NFFT70002203124 SF 18 Quote Form.pdf

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NFFT70002203124 July 26, 2022 NFFT70002203124

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BASE YEAR: Services, non‐personal, to provide all labor, equipment and materials necessary for development of species‐specific qPCR assays for the detection of Hawaiian bottomfish species from environmental DNA samples 1 ‐ 4 in accordance with the Statement of Work.

Target Species 1: Aprion virescens

Target Species 2: Pristipomoides filamentosus

Target Species 3: Etelis coruscans

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Target Species 4: Aphareus rutilans

Period of Performance: 9/1/2022 – 8/31/2023

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Services, non‐personal, to provide all labor, equipment and materials (unless otherwise provided herein) necessary for development of species‐specific qPCR assays for the detection of Hawaiian bottomfish species from environmental DNA samples Species 5 – 8 in accordance with the Statement of Work.

Target Species 5: Etelis carbunculus

Target Species 6: Pristipomoides zonatus

Target Species 7: Pristipomoides sieboldii

Target Species 8: Hyporthodus quernus

Period of Performance: 9/1/2023 – 8/31/2024

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STATEMENT OF WORK

Project Title: Development of species-specific qPCR assays for the detection of Hawaiian bottomfish species from environmental DNA samples.

Background/Overview:

Environmental DNA (eDNA) is a powerful and emerging tool that can complement existing surveys to expand and refine our understanding of fish populations and habitat use in marine ecosystems. Currently, eDNA techniques are in the process of becoming a part of the standard fishery and wildlife management toolkit for population detection, assessment, and monitoring.

Large-scale eDNA surveys can be cost-effective ways for assessing occupancy and relative abundance of target fisheries species in habitats and locations not currently surveyed by other techniques. eDNA studies commonly use quantitative polymerase chain reaction (qPCR) for the detection of low levels of target species' eDNA found in complex environmental samples such as a water sample (e.g., Shelton et al. 2022). However, robust application of these survey methods requires development of novel species-specific genomic resources, such as quantitative polymerase chain reaction (qPCR) assays, which do not currently exist for Hawaiian bottomfish species, an ecologically and commercially important group of fishes. In order to reliably detect and quantify environmental DNA from particular target species from a water sample, species-specific quantitative PCR assays must be developed and optimized.

qPCR is one of the most common techniques for quantification of nucleic acid molecules in biological and environmental samples. In real-time PCR, the accumulation of amplification product is measured as the reaction progresses, in real time, with product quantification after each cycle. First, amplification reactions are set up with PCR reagents and unique custom-designed primers. Reactions are then run in real-time PCR instruments. Detection of PCR products is enabled by the inclusion of a fluorescent reporter molecule in each reaction that yields increased fluorescence with an increasing amount of product DNA. Fluorescently-labeled sequence-specific probes are designed to particular gene regions that selectively amplify target species. When primers and probes are designed to be species-specific, fluorescence occurs only when the forward primer, reverse primer and probe hybridize to the target species DNA. There are a number of steps and reagents that require optimization and validation to ensure reproducible data.

Specialized thermal cyclers equipped with fluorescence detection modules are used to monitor the fluorescence signal as amplification occurs. The measured fluorescence is proportional to the total amount of amplicon; the change in fluorescence over time is used to calculate the amount of amplicon produced in each cycle. The main advantage of real-time PCR over PCR is that real-time PCR allows you to determine the initial number of copies of template DNA (the amplification target sequence) with accuracy and high sensitivity. Real-time PCR results can either be qualitative (the presence or absence of a sequence) or quantitative (copy number). Quantitative real-time PCR is thus also known as qPCR analysis. Probe-based qPCR provides the opportunity to process multiple samples in a single reaction (multiplex).

Objectives:

To meet our objectives, The Pacific Islands Fisheries Science Center (PIFSC) is seeking to contract an external commercial laboratory to develop, validate, and optimize species-specific probe-based qPCR assays for the identification of eight target Hawaiian Bottomfish species including Uku (Aprion virescens) and each of the Deep7 species: 'Ōpakapaka (Pristipomoides filamentosus), Onaga (Etelis coruscans), Lehi (Aphareus rutilans), Ehu (Etelis carbunculus), Gindai (Pristipomoides zonatus), Kalekale (Pristipomoides sieboldii), and the Hawaiian Grouper, Hapuʻupuʻu (Hyporthodus quernus). Multiple sets of species-specific primers will be designed, tested, and selected to achieve maximum sensitivity and reliability of each target species. The specificity of each primer set will be tested against DNA extractions of taxonomically-verified specimens of related and co-occurring species including all locally occurring Snapper and relevant Grouper species found in the Hawaiian Islands to ensure there is no cross-reactivity with non-target species. Best performing assays will be optimized and combined into multiplexed reactions, which will dramatically reduce per sample cost and increase scalability. The overall objectives of this contract is to develop quantitative assays that are reliable, sensitive and reproducible and can subsequently be used to conduct accurate and consistent detections and relative quantitative assays for each target fish species from eDNA water samples.

The intent of this acquisition is to obtain a two-year contract (base period with one one-year renewable option years) in accordance with this Statement of Work to purchase laboratory processing services including development of species-specific quantitative PCR primer-probe assays that are optimized to allow for the identification of eight target fisheries species in the Hawaiian Islands. qPCR primer-probe sets developed in this contract will ultimately be used as an efficient and cost-effective means to identify target fish species from low abundance environmental DNA samples. These novel resources will be used to support large-scale at-sea eDNA sampling efforts for the simultaneous detection (presence/absence) and quantification (relative abundance) of multiple targeted fish species from environmental water samples (e.g., Ramon-Laca et al. 2021; Shelton et al. 2022).

Scope of Work:

qPCR assay development services include qPCR assay design, assay validation, sample testing, and technology transfer. “Assay Design” shall mean the sequence of primers and probes designed and optimized by the Contractor.

Multiple sets of species-specific primers should be designed, tested, and selected to achieve reliable amplification of each target species. The specificity of each primer-probe set will be tested against DNA extractions of taxonomically verified specimens of related and co-occurring species including all 15 snapper species found in Hawaii to ensure there is no cross-reactivity with non-target species. PIFSC will provide tissue samples for all target species and co-occurring non-target species. The sensitivity of each assay will be tested and conditions optimized to allow the lowest limit of detection (LOD) possible while maintaining high specificity. Each of the eight species-specific primer-probes (assays) should be developed and optimized in singleplex reactions. Well-performing (high-sensitivity, reproducible) singleplex assays are the first priority. When possible prime-probe pairs should be grouped into sets of minimum 2 and maximum 4 primer-probe pairs in order to maximize the multiplexing capacity of the qPCR machine while minimizing costs required for reagents.

Tasks:

PIFSC will provide preserved tissue samples from morphologically identified fish samples for all 8 target species outlined in the Objectives and locally-occurring snapper species (see list in Miscellaneous). PIFSC will provide reference sequences for target mitochondrial genes.

The Contractor will develop, validate, and optimize up to eight sets of quantitative PCR assays one for each target species. This assay design process includes the following steps:

1. Generate sequence database and species alignment: Select target sequence for each species using alignments of available mitochondrial DNA reference sequences from public databases (e.g., GenBank, BOLD). PIFSC will provide species list and tissue samples from species of interest and co-occurring non-target species within the same family.

2. Primer Design: Primer design should follow standard practice with the goal of producing primers that are highly conserved in target taxa, but incorporate multiple mismatches, especially at the 3’ ends, with sequences from non-target taxa. Regions should be chosen from available data to maximize divergence among species and minimize variation within species. This will provide greater gene regions to choose from and therefore increase the likelihood that primers and probes designed will be able to discriminate the target from non-target species, while also ensuring intraspecific variants will amplify with the assay.

3. Assay Screening and Optimization: Test primer amplification in-silico to ensure specificity and compare against available sequences. In-vitro assay optimization. Primer probe-pairs should be evaluated for efficiency of amplification, cross-reactivity with closely related species.

4. Condition Optimization: Determine optimal reaction conditions for singleplex and multiplex assays including optimized reagent concentrations and volumes, primer-probe concentrations, cycling conditions, and annealing temperatures.

5. Experimentation using Standard Curves: Synthetic target gene fragments (e.g., gBlocks as in Conte et al. 2018) should be synthesized to provide standards for the target species. The performance of the standards from both genomic DNA and the synthetic fragments (e.g., gBlocks) should be compared in the multiplexed reactions and used to build standards (e.g., 10-fold dilutions down to 1 copy).

6. Validation of species-specific assays: Both singleplex and multiplex qPCR assays should be examined in-vitro for robustness, accuracy and specificity by testing genomic DNA from multiple individuals for each species (provided by PIFSC). To corroborate the specificity of each species-specific qPCR assay, qPCR detections should be verified by sequencing amplicons (e.g., Sanger sequencing) to confirm that the signal obtained in the qPCR assay was from the target species.

7. Multiplexing: selected singleplex primer-pair probes should be combined into multiplexes (each with up to a maximum of four primer-probe sets). Ideally, two multiplex reactions will provide simultaneous quantification of all eight target species.

Provide a comparison of performance of Singleplex vs. Multiplex assays and ensure resulting multiplexing does not affection amplification of target DNA. Multiplexing assays into as few as two 4-primer sets is a goal of this contract, however not strictly required. Target should be to combine optimized primer-probe pairs into multiplexes in as few sets as is feasible while maintaining high performance. If multiplexes are unfeasible, the Contractor will need to provide evidence of testing.

8. Determine specificity of detections: Specificity rates from in-vitro specificity tests should be conducted to determine the Limit of detection (LOD) and limit of quantification (LOQ) using lowest detectable standards. LOD can be defined as the lowest concentration of target DNA that can be detected with a defined level of confidence, with a 95% detection rate as the standard confidence level (Klymus et al.

2019). This should be accomplished by running a large number of replicate standard curves including low-concentration standards and determining the lowest standard concentration at which 95% of the replicates produce positive amplification of the target DNA. LOQ is defined as the lowest amount of target DNA in a sample that can be quantitatively determined with a stated precision, under stated experimental conditions (CLSI 2012). Precision can be assessed using the coefficient of variation (CV) of the measured concentrations of standards. The concentration of target DNA at an assay's LOQ may be greater than or equal to the concentration identified as the LOD but cannot be less than the LOD.

9. Comprehensive report providing all technical details of species-specific primer-probe pairs for each target species including methods used, performance and conditions must be disclosed, as would be expected for publication in the peer-reviewed literature. Reporting will include results of in-vitro specificity and reproducibility experiments of each assay, validation of efficiency and replication, limit of detection (LOD), limit of quantification (LOQ), and comparison of the performance of standards.

Period of Performance:

Base Period: September 1, 2022 – August 31, 2023

Option Period: September 1, 2023 – August 31, 2024

Place of Performance:

The contract will be performed at the Contractor’s facility. The Contractor’s facility needs to be a commercial molecular laboratory with separate dedicated purpose-built rooms for each stage of genetic processing procedures (DNA extraction, PCR, and post-PCR processes) in order to minimize the risk of sample cross-contamination. In addition, the facility must possess dedicated and up-to-date quantitative PCR (qPCR) equipment. The facility must be able to receive, store and process sensitive eDNA samples.

Government-Furnished Property (GFP): There will be no Government-Furnished Property with this Contract.

Security Requirements: There are no security requirements with this contract.

Applicable Standards:

These targeted assays should meet the validation and reporting standards outlined in Thalinger et

al. (2020) 5-level validation scale. The proposed contract should generate assays that meet the criteria and standards up to Level 4 on Thalinger et al. (2020)’s 5-level validation scale. These levels include:

● Level 1 (Assay Designed and tested on target tissue)

● Level 2 (Assay Optimized and tested on closely related non-target species)

● Level 3 (assay tested on eDNA samples, provided by PIFSC)

● Level 4 (Limit of detection established using in-vitro testing of synthetic standards). Level 4 expectations will be limited to in-vitro testing and will not be expected to include field testing. This contract does not include any field testing and is limited to in-silico and in-vitro testing of assays using tissues and samples provided by PIFSC.

Contractors meet or exceed existing guidelines for best practices along the validation workflow prior to assay design (Goldberg et al. 2016; Klymus et al. 2019; MacDonald & Sarre 2016). We recommend using guidelines established by Klymus et al. (2019) for standards of reporting limits of detection and quantification. Raman-Laca et al. (2021) provide helpful methodological workflow for developing qPCR assays for the simultaneous detection and quantification of multiple marine fish species using eDNA.

Special Requirements:

The Contractor must possess:

o Strong experience in qPCR assay design, development and validation o Experienced scientists functioning as project managers or study directors o Controlled laboratory environment with a systematic approach in preventing cross-contamination from environment, reagents, and supplies.

o Quantitative PCR (qPCR) system, preferably QuantStudio RealTime PCR System from Applied Biosystems by Thermo Fisher.

o Equipment for quality and quantity assessment as appropriate for DNA extractions, quantification, qPCR.

● All assays must be performed using the same techniques/kits and tested on the same qPCR platform. The contractor must use the Applied Biosystems QuantStudio RealTime PCR System using TaqMan Environmental Master Mix and TaqMan multiplex kits, unless negotiated with and approved by the PIFSC Point of Contact.

Deliverables Schedule:

Due Date Deliverable Activities % of Payment

Base Year:

August 31, 2023 Completed qPCR Assay for Target Species 1:

Aprion virescens

10%

August 31, 2023 Completed qPCR Assay for Target Species 2:

Pristipomoides filamentosus

8%

August 31, 2023 Completed qPCR Assay for Target Species 3: Etelis coruscans

8%

August 31, 2023 Completed qPCR Assay for Target Species 4:

Aphareus rutilans

8%

August 31, 2023 Acceptance of Report (Target species 1-4)

10%

August 31, 2024 Completed Multiplexed Assays (Target species 1- 4)

6%

Option Year 1

August 31, 2024 Completed qPCR Assay for Target Species 5:

Etelis carbunculus

8%

August 31, 2024 Completed qPCR Assay for Target Species 6:

Pristipomoides zonatus

8%

August 31, 2024 Completed qPCR Assay for Target Species 7:

Pristipomoides sieboldii

8%

August 31, 2024 Completed qPCR Assay for Target Species 8:

Hyporthodus quernus

7%

August 31, 2024 Completed Multiplexed Assays 5%

August 31, 2024 Acceptance of Final Report 14%

Acceptance Criteria:

Quality of Assays will be evaluated based on comprehensive reporting of conditions, optimizations, and validation experiments. In general, Contractors should report as much information as possible on the conducted development and validation steps.

Assays will be evaluated using the following criteria:

● Reproducibility

● Comprehensive specificity testing

● High specificity

● Low/No false positives and false negatives.

● High amplification efficiency

● Low limit of detection (LOD) – assay should perform well at low target DNA concentrations as found in environmental water samples.

● Robust assessments of parameters associated with low-concentration detection by each qPCR assay.

● Multiplexing assays into as few as two 4-primer sets is a goal of this contract. Target should be to optimize multiplexes in a as few sets as is feasible while maintaining high performance.

Miscellaneous:

List of target and non-target species to be subjected to in-silico and in-vitro specificity tests

Species Family Target/Non-target

Aphareus furca Lutjanidae (Snapper)

Non-target co-occurring

Aphareus rutilans Lutjanidae

Target Species

Aprion virescens Lutjanidae (Snapper)

Target Species

Etelis boweni Lutjanidae

Etelis carbunculus Lutjanidae

Etelis coruscans Lutjanidae

Lutjanus fulvus Lutjanidae

Lutjanus gibbus Lutjanidae

Lutjanus kasmira Lutjanidae (Snapper)

Lutjanus sebae Lutjanidae

Pristipomoides auricilla Lutjanidae

Pristipomoides filamentosus

Lutjanidae

Pristipomoides sieboldi Lutjanidae

Pristipomoides zonatus Lutjanidae

Randallichthys filamentosus

Lutjanidae

Hyporthodus quernus Serranidae (Grouper)

References:

Conte J, Potoczniak MJ, Tobe SS. Using synthetic oligonucleotides as standards in probe-based qPCR.

BioTechniques. 2018; 64(4):177–9. https://doi.org/10.2144/btn-2018-2000 PMID: 29661012

Klymus, K. E., Merkes, C. M., Allison, M. J., Goldberg, C. S., Helbing, C. C., Hunter, M. E., Jackson, C.

A., Lance, R. F., Mangan, A. M., Monroe, E. M., Piaggio, A. J., Stokdyk, J. P., Wilson, C. C., & Richter, C. A. (2019). Reporting the limits of detection and quantification for environmental DNA assays.

Environmental DNA, 2, 271–282. https://doi.org/10.1002/edn3.29.

Goldberg, C. S., Turner, C. R., Deiner, K., Klymus, K. E., Thomsen, P. F., Murphy, M. A., & Taberlet, P.

(2016). Critical considerations for the application of environmental DNA methods to detect aquatic species. Methods in Ecology and Evolution, 7(11), 1299–1307. https://doi.org/10.1111/2041-210X.12595

MacDonald, A. J., & Sarre, S. D. (2017). A framework for developing and validating taxon-specific primers for specimen identification from environmental DNA. Molecular Ecology Resources, 17(4), 708–720. https://doi.org/10.1111/1755-0998.12618

Ramon-Laca A, Wells A, Park L (2021) A workflow for the relative quantification of multiple fish species from oceanic water samples using environmental DNA (eDNA) to support large-scale fishery surveys. PLoS ONE 16(9): e0257773. https://doi.org/10.1371/journal.pone.0257773

Shelton AO et al. 2022 Environmental DNA provides quantitative estimates of Pacific hake abundance and distribution in the open ocean. Proc. R. Soc. B 289: 20212613.

https://doi.org/10.1098/rspb.2021.2613.

Thalinger B, Deiner K, Harper LR, et al. A validation scale to determine the readiness of environmental DNA assays for routine species monitoring. Environmental DNA. 2021;3:823– 836.

https://doi.org/10.1002/edn3.189