Breaking the cold chain: solutions for room temperature preservation of mosquitoes leading to high quality reference...

The Earth BioGenome Project (EBP) is a global endeavour to produce reference genomes for all described eukaryotic species. The majority of described species are arthropods, which tend to be small and require taxonomic expertise to identify at species level. Therefore, the ability to collect and preserve specimens in a suitable way for long read and Hi-C data generation using very simple approaches with minimal infrastructure will be important in scaling up reference genome generation. Field collections requiring dry ice or dry shippers can be logistically challenging to arrange, are notoriously expensive, and DNA degrades rapidly if ultra-cold temperature is not maintained, which is devastating given how expensive and time consuming fieldwork can be.

Using Anopheles mosquitoes as an insect representative, we evaluate how well different preservation liquids protect high molecular weight DNA, RNA, and nuclei for Hi-C at room temperature when mosquitoes are held intact versus slightly squished. We find that squished samples stored in 100% ethanol and Allprotect held at room temperature for 1 week result in excellent preservation of both high molecular weight DNA and nuclei for Hi-C. Other tested buffers, including RNAlater, EDTA at several pHs, and dimethyl sulfoxide EDTA salt solution (DESS), performed satisfactorily for long read data generation and RNA retrieval, but less ideally for Hi-C, which may have bigger negative impacts when aiming to generate data for organisms with larger genomes.

We present multiple viable options for room temperature collection and/or shipment for arthropod samples, with direct suggestions for shipment and storage of mosquitoes and similarly sized specimens. Further exploration across a broader range of species will hopefully enable cheaper and more widely available reference genome generation globally.