Alaska Sustainable Salmon Fund
Statement of Work
I.
Project Title: Monitoring Metapopulations of Chinook Salmon in the Kuskokwim and
Nushagak Drainages - Phase 2
II.
Project Number: 44913
III.
Principal Investigator
James E. Seeb, Research Professor
University of Washington (UW)
School of Aquatic and Fishery Sciences
1122 NE Boat Street Box 355020
Seattle, WA 98195
206-685-2097
jseeb@uw.edu
Co-Principal Investigators
William D. Templin, Principal Geneticist
Alaska Department of Fish and Game (ADF&G)
Commercial Fisheries Division (CF)
333 Raspberry Road
Anchorage, AK 99518
907-267-2234
bill.templin@alaska.gov
Lisa W. Seeb, Research Professor
UW, School of Aquatic and Fishery Sciences
1122 NE Boat Street Box 355020
Seattle, WA 98195
206-685-3723
lseeb@uw.edu
IV.
Project Period: 7/1/14 – 6/30/16
V.
AKSSF Objective: 2B-1
VI.
Project Description
1. Synopsis
Populations of Chinook salmon originating from coastal western Alaska, particularly
the Kuskokwim and Nushagak drainages, are a highly valuable resource for
subsistence fisheries. However, the metapopulation structure of these drainages is
poorly understood. This project will use high-density DNA sequencing to discover
novel high resolution single nucleotide polymorphisms (SNPs) to more precisely and
accurately differentiate populations of Chinook salmon from the Kuskokwim and
Nushagak drainages and provide genetic resources for mark-recapture studies.
PCSRF Objective: RM&E
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Additionally, this project will improve GSI estimates of origin for Chinook salmon
caught as bycatch intercepted in the Bering Sea walleye pollock fishery. This project
continues work begun in AKSSF project 44812.
2. Introduction
The use of genetic markers to identify discrete populations of salmon has become a
key feature of conservation and management activities in Alaska. Panels of up to 96
SNPs distinguish stock of origin of sockeye and chum salmon harvested in migrating
mixtures (e.g., the Western Alaska Salmon Stock Identification Program).
Discriminating stocks of Chinook salmon using SNPs has also worked well with the
exception of those inhabiting coastal western Alaska (defined as stocks inhabiting
Norton Sound, the lower Yukon River, the lower Kuskokwim River, and Bristol
Bay). Resolving these groups of stocks is increasingly important given the
management challenges of the federally declared fisheries disasters of 2011 and 2012.
AKSSF project 44515, High Resolution SNPs for Chinook, used next-generation
sequencing (restriction site associated DNA; RAD sequencing) to identify 10,944
total SNPs from western Alaska Chinook salmon. The results greatly improved
assignment to populations and also showed that individual assignment to population
of origin was possible with a large number of SNPs. However, populations of the
Kuskokwim and Nushagak drainages were not adequately resolved. Phase I of this
project (AKSSF project 44812, Monitoring Metapopulations of Chinook Salmon in
the Kuskokwim and Nushagak Drainages: Phase 1) targets specific populations to
develop high resolution SNPs to differentiate the two drainages and develop
resources for genetic mark-recapture studies. In Phase 2, the first objective is to
strengthen SNP discovery begun in Phase I by RAD sequencing four additional
ascertainment populations.
The second objective will represent the largest proportion of the project’s effort.
Results have shown that large numbers of SNPs are desirable for differentiating
closely related populations and conducting individual assignment. The goal of this
objective is to develop cost efficient panels of large numbers of SNPs that have the
power to be used not only in traditional GSI studies, but also in emerging applications
of parentage based tagging (PBT) and genetic mark-recapture (GMR) as well as to
refine estimates of important population parameters such as effective population size
(Ne). It is important to note that data has shown that different sets of SNPs provide
resolution at different scales. Some SNPs offer fine scale resolution of populations
within drainages while other SNPs provide improved differentiation on broader
geographic scales. This large amplicon panel will be composed of subpanels of SNPs
designed to offer resolution for interrogating inriver mixtures, nearshore mixtures,
and high seas mixtures (bycatch).
The third objective will transfer the technologies of next-generation sequencing
including RAD sequencing, amplicon sequencing, and comprehensive bioinformatics
analyses to ADF&G’s Gene Conservation Laboratory. This will be accomplished
through the graduate training of an ADF&G staff geneticist at the UW Seattle campus
and Aleknagik Field Camp in Bristol Bay.
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3. Location
Site: Aleknagik Field Camp
Latitude: 59.278333 N
VII.
Longitude: 158.623056 W
Objectives
1. Add SNP discovery populations to broaden representation of high resolution SNPs
for differentiating river of origin of individual fish (within and between the
Kuskokwim and Nushagak rivers)
2. Develop an amplicon panel for genetic stock identification of in-river, near-shore, and
high-seas mixtures
3. Transfer knowledge of high-throughput technologies and procedures to the ADF&G
Gene Conservation Laboratory
VIII. Methods
SNP Discovery (Objective 1)
The additional ascertainment populations to be used in RAD sequencing will be chosen
by ADF&G from their archives. Four populations from the Nushagak and Kuskokwim
rivers will be included to represent the diversity of life history types and geography. This
study will sequence 48 individuals from each population.
High density genotyping will be carried out by sequencing RAD tags using the
HiSeq2000 genome analyzer at the University of Oregon High Throughput Sequencing
Facility. Bioinformatic analysis will be conducted using locally developed PERL scripts
and the program Stacks which is freely available from the University of Oregon. It is
anticipated that between 5,000 and 10,000 SNPs will be discovered based upon
experience from existing efforts. Standard population genetic analyses will be conducted
on the full set of SNPs for all 48 individuals from the four ascertainment populations.
Development of Amplicon Panels (Objective 2)
The “amplicon” method is conceptually simple: genetic regions of interest are targeted
with specific primers or probes and enriched via Polymerase chain reaction (PCR) or
subtractive hybridization. Barcodes and adapters are then added with a second PCR or
ligation reaction. For PCR-based methods, these two steps may be combined by using
“fusion primers” which comprise (from 5’ to 3’) the adapter sequence for the sequencing
platform to be used, a barcode sequence, and the normal reverse-complemented
recognition sequence for PCR amplification. Multiplexing multiple loci in a single PCR
reaction can provide further efficiencies. Following PCR amplification, purification, and
quantification, individual libraries can be pooled in equimolar amounts and sequenced.
Given the number of reads provided by any given sequencing platform, the method can
be applied to any combination of individuals × loci, as long as adequate sequencing depth
at each locus is allowed. The sequence data for each locus are limited only by the read
length of the sequencing platform; they may contain multiple SNPs, insertion/deletion
markers (indels), or other informative genetic variation. Because next-generation
sequencing (NGS) methods read from single-stranded molecules, the haplotypes of each
gene copy are easily inferred without further cloning or statistical haplotype
reconstruction.
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Amplicon sequencing will be done on an Illumina Hi-Seq at the University of Oregon.
This study anticipates standardizing amplicon panels with other Pacific Salmon Treaty
genetics laboratories in the United States and Canada. The goal will be to develop an
amplicon panel of up to 768 SNPs. This panel will be composed of subpanels of 96 SNPs
that will be selected for resolution at three different scales: inriver, facilitating ADF&G
studies of migrating fry and smolts or returning adults; nearshore for studies of
emigrating smolts or composition of nearshore fisheries; and high seas for study of
migrating subadults or pollock bycatch.
Technology Transfer to ADF&G (Objective 3)
Tyler Dann from the ADF&G Gene Conservation Laboratory has been accepted for
admission into a Ph.D. program at the UW School of Aquatic and Fishery Sciences and
will spend one year on campus during the completion of this project. As a part of his
research, he will: 1) become proficient in RAD sequencing and bioinformatics, 2)
develop SNP panels, evaluate their resolving power, and aggregate them into larger
amplicon panels for high resolution genotyping, and 3) evaluate potential instrumentation
for amplicon sequencing to better inform decision making by ADF&G.
IX.
Benefits
This project will assist in identifying and cataloging the population structure of wild
Chinook salmon stocks, resulting in a significant advancement in the understanding of
the geographic and temporal diversity of Chinook salmon in Western Alaska. These data
will improve management of Chinook salmon populations of the Kuskokwim and
Nushagak regions and adjacent drainages that support important subsistence fisheries.
X.
Products, Milestones, and Timelines
 July 2014 – March 2015: SNP discovery sample coordination; DNA extraction;
library preparation; amplicon sequencing
 March – July 2015: Evaluation of amplicon subsets of 96 SNPs on varying scales
 August – December 2015: Bioinformatics analysis of amplicon sequence data;
identification of panel of SNPs
 January – April 2016: Statistical analysis of panel results
 March – May 2016: Draft report and manuscripts
 June 2016: Communicate data and final reporting to AKSSF
XI.
Budget
UW Budget
100 Personnel
200 Travel
300 Contractual
400 Supplies
500 Equipment
Subtotal
600 Indirect @ 26%
Total
Total
$159,809
$10,600
$46,682
$86,144
$0
$303,235
$78,841
$382,076
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UW Budget Narrative:
Line 100 Personnel ($159,809)
 Jim Seeb, Research Professor, will oversee all aspects of the project including
reporting for 2.5 months each year of the project: 5 months @ $10,000/month =
$50,000 + benefits @ 25% = $62,500.
 Lisa Seeb, Research Professor, will manage NGS sequencing, SNP discovery and
validation, assay selection, and contribute to reporting for 2.5 months each year of the
project: 5 months @ $10,000/month = $50,000 + benefits @ 25% = $62,500.
 Carita Pascal, Research Scientist IV, will conduct and supervise laboratory activities
including DNA library preparation for 1.5 months each year of the project: 3 months
@ $5,804/month + benefits @ 30% = $22,792.
 Garrett McKinney, Research Scientist III, Postdoctoral Fellow, will oversee field
collections and conduct DNA library preparation and bioinformatics of SNPs for 1
month each year of the project: 1 month @ $4,400 + 1 month @ $4780 + benefits @
30% = $12,017.
Line 200 Travel ($10,600)
The postdoctoral candidate will travel to the Alaska Salmon Program camps in Bristol
Bay during the summer field season for 30 days each year to assist ADF&G with sample
collection on the Nushagak or Kuskokwim rivers as well as DNA extraction at the camps.
The Alaska Salmon Program charges $150/day for camp fees which cover lodging and
meals.
 Airfare Seattle-Dillingham: 2 tickets @ $800/ticket = $1,600
 Camp fees: 30 days/year @ $150/day x 2 years = $9,000
Line 300 Contractual ($46,682)
Number
University Cost Centers Supplies
of items
UW HT Sequence Center (Sanger)
16
UW DNA extraction per 96
8
UW AB 7900 per 384
8
UW DNA preamp per 384
4
UW BioMark per 96
10
University of Oregon NGS
sequencing
14
Total
Unit
price
$448
$465
$164
$133
$1,015
$1,700
Total
$7,168
$3,720
$1,312
$532
$10,150
$23,800
$46,682
Line 400 Supplies ($86,144)
Illumina library prep for RAD
Number
of items
96
Unit
price
89
Total
$8,544
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Custom primers for 96 SNPS
Illumina Indices (bar codes)
Nextera Amplicon kit /96 samples
Total
8
16
16
$2,000
$950
$2,900
$16,000
$15,200
$46,400
$86,144
Line 600 Indirect ($73,681)
UW’s off-campus federally negotiated indirect rate through June 30, 2014, is 26%
excluding, among other costs, equipment, tuition remission, scholarships, fellowships,
and the portion of subgrants and subcontracts in excess of $25,000.
XII.
Match Budget
Summary (35%)
100 Personnel
200 Travel
300 Contractual
400 Supplies
500 Equipment
Subtotal
600 Indirect
Total
Total
$126,927
$2,900
$0
$0
$0
$129,827
$3,900
$133,727
The following entities will provide match:
 UW: $18,900
 ADF&G: $114,827
UW Match Budget
UW Match Budget
100 Personnel
200 Travel
300 Contractual
400 Supplies
500 Equipment
Subtotal
600 Indirect @ 26%
Total
Total
$15,000
$0
$0
$0
$0
$15,000
$3,900
$18,900
UW Match Budget Narrative:
Line 100 Personnel ($15,000)
 Jim Seeb, Research Professor, will oversee all aspects of the project including
reporting: .6 months @ $10,000/month + benefits @ 25% $7,500.
 Lisa Seeb, Research Professor, will manage NGS sequencing, SNP discovery and
validation, assay selection, and contribute to reporting: .6 months @ $10,000/month +
benefits @ 25% $7,500.
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Line 600 Indirect ($3,900)
UW’s off-campus federally negotiated indirect rate through June 30, 2014, is 26%
excluding, among other costs, equipment, tuition remission, scholarships, fellowships,
and the portion of subgrants and subcontracts in excess of $25,000.
ADF&G CF Match Budget
ADF&G CF Match Budget
100 Personnel
200 Travel
300 Contractual
400 Supplies
500 Equipment
Total
Total
$111,927
$2,900
$0
$0
$0
$114,827
ADF&G CF Match Budget Narrative:
Line 100 Personnel ($111,927)
Salaries are based on the State of Alaska salary calculator.
A Fishery Geneticist III (PCN 11-1964) will oversee coordination between the labs,
preparation of written products, and collaboration with UW researchers:
 FY15: 0.5 months @ $13,904/month = $6,952
A Fishery Geneticist II (PCN 11-1823) will conduct laboratory and statistical analysis,
bioinformatics, and preparation of written products:
 FY15: 2.5 months @ $9,025/month = $22,562
 FY16: 2.5 months @ $9,657/month = $24,142
A Fishery Biologist III (PCN 11-5073) will conduct selection and transfer of samples,
statistical analysis, selection of SNP loci, coordination with regional staff, and
preparation of written products:
 FY15: 2 months @ $9,379/month = $18,758
 FY16: 2 months @ $10,035/month = $20,071
A Fishery Biologist II (PCN 11-1375) will standardize laboratory methods and review
newly developed methods and implementation within the ADF&G laboratory:
 FY15: 1.75 months @ $8,592/month = $15,037
 FY16: 0.5 months @ $9,194/month = $4,597
Line 200 Travel ($2,900)
Tyler Dann will travel from Anchorage to Seattle for training and analysis:
FY15:
 Airfare: $700
 Per diem: 5 days @ $60/day = $300
 Hotel: 4 nights @ $150/night = $600
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
FY16:




Ground transportation: $100
Airfare: $700
Per diem: 3 days @ $60/day = $180
Hotel: 2 nights @ $150/night = $300
Ground transportation: $20
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