchery brood stocks in BC and provide information critical to aid in implementation of Canada’s Policy for Conservation of Wild Pacific Salmon.

A GT-seq panel for walleye (Sander vitreus) provides a generalized workflow for efficient development and implementation of amplicon panels in non-model organisms

Targeted amplicon sequencing methods, such as genotyping-in-thousands by sequencing (GT-seq), facilitate rapid, accurate, and cost-effective analysis of hundreds of genetic loci in thousands of individuals, but studies describing detailed workflows of GTseq panel development are rare. Here, we develop a dual-purpose GT-seq panel for walleye (Sander vitreus) and discuss trade-offs associated with different development and genotyping approaches. Our GT-seq panel was developed using restriction site-associated DNA data from 954 individuals sampled from 23 populations in Minnesota and Wisconsin, USA. We then conducted simulations to test the utility of loci for parentage analysis and genetic stock identification and designed 600 primer pairs to maximize joint accuracy for these analyses. We conducted three rounds of primer optimization to remove loci that overamplified and our final panel consisted of 436 loci. Optimization focused on reducing variation in amplification rate among loci and minimizing the proportion of off-target sequence, both of which are important considerations for developing large GT-seq panels. We also explored different approaches for DNA extraction, multiplexed polymerase chain reaction (PCR) amplification, and cleanup steps during the GT-seq process and discovered the following: (1) inexpensive Chelex extractions performed well for genotyping, (2) the exonuclease I and shrimp alkaline phosphatase (ExoSAP) procedure included in some current protocols did not improve results substantially and was likely unnecessary, and (3) it was possible to PCR amplify panels separately and combine them prior to adapter ligation. Well-optimized GT-seq panels are valuable resources for conservation genetics and our findings should aid in their construction in myriad taxa.