QUESTIONS.pdf

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Development of species-specific qPCR assays for the detection of Hawaiian bottomfish species from environmental DNA samples
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NFFT70002203124
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QUESTIONS

RFQ #NFFT70002203124

8/8/22

1) Question: Does the project truly require qPCR testing of the fish species, or is metabarcoding a suitable substitute?

Answer: The overarching intent of the project is to conduct large‐scale eDNA surveys of these target species at the island‐scale. For example, for Uku an ultimate goal is to generate a reliable index of detections and relative abundance in a range of habitats across the Hawaiian archipelago.

Metabarcoding was our first approach, but did a poor job of detecting snapper (16S and COI). An unpublished study was done collecting water (from Niskins) attached to a baited camera system, water filtered and amplified at 16S and COI. Neither assay resulted in any snapper detections ‐ even from samples taken directly adjacent to abundant schools of snapper (confirmed with video). The 12S

MiFishU assay was also attempted but had unreliable amplification and therefore was inconclusive.

While there remains much promise with the metabarcoding approach, we began exploring the development of qPCR primers that could more reliably detect target species, particularly if rare.

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