Aquaculture America 2024

February 18 - 21, 2024

San Antonio, Texas

SEQUENCING ALL THE FISHES IN THE DEEP BLUE SEA: IMPACTS OF SAMPLE STORAGE TIME, TEMPERATURE, AND BUFFER TYPE ON DNA QUALITY VALIDATED THROUGH LONG READ SEQUENCING AND ASSEMBLY

Jeremiah J Minich*, Malia L Moore* , Nicholas A Allsing , Emily R Murray, Loi Tran, Todd P Michael

Salk Institute for Biological Studies
10010 N Torrey Pines Road, La Jolla CA 92037

jminich@salk.edu



 There are over 1.6 million species of animals and 435 thousand species of plants on Earth. Long-read sequencing technology is enabling biologists to digitally archive the genomes of these organisms to understand fundamental basic questions in evolution, bolster conservation strategies in the midst of massive extinctions, and improve food production through crop and livestock genomics. One of the primary challenges however, is obtaining samples from field sites and preserving them in a way that ensures high quality DNA for long read sequencing. Here we demonstrate the impacts of storage time (0 days, 7 days, 21 days, and 42 days), storage temperature (4 Celsius , 22 Celsius), and storage buffers (95% EtOH and RNAlater )  on the preservation of fish blood across ten species of fish. We show that high quality DNA as measured by yield, purity, and fragment size can be obtained from samples stored at 95% EtOH at 4 Celcius for up to 6 weeks and RNAlater at room temperature for up to 3 weeks. We compare these storage impacts on sequencing read length, genome assembly quality, and methylation variation. We compare the impact of storage metrics on sequencing read length, quality and yield across  10 species of fishes sequenced using the gold standard ‘snap frozen’ method and demonstrate the feasibility of using this new storage method.  We validate by doing deep sequencing on three fish and compare assembly metrics. Here we show 95% ethanol protects against degradation for fish blood (22°C, ≤6 weeks). Using Nanopore, we assemble high quality reference genomes from three fish and two plant species (contig N50: 6.5-13.8Mb; BUSCO completeness: 94.4-99.2%; QV: 43.8 for M. esculenta).