April 2015

Restoring and Managing Historical Columbia River
Basin Fish Populations with Genomics
Novel GT-Seq method using the HiSeq® System reduces genotyping costs and enables analysisof tens of thousands of fish.
Introduction
Hydropower dam construction and other environmental incursions
have suppressed and sometimes decimated fish populations aroundthe world. In the Columbia River Basin, an area approximately thesize of France covering portions of Washington, Oregon, Idaho, andBritish Columbia, the survival of some species was imperiled, including
populations of coho, sockeye, and Chinook salmon. The catastrophic
loss of fish in this region represented a marked infringement of therights of Native American tribes, which include the guaranteed right toharvest fish in all “usual and accustomed” areas from treaties signedwith the US government in 1855.
Shawn Narum, PhD, is part of a team of geneticists and otherresearchers that are helping Native American tribes revitalize fishpopulations. They’re providing the technical information needed byfisheries, hatcheries, hydropower operators, and Native Americans
to restore Columbia River Basin salmon and other fish populations
to their historical population levels.
iCommunity recently spoke with Dr. Narum about his work at theUniversity of Idaho’s Hagerman Fish Culture Experiment Station
for the Columbia River Inter-Tribal Fish Commission (CRITFC). His
research sparked the development of Genotyping-in-Thousandsby sequencing (GT-Seq), a new next-generation sequencing (NGS)
method for cost-effectively genotyping thousands of individual fish
simultaneously in a single Illumina HiSeq® System lane.1

Dr. Shawn Narum with a Chinook salmon he caught on a recent
fishing trip. NGS methods are used to identify fish and their
population of origin, enabling researchers to determine theoverall proportion of fish harvested from different regions.

Q: What is CRITFC and what is its mission?
Shawn Narum (SN): CRITFC is an organization that supports 4
Native American tribes in the US Pacific Northwest. That includes
the Nez Perce Tribe, which is here in the State of Idaho where Iwork, the Yakima Nation in Washington, and the Warm Springs andUmatilla tribes in Oregon. A shared concern among these tribes is theconservation and recovery of salmon. Salmon is a subsistence foodand has been an important part of their tribal cultures for thousands ofyears. Restoring salmon to the Columbia River Basin and being able topartake in harvesting the fish is a way of life for them. CRITFC provides
the tribes with technical information to support fish conservation
efforts. This includes promoting responsible management of theColumbia River Basin watershed, encouraging productive hatcherypractices, and coordinating with co-managers to determine the best
times and places for tribal and nontribal fisheries to harvest fish.
Q: What is your role at CRITFC?
SN: Our group performs genetic research on many of the fish species
found in the Columbia River. Salmonids (salmon and trout) make up
the largest focus of our work, with specific emphasis on Chinook
salmon and steelhead trout. We also study coho and sockeye
salmon, sturgeon, lamprey, and other resident trout species. We
use genetic methods and tools to monitor harvest operations ofcommercial, sport, and tribal fisheries, and to study the genetic effectsof hatchery practices and the genetic adaptation of fish populations tolocal environments.

Nathan Campbell developed the GT-Seq method, an ampliconsequencing-based approach that the Hagerman Genetics
Laboratory team uses to genotype thousands of fish, suchas the steelhead trout he’s holding.

April 2015
Q: What is the role of genetic analysis in restoring fish populations?
SN: In the last 2 decades, there has been a substantial effort to reform
hatchery practices. Tribal hatcheries strive to operate “integrated”
hatcheries that use local wild fish as broodstock, and other techniques
to produce genetically diverse fish that best represent local wild
populations. Genetic analysis enables maintenance of wild broodstock
diversity and mating success evaluation of hatchery-reared offspring.
We can also investigate whether domestication selection is occurring
even within the short generational time frame of a hatchery rearing
program. For most studies where they’re using primarily broodstock ofwild origin, we see that indeed the hatchery offspring are similar to theirwild counterparts and provide a demographic boost to help recovernative populations. This is in contrast to “segregated” hatcheries that are
divergent from local stocks and have greater potential for domestication
selection to accumulate over generations. These segregated stocks
are intended to increase harvest opportunities, but it is very difficult
to keep these fish entirely isolated from wild fish. We can use genetic
tools, such as parentage analysis, to identify mating events in thenatural environment and see if any negative genetic consequences areoccurring from hatchery practices.

Genetic analysis enables
maintenance of wild broodstock
diversity and mating successevaluation of hatchery-rearedoffspring.
Our work with coho salmon provides an interesting example of how
we use genetic analysis. Coho salmon were considered functionallyextinct throughout much of the Columbia River by the 1980s or earlier.
They were reintroduced successfully by Native American tribes into
multiple river systems, and large spawning runs have returned inrecent years (approximately 280,000 coho salmon in 2014). In coho
salmon and other fish species, we can study genetic adaptation of fish
populations to their local environments. In particular, we have used
NGS methods such as Restriction site Associated DNA sequencing(RAD-Seq) and RNA-Seq to detect signals of evolutionary adaptation
to environmental stressors such as warmer conditions. These methods
enable us to separate our analyses of adaptive variation affecting
functional genes from neutral variation appearing in noncoding partsof the genome. Neutral variations are better indicators of interbreeding
and genetic relationships between different populations.
Another role for genetic analysis is genetic tagging and monitoring ofstocks. There are several distinct salmonid stocks in the ColumbiaRiver Basin. Some of these stocks are fairly abundant, but there
are others that are listed under the Endangered Species Act. At
multiple points in their lifetimes, these healthy and endangered stocks
intermingle. Using genetic tagging and monitoring, we can determine
which stocks are prevalent in various harvest locations and at whattimes. This information is useful in fishery management to assist withcoordinating harvest seasons and locations.

Q: How do you obtain fish DNA samples for analysis?
SN: We need only approximately 10 nanograms of DNA, which we
can obtain from a very tiny piece of fin tissue. This sampling is easy
to do and is nonlethal. For example, as adult salmon return from theocean to their natal streams, the first dam they encounter is BonnevilleDam. We can divert some of those fish into a trap where we take atissue sample and release them back into the water quickly.
Q: What are some of the methods you have used for genetic
analysis of fish?
SN: When I started in 2002, we were using capillary gelelectrophoresis to look at about a dozen microsatellite markers.
In 2008, we started performing more SNP discovery and made thetransition to using whole-genome information. At that point, wewere still using low-throughput technologies. It wasn’t until 2010that we started using NGS. It became clear to us that some of theRAD-Seq methods being presented at the time could be very usefulfor our studies.2 We performed the first few runs on the Illumina
Genome Analyzer® II sequencing system, and did some more runs
at a core lab with a HiSeq® 2000 System. It was about 3 years ago
when we purchased the HiSeq 1500 System for our own lab. Since
then, we’ve been using the HiSeq System extensively for RAD-Seq,RNA-Seq, and now GT-Seq.
Q: Is RAD-Seq a productive approach for your studies?
SN: Yes, it’s very productive. RAD-Seq enables us to sequence aroundcertain restriction enzyme sites to obtain a reduced representation of thegenome. In salmonids, RAD-Seq enables us to genotype somewherearound 10,000 SNPs for about 50 individuals in a single lane of a HiSeq
run. After we began using RAD-Seq, we realized that genotyping by
sequencing was widely applicable for our work.

GT-Seq enables us to genotype
thousands of fish at relatively
low cost.
Q: What is GT-Seq and how did you develop it on theHiSeq platform?
SN: I have to give credit to Nathan Campbell, a scientist here in theHagerman Genetics Lab and first author on our paper describing thetechnique. He came up with the idea to use amplicon sequencing withIllumina technology. He developed the subsequent lab protocols andbioinformatic pipelines and deserves the recognition for developing
the GT-Seq method.
As Illumina NGS system users know, the HiSeq flow cell has alarge capacity for sequencing DNA fragments. In our RAD-Seq
studies, we were looking at 10,000 SNPs for 50 individual fish. We
didn’t need that many SNPs for high-throughput screening, andinstead used 5’ exonuclease fluorescence allelic discrimination
assays to genotype about 200 SNPs on 96 fish at a time. We
were satisfied with the quality of the data, but we realized that thisapproach wouldn’t be viable from a cost standpoint for the highthroughput types of studies we envisioned. There are hatcheriesApril 2015
throughout the Columbia River Basin, and from Alaska to California.
If we could genotype all the parents, we could efficiently tag themillions of offspring they produce. However, that means we’dneed to genotype tens to hundreds of thousands of individuals. In
developing the GT-Seq method, this was the throughput we werelooking to achieve within a realistic cost scenario.
One way to understand GT-Seq is that it is an inversion of the RADSeq ratio between the number of individuals and the number ofsequenced loci. Instead of sequencing a high number of RAD loci ona low number of individuals, GT-Seq uses the HiSeq System flow cell
capacity to sequence a reduced number of SNP loci (approximately100–500) on thousands of individuals per lane.

The HiSeq System has enabled
us to move from genetic researchusing just a handful of markers, togenomics where we’re coveringmuch more of the genome.
Q: How does GT-Seq reduce the cost of genotyping thousands of fish?
SN: GT-Seq is an amplicon sequencing method that uses 2 roundsof tailed PCR before the individual samples are mixed. In our initialdevelopment, we used 192 SNP loci to perform genotyping. The firstround of PCR amplifies all 192 SNP sites simultaneously and adds thesequencing primer sites. The second round of PCR adds 2 uniquebarcode sequences and the Illumina capture sites. The first barcode
maps back to 1 of the 96 individual wells in a microplate, and thesecond barcode identifies which microplate is holding that well. At
that point, we can mix the amplification products of all the individuals
together for loading on the HiSeq System, because we’ll be able toread the barcodes and tie all the resulting sequence reads back to
the individuals they came from. In addition to enabling us to genotype
thousands of fish in a single HiSeq lane, we realized even greater
economy from the dual barcode, well-and-plate identification system.
The number of individual barcodes we need to order is only the sumof 96 and the number of plates we’re going to use, rather than aseparate barcode for each 1 of the thousands of individuals.
Q: What is the principal benefit of GT-Seq?
SN: The main benefits are economy and throughput. Many of ourpopulation studies require analysis of many individuals. Relative toarray genotyping or the 5’ exonuclease assays we used previously,
GT-Seq enables us to genotype thousands of fish at relatively low cost.
We wouldn’t be able to carry out these increasingly large population
studies without GT-Seq.
Q: What were the technical challenges you and Nathan Campbell
encountered in developing GT-Seq?
SN: In a GT-Seq experiment, the sequencing objective is to covera panel of 100–500 custom amplicons containing targeted SNPs.
Initially, some of the loci were amplified more efficiently than others.
Without a way of balancing out amplicon production from all thetargeted loci, we would have seen a lot of sequencing coverage forthe most abundant amplicons, and inadequate coverage of the lessabundant ones. For our steelhead trout genotyping experiment, we
eventually came up with a protocol that would amplify our 192 SNP
genotyping loci relatively evenly. The panel is well-suited for highthroughput screening applications like the ones we’re performingat CRITFC.
After the amplification steps, there are thousands of individual
samples in microplate wells, each with 192 amplification products.
This is another step where the amount of DNA needs to bebalanced out. In this case, the objective is to prevent over- or
under-representation of individuals (rather than amplicons) in theHiSeq flow cell and the resulting sequencing data. We looked
at several methods to make sure that we would be loading asimilar amount of amplicon DNA from every individual. We founda commercial product, SequalPrep, that was compatible with
our throughput requirements and normalized the amount of PCR
product DNA across all the individuals.
Q: Have you tried GT-Seq in species other than steelhead trout?
SN: We’ve used the technique with steelhead trout and have alreadydeveloped panels for Chinook, coho, and sockeye salmon, as well
as Pacific lamprey. We’re genotyping large numbers of individuals of
those 4 salmonids from various populations in the Columbia River.
Our lamprey study is now underway as well.
Q: Would GT-Seq be useful for other species and applications?
SN: There is intense interest in performing amplicon sequencing infish and many other organisms as well. GT-Seq changes the cost
structure of genotyping by sequencing, where you want to look at
relatively few SNP markers across many individuals. Usually there area few hundred SNPs that are high targets of interest or candidates
for a particular trait. If you’re talking about cows, it might be milk
production. If you’re talking about aquaculture production of Atlantic
salmon, scientists are looking for a fast-growing fish with high-quality
filets. These are all scenarios where people might want to use ahigh-throughput, low-cost genotyping platform.

From our perspective, the numberof sequence reads per run isprobably the greatest benefit of
the Illumina HiSeq platform.
Q: What role did the HiSeq System play in your research?
SN: The HiSeq System has enabled us to move from genetic
research using just a handful of markers, to genomics where we’re
covering much more of the genome. We use the HiSeq System
extensively for RAD-Seq and also for RNA-Seq, which allows us
to look at gene expression across the transcriptome in thermal
adaptation studies. We’re now using the HiSeq System evenmore extensively for high-throughput genotyping with the GT-Seq
method. From our perspective, the number of sequence reads per
run is probably the greatest benefit of the Illumina HiSeq platform.
Two-hundred million reads per lane is a tremendous numberof reads that can be used to obtain the coverage needed forgenotyping by sequencing applications.

April 2015

References
1.

2.

Campbell NR, Harmon SA, Narum SR. Genotyping-in-Thousands bysequencing (GT-Seq): A cost effective SNP genotyping method based on
custom amplicon sequencing. Mol Ecol Resour. 2014; doi:
10.1111/1755-0998.12357. [Epub ahead of print]
RAD-Seq Genotypes Less, But Offers More, www.illumina.com/content/dam/illumina-marketing/documents/icommunity/article_2011_10_
Floragenex_RADseq.pdf

Learn more about the Illumina system mentioned inthis article:•HiSeq System, www.illumina.com/systems/hiseq_2500_1500.html

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